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1 THE DEVELOPMENT OF ANTIMICROBIAL AGENTS, PAST, PRESENT AND FUTURE 1

1 THE DEVELOPMENT OF ANTIMICROBIAL AGENTS, PAST, PRESENT AND FUTURE 1
1.1 The social and economic importance of antimicrobial agents 1
1.2 All outhne of the historical development of antimicrobial agents 2
1.3 Reasons for studying the biochemistry and molecular biology of antimicrobial compounds 9
1.4 Uncovering the molecular basis of antimicrobial action iO
1.5 Current trends in the discovery of antimicrobial drugs 14
1.6 .Scope and layout of the book 15
2 VULNERABLE SHIELDS—THE CELL WALLS OF BACTERIA AND FUNGI 17
2.1 Functions of the ceU wall 17
2.2 .Structure of the bacterial wall 17
2.3 Structure and biosynthesis of peptidoglycan 22
2.4 Antibiotics that inhibit peptidoglycan biosynthesis 29
2.5 Drugs that interfere with the biosynthesis of the cell wall of mycobacteria 39
2.6 The fungal cell wall as a target for antifungal drugs 41
3 ANTIMICROBIAL AGENTS AND CELL MEMBRANES 47
3.1 Microbe killers: antiseptics and disinfectants 47
3.2 Cationic peptide antibiotics 52
3.3 lonophoric antibiotics 54
3.4 Antifungal agents that interfere with the function and biosynthesis of membrane sterols 59
4 INHIBITORS OF NUCLEIC ACID BIOSYNTHESIS 65
4.1 Compounds affecting the biosynthesis and utilization of nucleotide precursors 66
4.2 Nucleoside analogues 70
4.3 Inhibitors of the reverse transcriptase of the human immunodeficiency virus 72
4.4 Antibacterial inhibitors of topoisomerases 75
4.5 Inhibitors of DNA-dependent RNA polymerase 79
4.6 Inhibition of nucleic acid synthesis by interaction with DNA 80
5 INHIBITORS OF PROTEIN BIOSYNTHESIS 85
5.1 Ribosomes 85
5.2 Stages in protein biosynthesis 87
5.3 Puromycin 90
Contents
5.4 Inhibitors of aminoacyl-tRNA formation 91
5.5 Intiibitors of initiation and translation 92
5.6 Inhibitors of peptide bond formation and translocation 98
6 ANTIMICROBIAL DRUGS WITH OTHER MODES OF ACTION 107
6.1 Nitroheterocyclic antimicrobial agents 107
6.2 A unique antifungal antibiotic—griseofulvin 108
6.3 Antiviral agents 109
6.4 Antiprotozoal agents 113
7 ATTACK AND DEFENSE: DRUG TRANSPORT ACROSS CELL WALLS AND
MEMBRANES 121
7.1 Cellular permeability barriers to drag penetration 121
7.2 Multidrug efflux 126
7.3 Facilitated uptake of antimicrobial drugs 129
8 THE GENETIC BASIS OF RESISTANCE TO ANTIMICROBIAL DRUGS 135
8.1 Mutations and the origins of drug-resistance genes 136
8.2 Gene mobility and transfer in bacterial drug resistance 140
8.3 Global regulators of drug resistance in Gram-negative bacteria 147
8.4 Genetic basis of resistance to antifungal drugs 147
9 BIOCHEMICAL MECHANISMS OF RESISTANCE TO ANTIMICROBIAL DRUGS 149
9.1 Enzymic inactivation of drugs 149
9.2 Loss or downregulation of drag activation 161
9.3 Modification of drug targets 162
9.4 Drug efflux pumps 167
9.5 Other mechanisms of resistance 170
9.6 Drug resistance and the future of antimicrobial chemotherapy 172
Index 175
Preface
The rapid advances made in the study of the synthesis, stractiire and ftinction of biological macromolecules in
the last fifteen yeai^s have enabled scientists concerned with antimicrobial agents to achieve a considerable meas-ure of understanding of how these substances inhibit cell growth and division. The use of antimicrobial agents
as highly specific inhibitors has in turn substantially assisted the investigation of complex biochemical processes.
The literature in this field is so extensive, however, that we considered an attempt should be made to draw to-gether in an introductory book the more significant studies of recent years. This book, which is in fact based on
lecture courses given by us to undergraduates at Liverpool and Manchester Universities, is therefore intended as
an introduction to the biochemistry of antimicrobial action for advanced students hi many disciplines. We hope
that it may also be useful to established scientists who are new to this area of research.
The book is concerned with a discussion of medically important antimicrobial compounds and also a num-ber of agents that, although having no medical uses, have proved invaluable as research tools in biochemistry.
Our aim has been to present the available information hi a simple and readable way, emphasizing the established
facts rather than more controversial material. Whenever possible, however, we have indicated the gaps in the
present knowledge of the subject where further hifomiation is required. We have avoided the use of literature ref-erences in the text; instead we have included short lists of key articles and books for further reading at the end
of each chapter.
We have drawn on the work of many scientists and we are especially pleased to express our thanks to those
who have given us permission to reproduce their original diagrams and photographs. We are also grateful to the
Pharmaceuticals Division of Imperial Chemicals Industries Ltd, for providing the necessary facilities for the
preparation of this book.
Abbreviations used without definition for common biochemical substances are those recommended by the
Biochemical Journal (1970).
T. J. FRANKLIN
June 1970 G. A. SNOW
Preface to the
sixth edition
Since the publication of the previous edition, the problems posed by infectious diseases afflicting human beings
and their domestic animals have continued to attract worldwide concern. The menace of AIDS remains unabated
and Is of epidemic proportions in parts of the developing world. The spread of multidrug-resistant bacteria, spo-radic outbreaks of meningitis, bacteriaily mediated food poisoning and dangerous new viral infections regularly
alarm the public. The mysterious emergence of bovine spongiform encephalitis, or mad cow disease, and its
human equivalent, new variant Creuzfeldt-Jacob disease, presents a major challenge to medical and veterinary
science. The potential of both wild-type and genetically modified Infectious micro-organisms for bioterrorlsm is
especially worrying. Fortunately, against this rather gloomy picture can be set some significant advances. Rapid
nucleic acid and protein sequencing technology, sophisticated computer software to organize and analyze the
huge amounts of emerging sequence data and spectacular advances in the X-ray crystallographlc and nuclear
magnetic resonsance spectroscopic Investigation of macromolecular structures have all contributed to advances
In our understanding of the mechanisms of antimicrobial action and drug resistance. These encouraging devel-opments should facilitate the discovery of new drugs. Some valuable new antimicrobial drugs have emerged, in-cluding novel agents against influenza and further developments in the treatment of AIDS where combinations
of anti-IIIV drugs continue to bring hope to victims of this appalling disease.
Sadly, my fornier co-author, Alan Snow, died In 1995 and although I must therefore take sole responsibil-ity for the contents of this new edition, I have been greatly helped by the Incisive comments of the following sci-entists; Drs. Boudewijn de Jonge, John Rosamond, Thomas A. Keating, Peter Doig, Ann Ealdn and Wright W.
Nichols. Dr. Paul M. O’Neill of Liverpool University kindly provided me with an advance copy of a comprehen-sive review of artemlslnln and related endoperoxldes co-authored by himself and Dr. Gary II. Posner of The
Johns Hopkins University. Over the years many helpful comments and criticisms from our readers have been in-valuable in planning future editions. I hope they will continue to let me have their views. As in the previous edi-tions, the discussions are mainly concerned with antimicrobial agents In medical. Industrial and domestic use.
Finally, I would like to express my thanks to AstraZeneca for the provision of facilities which have made
this new edition possible and especially to the Mereside Library staff for their help and advice on many
occasions.
TREVOR J. FRANKLIN
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sntiimicroljisl 3ci6nts
Few developments in the history of medicine have had
such a profound effect upon human life and society as
the development of the power to control infections by
micro-organisms. In 1969 the Surgeon General of the
United States stated that it was time ‘to close the book
on infectious diseases’. His optimism, which was then
shared by many, seemed justified at the time. In the
fight against infectious disease, several factors had
combined to produce remarkable achievements. The
first advances were mainly the result of improved san-itation and housing. These removed some of the worst
foci of infectious disease and limited the spread of in-fection ttaough vemiin and insect parasites or by con-taminated water and food. The earliest effective direct
control of infectious diseases was achieved through
vaccination and similar immunological methods
which still play an important part in the control of in-fection today. The use of antimicrobial drugs for the
control of infection was almost entirely a development
of the twentieth century, and the most dramatic devel-opments have taken place only since the 1930s.
Surgery is no longer the desperate gamble with human
life it had been in the early nineteenth century. By the
late nineteenth century, the perils of childbirth had
been greatly lessened with the control of puerperal
fever. The death of children and young adults from
meningitis, tuberculosis and septicemia, once com-monplace, was, by the late 1960s, unusual in the devel-oped world.
Unfoituoately, since the heady optimism of the
1960s we have learned to our cost that microbial
pathogens still have the capacity to spring unpleasant
surprises on the world. The problem of acquired bacte-rial resistance to drugs, recognized since the very be-ginning of antimicrobial chemotherapy, has become
ever more menacing. Infections caused by the tubercle
bacillus and Staphylococcus aureus, which were once
readily cured by drug therapy, are now increasingly
difficult or even impossible to treat because of wide-spread bacterial resistance to the available drugs. Nor
is resistance confined to these organisms; many other
species of bacteria as well as fungal pathogens, viruses
and protozoa, have also become drug-resistant. The
ability of micro-organisms to kill or disable the more
vulnerable members of society, especially the very
young and old and patients with weakened immune de-fences, is reported io the media almost daily. Alarming
reports of lethal enteric infections, meningitis and
‘flesh-eatiog’ bacteria have become depressingly fa-miliar. If this were not enough, the spectre of the virus
(HIV) infection which leads to AIDS (acquired im-mune deficiency syndrome) threatens human popula-tions around the world, in nations both rich and poor.
Dewelopment of antimicrobial agents
Drug therapy for AIDS can, initially at least, be very
effective, but for the occasional outbreaks of terrifying
viral infections such as Ebola and Lassa fever, there
are no treatments,. Perhaps more worrying than these
sporadic African hemorrahgic fevers, however, is the
perceived risk of epidemic or pandemic infections
caused by the recently discovered severe acute respira-tory syndrome (SARS) vims, or novel recombinations
of highly virulent influenza viruses with the potential
for causing severe illness and death on a catastrophic
scale. In recent years the mosquito-bome West Nile
virus, which in some cases can cause a potentially fatal
encephalitis, has been the subject of increasing con-cern in North America. Throughout much of the trop-ical and subtropical world, malaria continues to exact
a dreadful toll on the health and lives of inhabitants.
Although mass movements of populations and the fail-ure to control the anophelene mosquito insect vector
are major factors in the prevalence of malaria, the in-creasing resistance of the malarial protozoal parasite to
drag treatment is of the greatest concern.
Thirty years ago serious infections caused by
fungi were relatively rare. More common infections
like thrash and ringworm were more of an unpleasant
nuisance than a serious threat to health. Today, how-ever, many patients with impaired immunity caused by
HIV infection, cytotoxic chemotherapy for malignant
disease, or the immunosuppressive treatment associ-ated with organ graft surgery, are at risk from danger-ous fungal pathogens such as Pneumocystis carinii and
Cryptococcus neoformans. Less viralent organisms
like Candida albicans can also be devastating in im-munocompromised patients. Inevitably, the increasing
use of antifungal drugs to control these infections re-sults in the emergence of drug-resistant pathogens.
Fortunately, despite the threats posed by drag-resistant bacteria, virases, protozoal parasites and fun-gal pathogens, the current scene is not one of unre-lieved gloom. Most bacterial and fungal infections can
still be treated successfully with the remarkable array
of drugs available to the medical (and veterinary) pro-fessions. Work continues to develop drags effective
against resistant pathogens, and there has been major
progress against virases causing AIDS, influenza and
herpes infections. Vaccines are remarkably successful
in preventing some bacterial and viral infections. In-deed, outstanding amongst the medical achievements
of the twentieth century was the eradication of small-pox and the dramatic reduction in the incidence of
poliomyelitis through mass vaccination programmes.
A further incentive to the discovery and development
of novel antimicrobial drugs and vaccines is the threat
of bioterorrism, which could exploit conventional
lethal pathogens such as anthrax and smallpox or even
micro-organisms genetically manipulated to extraordi-nary levels of virulence and drag resistance.
Finally, mention must be made of the recent and
unexpected emergence of infectious prions which are
associated with such devastating neurological patholo-gies as Creuzfeldt-Jacob disease (CJD) and new vari-ant CJD, or mad cow disease in humans. It is now al-most universally accepted that the heat and chemically
resistant prion particles are proteinaceous and contain
no detectable nucleic acid-encoded information. Infec-tion is transmitted by various routes, including oral
ingestion, for example, in contaminated food, by injec-tion, or during surgery and possibly by blood transfu-sion. At present there is no effective drag treatment to
arrest or delay the relentless progression of the infec-tion, which involves the conversion of a normal neu-ronal protein of unknown function to an insoluble and
newly infectious form through interaction with the in-vading, closely related prion protein. The disturbing
possibility of a slowly developing epidemic of new-variant CJD is spurring efforts to find drags or vac-cines to control prion infections.
1.2 An outline of the historical development
of antimicrobial agents
1.2.1 Early remedies
Among many traditional and folk remedies, three
sources of antimicrobial compounds have survived to
the present day. These are cinchona bark and Artemisia
annua (Chinese quing hao su) for the treatment of
malaria and ipecacuanha root for amebic dysentery.
Cinchona bark was used by the Indians of Peru for
treating malaria and was introduced into European
medicine by the Spanish in the early seventeenth cen-tury. The active principle, quinine, was isolated in
1820. Quinine remained the only treatment for malaria
until well into the twentieth century and still has a
1.2 An outline of the historical dewelopment of antimicrobial agents
place in chemotherapy. The isolation of artemisinin,
the active compound in Artemisia annua, by Chinese
scientists is much more recent and only in recent years
has its therapeutic potential against malaria been fully
appreciated. Ipecacuanha root was known in Brazil
and probably in Asia for its curative action in diarrheas
and dysentery. Emetine was isolated as the active con-stituent in 1817 and was shown in 1891 to have a spe-cific action against amebic dysentery. In combination
with other drugs, it is still used for treating this disease.
These early remedies were used without any under-standing of the nature of the diseases. Malaria, for ex-ample, was thought to be caused by ‘bad air’ (mal’aria)
arising from marshy places; the significance of the
blood-borne parasite was not recognized until 1880,
and only in 1897 was the anophelene mosquito proved
to be the specific insect vector when the developing
parasite was observed in the intestine of the mosquito.
1.2.2 Antiseptics and disinfectants
The use of disinfectants and antiseptics also preceded
an understanding of their action, and seems to have
arisen from the obsen’ation that certain substances
stopped the putrefaction of meat or the rotting of
wood. The temi ‘antiseptic’ itself was appai^ently first
used by Pringle in 1750 to describe substances that
prevent putrefaction. The idea was eventually applied
to the treatment of suppurating wounds. Mercuric
chloride was used by Arabian physicians in the Middle
Ages to prevent sepsis in open wounds. However, it
was not until the nineteenth century that antiseptics
came into general use in medicine. Chlorinated soda,
essentially hypochlorite, was introduced in 1825 by
Labarraque for the treatment of infected wounds, and
tincture of iodine was first used in 18.39. One of the
earliest examples of disinfection used in preventing the
spread of infectious disease was recorded by Oliver
Wendell Holmes in 1835. He regularly washed his
hands in a solution of chloride of lime when dealing
with cases of puerperal fever and thereby greatly re-duced the incidence of fresh infections, as did Ignaz
Semmelweiss in Vienna a few years later. These pio-neer attempts at antisepsis were not generally accepted
until Pasteur’s publication in 1863 identifying the mi-crobial origin of putrefaction. This led to an under-standing of the origin of infection and suggested the
rationale for its prevention. As so often in the history
of medicine, a change in practice depended upon the
personality and persistence of one man. In antiseptics
this man was Lister. He chose phenol, the antiseptic
which had been introduced by Lemaire in 1860, and
applied it vigorously in surgery. A 2.5% solution was
used for dressing wounds and twice this concentration
for sterilizing instruments. Later he used a spray of
phenol solution to produce an essentially sterile envi-ronment for canying out surgical operations. The pre-vious state of surgery had been deplorable; wounds
usually became infected and the mortality was ap-palling. The effect of Lister’s measures was revolu-tionary, and the antiseptic technique opened the way to
great surgical advances. Even at this time, about 1870,
the use of antiseptics was still empirical. An under-standing of their function began with the work of
Koch, who from 1881 onwards introduced the tech-niques on which modem bacteriology has been built.
He perfected methods of obtaining pure cultures of
bacteria and growing them on solid media, and he
demonstrated practical methods of sterile working.
Once it became possible to handle bacteria in a con-trolled environment, the action of disinfectants and an-tiseptics could be studied. The pioneer work on the sci-entific approach to this subject was published by
Kronig and Paul in 1897.
Since that time, the history of antiseptics has been
one of steady but unspectacular improvement. Many of
the traditional antiseptics have continued in use in re-fined fomis. The phenols have been modified and
made more acceptable for general use. Acriflavine, in-troduced in 1913, was the first of a number of basic an-tiseptics. It had many years of use but was displaced by
colourless cationic antiseptics and the non-ionic tri-closan (acriflavine is bright orange). In surgery the an-tiseptic era gave way to the aseptic era in which the em-phasis is on the avoidance of bacterial contamination
rather than on killing bacteria already present. All the
same, infection of surgical wounds remains a constant
risk and antiseptics are still used as an extra precaution
or second line of defence. Surgical staff also ‘scrub up’
with mild antiseptic solutions before entering the oper-ating theatre. Disinfectants play an important pait in
the hygiene of milking sheds, broiler houses and other
places where strict asepsis is impracticable.
Dewelopment of antimicrobial agents
1.2.3 The beginnings of chemotherapy
The publications of Pasteur and Koch firmly estab-lished that micro-organisms are the cause of infectious
disease, though for some diseases the causative organ-isms still remained to be discovered. It was also known
that bacteria are killed by various antiseptics and dis-infectants. Not surprisingly, attempts were made to kill
micro-organisms within the body and so end the infec-tion. Koch himself carried out some experiments with
this aim. He had shown that mercuric chloride is one of
the few disinfectants that kill the particularly tough
spores of the anthrax bacillus. Koch therefore tried to
cure animals of anthrax infection by injecting them
with mercuric chloride. Unfortunately the animals
died of mercury poisoning and their organs still con-tained infectious anthrax bacilli. A slightly more suc-cessful attempt to cure an infection with a toxic agent
was made by Lindgard in 1893. He treated horses suf-fering from surra, a disease now known to be caused
by trypanosomes, with arsenious oxide. There was
some improvement of the disease, but the compound
was too toxic to be generally useful.
Chemotherapy, however, really began with Paul
Ehrlich. During the ten years from 1902 onwards
Ehrlich’s work foreshadowed many of the concepts
which have governed subsequent work on antimicro-bial agents. His first ideas arose from studies with
‘vital stains’, dyestuffs that were taken up selectively
by living tissue. One such dye was methylene blue,
which in the animal body is concentrated in nervous
tissue. Ehrlich showed that the same dye is readily
taken up by malaria parasites in the blood so that they
become deeply stained. Consequently methylene blue
was tried against human malaria and showed some ef-fect, though not sufficient to make it a useful treat-ment. Nevertheless, this minor success started a line of
thought that was to prove of the greatest significance.
Ehrlich believed that antimicrobial agents must be es-sentially toxic compounds and that they must bind to
the micro-organism in order to exert their action. The
problem was to discover compounds having a selective
action against the microbial cell rather than the cells of
the host animal. Starting from methylene blue, Ehrlich
began to search for other dyestuffs that would affect
protozoal diseases. In 1904, after testing hundreds of
available dyes, he eventually found one that was effec-tive against trypanosomiasis in horses. This com-pound, called trypan red, was a significant landmark in
the treatment of microbial infections since it was the
first man-made compound that produced a curative ef-fect.
However, it was not in the field of dyestuffs that
Ehrlich achieved his greatest success. Following the
early work on the treatment of trypanosomiasis with
arsenious oxide, Koch tested the organic arsenical,
atoxyl (Figure 1.1). This compound produced the first
cures of sleeping sickness, a human trypanosomal dis-ease. Unfortunately, however, the compound produced
serious side effects, with some patients developing
optic atrophy. The curative effect of this compound
stimulated Ehrlich to make other related arsenicals. He
tested these on mice infected experimentally with try-panosomiasis and showed that curative action did not
parallel toxicity to the mice. This suggested that if
enough compounds were made, some would have suf-ficiently low toxicity to be safe as chemotherapeutic
agents. Elhrlich continued his search for compounds
active against various micro-organisms and showed
that arsenicals were active against the causative organ-ism of syphilis. He began a massive search for an
organoarsenical compound that could be used in the
treatment of this disease and eventually in 1910 dis-covered the famous drug salvarsan (Figure 1.1). This
0=As ‘  AsO
p-Aminophenylarsenoxide
AsO
Marpharsen
FIGURE 1.1 Arsenical compounds used in the early treat-ment of trypanosomiasis or syphilis.
1.2 An outline of the historical dewelopment of antimicrobial agents
dmg and its derivative, Neosalvarsaii, became the stan-dard treatment for syphilis. Coupled with bismuth
therapy, they remained in use until supplanted by peni-cillin in 1945. This was the most spectacular practical
achievement of Ehrlich’s career, but scientifically he is
remembered at least as much for his wealth of ideas
that have inspired workers in the field of chemotherapy
down to the present day. These ideas are so important
that they deserve separate consideration.
1.2.4 The debt of chemotherapy to Ehrlich
The very term ‘chemotherapy’ was invented by
Ehrlich and expressed his belief that infectious disease
could be controlled by treatment with synthetic chem-icals. Successes since his day have entirely justified his
faith in this possibility. He postulated that cells possess
chemical receptors which are concerned with the up-take of nutrients. Drugs that affect the cell must bind to
one or other of these receptors. The toxicity of a drug
is determined partly by its distribution in the body.
However, in the treatment of an infection, the binding
to the parasite relative to the host cell determines the
effectiveness of the compound. Thus Ehrlich recog-nized the importance of Cjuantitative measurement of
the relationship between the dose of a compound re-cjuired to produce a therapeutic effect and the dose that
causes toxic reactions. Such measurements are still of
prime importance in chemotherapy today.
Ehrlich pioneered methods that have since be-come a mainstay of the search for new drugs. One as-pect of his approach was the use of screening. This is
the application of a relatively simple test to large num-bers of compounds in order to obtain evidence of
biological activity in types of chemical structure not
previously examined. Modern drug-discovery labora-tories usually employ sequences or cascades of screen-ing tests, often beginning with a purified enzyme from
the target organism, followed by test cultures of the
pathogen and sophisticated evaluation in experimental
animals. Vast numbers of compounds may be screened
in the primaiy in vitro test, with the succeeding screens
used to progressively filter out insufficiently active or
potentially toxic compounds until a very limited set of
compounds is considered to be sufficiently effective to
warrant evaluation in a model infection in experimen-tal animals.
The second of Ehrlich’s methods was the synthe-sis of chemical variants of a compound exhibiting an
interesting but not optimal level of activity. The new
compounds were examined for increased activity or
for improvements in some other property, such as re-duced toxicity. Any improvement found was used as a
guide to further synthesis, eventually arriving by a se-ries of steps at the best possible compound. These
methods are now so well accepted that their novelty in
Ehrlich’s day can easily be forgotten. They depend on
the thesis that a useful dmg possesses an ideal combi-nation of structural features which cannot be predicted
at the outset. A compound approximating this ideal
will show some degree of activity and can therefore act
as a ‘lead’ towards the best attainable structure.
According to Ehrlich, a chemotherapeutic sub-stance has two functional features, the ‘haptophore’ or
binding group which enables the compound to attach
itself to the cell receptors, and the ‘toxophore’ or toxic
group which brings about an adverse effect on the cell.
This idea has had a continuing influence in subsequent
years. In cancer chemotherapy it has frequently been
used in attempts to bring about the specific concentra-tion of toxic agents or antimetabolites in tumour cells.
In antimicrobial research it has helped to explain some
features of the biochemical action of antimicrobial
compounds.
Ehrlich also recognized that compounds acting
on microbial infection need not necessaiily kill the in-vading organism. It was, he suggested, sufficient to
prevent substantial multiplication of the infectious
agent, since the normal body defences, antibodies and
phagocytes, would cope with foreign organisms pro-vided that their numbers were not overwhelming. Flis
views on this topic were based in part on his obseiwa-tion that isolated syphilis-causing spirochetes treated
with low concentrations of salvarsan remained motile
and were therefore apparently still alive. Nevertheless
they were unable to produce an infection when they
were injected into an animal body. It is a striking fact
that several of today’s important antibacterial and anti-fungal drugs are ‘biostatic’ rather than ‘biocidal’ in ac-tion.
Another feature of Ehrlich’s work was his recog-nition of the possibility that drugs may be activated by
Dewelopment of antimicrobial agents
metabolism in the body This suggestion was prompted
by the observation that the compound atoxyl was ac-tive against trypanosomal infections but was inactive
against isolated trypanosomes. His explanation was
that atoxyl was reduced in the body to the much more
toxic /7-aminophenylarsenoxide (Figure 1.1). Later
work showed that atoxyl and other related arsenic
acids are not in fact readily reduced to arsenoxides in
the body, but local reduction by the parasite remains a
possibility. Ehrlich, surprisingly, did not recognize that
his own compound salvarsan would undergo metabolic
cleavage. In animals it gives rise to the arsenoxide as
the first of a series of metabolites. This compound
eventually was introduced into medicine in 1932 under
the name Mapharsen (Figure 1.1); its toxcity is rather
high, but it has sufficient selectivity to give it useful
chemotherapeutic properties. Other examples of acti-vation through metabolism have been discovered in
more recent times; for example, the conversion of the
antimalarial ‘prodrug’ proguanil to the active cy-cloguanil in the liver and the metabolism of antiviral
nucleosides to the inhibitory triphosphate derivatives.
Of course, it later emerged that metabolism in the body
or in the infecting micro-organism could also result in
the inactivation of drags.
Ehrlich also drew attention to the problem of re-sistance of micro-organisms towards chemotherapeu-tic compounds. He noticed it in the treatment of try-panosomes with parafuchsin and later with trypan red
and atoxyl. He found that resistance extended to other
compounds chemically related to the original three,
but there was no cross-resistance among the groups. In
Ehrlich’s view this was evidence that each of these
compounds was affecting a separate receptor. Indepen-dent resistance to different drugs later became a com-monplace in antimicrobial therapy. Ehrlich’s view of
the nature of resistance is also interesting. He found
that trypanosomes resistant to trypan red absorbed less
of the compound than sensitive strains, and he postu-lated that the receptors in resistant cells had a dimin-ished affinity for the dye. This mechanism corresponds
to one of the currently accepted types of resistance in
micro-organisms (Chapter 9) in which mutations af-fecting the target protein reduce or eliminate drug
binding to the target.
Several useful antimicrobial dmgs appeared in
later years as an extension of Ehrlich’s work. The most
notable (Figure 1.2) are suramin, developed from try-pan red, and mepacrine (also known as quinacrine or
atebrin) developed indirectly from methylene blue
(Figure 1.2). Suramin, introduced in 1920, is a color-less compound with useful activity against human try-panosomiasis. Its particular value lay in its relative
safety compared with other antimicrobial dmgs of the
period. It was the first useful antimicrobial drug with-out a toxic metal atom, and the ratio of the dose re-quired to produce toxic symptoms to that needed for a
curative effect is vastly higher than with any of the ar-senicals. Suramin is remarkably persistent, a single
dose giving protection for more than a month.
Mepacrine, first marketed in 1933, was an antimalarial
agent of immense value in the Second World War. It
was supplanted by other compounds partly because it
caused a yellow discoloration of the skin. Besides
these obvious descendants from Elhrlich’s work, the
whole field of drug therapy is pemieated by his ideas,
and many other important compounds can be traced di-rectly or indirectly to the influence of his thought.
1.2.5 The treatment of bacterial infections by
synthetic compounds
In spite of the successes achieved in the treatment of
protozoal diseases and the spirochetal disease syphilis,
the therapy of bacterial infections remained for many
years an elusive and apparently unattainable goal.
Ehrlich himself, in collaboration with Bechtold, made
a series of phenols which showed much higher anti-bacterial potency than the simple phenols originally
used as disinfectants. These compounds, however, had
no effect on bacterial infections in animals. Other at-tempts were equally unsuccessful and no practical
progress was made until 1935, when Domagk reported
the activity of prontosil ruhrum (Figure 4.1) against in-fections in animals. The discovery occun’ed in the
course of a widespread research programme on the
therapeutic use of dyestuffs, apparently inspired by
Ehrlich’s ideas. Trefouel showed that prontosil rubrum
is broken down in the body, giving sulfanilamide (Fig-ure 4.1), which was in fact the effective antibacterial
agent. The sulfonamides might have been developed
and used more widely if penicillin and other antibi-otics had not followed on so speedily. In fact, relatively
1.2 An outline of the historical dewelopment of antimicrobial agents
NaSO.
NaSO.
CH-fCHjls-N^
OCH3
CH3
CH3
Mepacrlne
Suramin
FIGURE 1.2 Early synthetic
compounds used for treating pro-tozoal infections: suramin for tiy-panosomiasis (African sleeping
sickness) and mepacrine for
malaria.
few wholly synthetic compounds have achieved suc-cess against the common bacterial infections mainly
because effective synthetic compounds have been dif-ficult to find. After some 60 years of effort, the syn-thetic antibacterial compounds in current use include,
in addition to a few sulfonamides, several drugs for the
treatment of tuberculosis, the quinolones, trimetho-prim, certain imidazoles such as metronidazole, nitro-furans such as nitrofurantoin, and most recently the
oxazolidinone, linezolid. There are also numerous
semisynthetic derivatives of naturally occurring anti-bacterial antibiotics, including P~lactams, aminogly-cosides, macrolides, streptogramins and
glycopeptides.
For several years after treatment was available for
streptococcal and staphylococcal infections, the my-cobacterial infections that cause tuberculosis and lep-rosy remained untreatable by chemotherapy. The first
success came with the antibiotic streptomycin, which
remains an optional part of the standard treatment for
tuberculosis. Later, several chemically unrelated syn-thetic agents were also found to be effective against this
disease. The best of these are isonicotinyl hydrazide
(isoniazid), ethambutol and pyrazinamide. Another an-tibiotic, rifampicin (rifampin), is usually included in
the current combination therapy for tuberculosis. The
synthetic compound, 4,4′-diaminodiphenylsulfoiie, is
regularly used in the treatment of leprosy.
1.2.6 The antibiotic revolution
Ever since bacteria have been cultivated on solid
media, contaminant organisms have occasionally ap-peared on the plate. Sometimes this foreign colony
would be surrounded by an area in which bacterial
growth was suppressed. Usually this was regarded as a
mere technical nuisance, but in 1928, observing such
an effect with the mold PeniciUimn notatum on a plate
seeded with staphylococci, Alexander Fleming was
struck by its potential importance. He showed that the
mold produced a freely diffusible substance highly ac-tive against Gram-positive bacteria and apparently of
low toxicity to animals. He named it penicillin. It was,
however, unstable and early attempts to extract it
failed, so Fleming’s obser\’ation lay neglected until
1939. By then the success of the sulfonamides had
stimulated a renewed interest in the chemotherapy of
bacterial infections. The search for other antibacterial
agents now seemed a promising and exciting project,
and Howard Florey and Ernst Chain selected Flem-ing’s penicillin for re-examination. They succeeded in
isolating an impure but highly active solid preparation
and published their results in 1940. Evidence of its
great clinical usefulness in patients followed in 1941.
Because of the extraordinary antibacterial potency of
penicillin and its minimal toxicity to patients, it was
apparent that a compound of revolutionary importance
Dewelopment of antimicrobial agents
in medicine had been discovered. Making it generally
available for medical use, however, presented formida-ble problems both in research and in large-scale pro-duction, especially under conditions of wartime strin-gency. Eventually perhaps the biggest chemical and
biological joint research programme ever mounted
was undertaken, involving 39 laboratories in Britain
and the United States. It was an untidy operation with
much duplication and overlapping of work, but it cul-minated in the isolation of pure penicillin, the determi-nation of its structure, and the establishment of the
method for its production on a large scale. The obsta-cles overcome in this research were enormous. They
arose mainly from the very low concentrations of peni-cillin in the original mold cultures and from the
marked chemical instability of the product. In the
course of this work the concentration of penicillin in
mold culture fluids was increased 1000-fold by the iso-lation of improved variants of PeniciUum notatum
using selection and mutation methods and by im-proved conditions of culture. This tremendous im-provement in yield was decisive in making large-scale
production practicable and ultimately cheap.
The success of penicillin quickly diverted a great
deal of scientific effort towards the search for other an-tibiotics. The most prominent name in this develop-ment was that of Selman Waksman, who began an in-tensive search for antibiotics in micro-organisms
isolated from soil samples obtained in many parts of
the world. Waksman’s first success was streptomycin,
and other antibiotics soon followed. Waksman’s tech-nique of screening soil organisms for antibiotics was
immediately copied in many other laboratories. Or-ganisms of all kinds were examined and hundreds of
thousands of cultures were tested. Further successes
came quickly. Out of all this research, several thousand
named antibiotics have been listed. Most of them,
however, have adverse properties that prevent their de-velopment as drugs. Perhaps 50 have had some sort of
clinical use and only a few of these are regularly em-ployed in the therapy of infectious disease. However,
among this select group and their semisynthetic vari-ants are compounds of such excellent qualities that
treatment is now available for most of the bacterial in-fections known to occur in humans, although as we
have seen, resistance increasingly threatens the effi-cacy of drug therapy.
Following the wave of discovery of novel classes
of antibiotics in the 1940s and 1950s, research focused
largely on taking antibiotics of proven worth and sub-jecting them to chemical modification in order to ex-tend their antibacterial spectrum, to combat resistance
and to improve their acceptability to patients. Re-cently, however, the pressure of increasing drug resist-ance has renewed efforts to discover novel chemical
classes of both naturally occurring and synthetic anti-bacterial compounds.
1.2.7 Antifungal and antiviral drugs
The diversity of fungal pathogens which attack man
and his domesticated animals is considerably smaller
than that of bacteria. Nevertheless, fungi cause infec-tions ranging from the trivial and inconvenient to those
resulting in major illness and death. Fungal infections
have assumed greater importance in recent years be-cause of the increased number of medical conditions in
which host immunity is compromised. Fungi as eu-karyotes have much more biochemistry in common
with mammalian cells than bacteria do and therefore
pose a serious challenge to chemotherapy. Specificity
of action is more difficult to achieve. Few antibiotics
are useful against fungal infections, and attention has
concentrated on devising synthetic agents. Some ad-vantage has been taken of the progress in producing
compounds for the treatment of fungal infections of
plants to develop from them reasonably safe and effec-tive drugs for human fungal infections.
Elnoniious strides have been made in the control
of viral infections through the use of vaccines. Small-pox has been eradicated throughout the world. In the
developed countries at least, the seasonal epidemics of
poliomyelitis that were the cause of so much fear and
suffering 50 years ago have disappeared. But despite
these and other vaccine-based successes against viral
infections, not all such infections can be so effectively
controlled by mass vaccination programmes. The be-wildering diversity of common cold viruses, the ever-shifting antigenic profiles of influenza viruses and the
insidious nature of the viras that leads to AIDS are just
three examples of diseases that may not yield readily
to the vaccine approach. Attention is therefore focused
on finding drags that specifically arrest or prevent viral
1.3 Reasons for studying the biochemistry and molecular biology of antimicrobial compounds
infection, a fomiidable cliallenge since vimses par-tially parasitize the biochemistry of the host cells.
Nevertheless, considerable success has been achieved
in devising effective drugs against several viruses, in-cluding HIV, herpes and cytomegalo-viruses and even
against influenza viruses. Recombinant forms of the
naturally occuning antiviral protein interferon~a
(IFN-a), have a useful role in combating the viruses
which cause the liver infections hepatitis B and C.
1.2.8 Antiprotozoal drugs
After the Second World War, several valuable new
drags were introduced in the fight against malaria, in-cluding chloroquine, proguanil and pyrimethamine.
For several years these drags were extremely effective
for both the prevention and treatment of malaria. How-ever, by the time of the outbreak of the war in Vietnam
in the 1960s it had become clear that, like bacteria, the
malaiial parasites were adept at finding ways to resist
drug therapy. The US government then launched a
massive screening project to discover new antimalarial
agents. Two compounds, mefloquine and halofantriiie,
resulted from this effort and ai^e still in use today. Nev-ertheless, the development of resistance to these drugs
seems inevitable and the search for new antimalarial
drugs continues. Several compounds currently offer
real promise: the naturally occuning compound,
artemisinin and its semisynthetic derivatives, and the
synthetic compound, atovaquone.
The treatment of other serious protozoal infec-tions, such as the African and South American forms
of trypanosomiasis remains relatively primitive. The
arsenical melarsoprol is still used for African try-panosomiasis (sleeping sickness), although the less
toxic difluorodimethyi ornithine is increasingly seen
as the drug of choice. South American trypanosomia-sis, or Chagas’ disease, is still very difficult to treat
successfully; control of the insect vector, the so-called
kissing bug which infests poor-quality housing, is very
effective. The only useful drugs against leishmaniasis
are such venerable compounds as sodium antimony
gluconate and pentamidine, neither of which is ideal.
Unfortunately, the parasitic diseases of the developing
world do not present the major pharmaceutical compa-nies with attractive commercial opportunities, and re-search into the treatment of these diseases is relatively
neglected.
a9k^ K 9 %^C ^ ^%J ‘ a a ^ 9 %flV 5 ^%<Qtif l vji Wel l %r« Q^S 5 % ^ &jS n %^%d ^ a a Qs? 5 5 5 i ^ ^ n W
and moleciilai’ biology of antimicrobial s
connpouiiiis
Following this brief survey of the discovery of the
present wide range of antimicrobial compounds, we
may now turn to the main theme of the book. We shall
be concerned with the biochemical mechanisms that
underlie the action of compounds used in the battle
against pathogenic micro-organisms. Where there is
sufficient information, this will also include general
descriptions of the interactions between drugs and
their primary molecular targets. Increasingh’, the de-tailed understanding of drug action at the molecular
level is now used to generate ideas for the design of en-tirely novel antimicrobial agents. Antimicrobial
agents, particularly the antibiotics, often have a highly
selective action on biochemical processes. They may
block a single reaction within a complex sequence of
events. The use of such agents has often revealed de-tails of biochemical processes that would othenvise
have been difficult to disentangle. Attempts to under-stand the biochemistry of antimicrobial action were
initially slow and painful, with many false starts and
setbacks. Progress began to accelerate in the early
1960s and accompanied the dramatic advances being
made at that time in the biochemistry and molecular
biology of bacteria. In the past few years, tmly remark-able developments in the genetic manipulation of bac-teria, the plentiful production of hitherto inaccessible
proteins and the elucidation of macomolecular struc-tures by X-ray crystallography and nuclear magnetic
resonance spectroscopy have taken our understanding
of antibacterial drug action to unprecedented levels of
detail.
Knowledge of the mechanism of action of an-tiprotozoal drugs, some of which were discovered long
before the antibacterial drugs, lagged well behind for
many years. This was due mainly to the difficulty in
isolating and working with protozoa outside the ani-mal body, but interest had also been concentrated on
bacteria because of their special importance in infec-tious disease and their widespread use in biochemical
Dewelopment of antimicrobial agents
and genetic research. However, advances in tlie molec-ular genetics of the major parasitic protozoans should
now facilitate the development of our understanding of
dmg action in these species. Rapid progress is also
being made in working out the biochemical and molec-ular basis of the action of antifungal and antiviral
drags.
1.4 Uncovering the molecular basis of
antimicrobial action
the real site of action of various antimicrobial com-pounds. The limiting factor then became the extent of
biochemical information about the nature of the target
site. From about 1960 onwards there have been contin-uing and remarkable advances in our understanding of
the structure, function and synthesis of macromole-cules. Most of the important antibiotics were found to
act by interfering with the biosynthesis or function of
macromolecules, and the development of new tech-niques provided the means of defining their site of ac-tion in ever-increasing detail.
Several steps in discovering the molecular basis of an-timicrobial action can be distinguished and will be dis-cussed separately.
1.4.1 Nature of the biochemical systems
affected
As long as antimicrobial compounds have been
known, scientists have attempted to explain their ac-tion in biochemical terms. Ehrlich made a tentative be-ginning in this direction when he suggested that the ar-senicals might act by combining with thiol groups on
the protozoal cells. He was, however, severely limited
by the elementary state of biochemistry at that period.
By the time the sulfonamides were discovered, the bio-chemistry of small molecules was more advanced and
a reasonable explanation of the biochemical basis of
sulfonamide action was soon available. However,
many of the antibiotics which followed presented very
different problems. Attempts to apply biochemical
methods to the study of their action led to highly con-flicting answers. At one stage a count showed that 14
diiferent biochemical systems had been suggested as
the site of action of streptomycin against bacteria.
Much of this confusion arose from a failure to distin-guish between primary and secondary effects. The bio-chemical processes of bacterial cells are closely inter-linked. Thus disturbance of any one important system
is likely to have eifects on many of the others. Meth-ods had to be developed that would distinguish be-tween the primary biochemical effect of an antimicro-bial agent and other changes in metabolism that
followed as a consequence. Once these were estab-lished, more accurate assessments could be made of
1.4.2 Methods used to study the mode of
action of antimicrobial compounds
Many of the early antimicrobial drugs were discovered
by the simple method of screening for antimicrobial
activity in collections of synthetic compounds and the
media in which micro-organisms suspected of antibi-otic production had been cultured. This approach pro-vided little or no information as to the likely mecha-nism of antimicrobial action. However, experience
over the past five decades has developed systematic
procedui-es for working out the primai7 site of action
for most of these empirically discovered compounds.
Once the primary site of action is established, the over-all effects of a drug on the metabolism of microbial
cells can often be explained and the precise details of
the interaction between the drug and its molecular tai”-get finally revealed. Many of the techniques are dis-cussed in later chapters, but it may be helpful to set
them out in a logical sequence.
1. The chemical stracture of the drug is studied
carefully to determine whether a structural
analogy exists with part, or the whole, of a bi-ologically important molecule, for example,
a metabolic intermediate or essential cofac-tor, or nutrient. An analogy may be immedi-ately obvious, but sometimes it becomes ap-parent only through imaginative molecular
model building or by hindsight after the tar-get site of the compound has been revealed
by other means. This approach revealed the
site of action of the sulfonamide antibacterial
drugs. Nevertheless, analogies of structure
10
1.4 Uncowering the molecular basis of antimicrobial action
can sometimes be misleading and should
only be used as a preliminary indication.
2. The next step is to examine tlie effects of the
compound on the growth kinetics and mor-phology of suitable target cells. A cytocidal
effect shown by a reduction in viable count
may indicate damage to the cell membrane.
This can be confirmed by observation of
leakage of potassium ions, nucleotides or
amino acids from the cells. Severe damage
leads to cell lysis. Examination of bacterial
and fungal cells by electron microscopy may
show morphological changes which indicate
interference with the synthesis of one of the
components of the cell wall. Many antibi-otics have only a cytostatic action and do not
cause any detectable nioiphological changes.
3. Usually attempts are made to reverse the ac-tion of an inhibitor by adding vaiious supple-ments to the medium. Cellular nutrients, in-cluding oxidizable carbon sources, fatty
acids, amino acids, the nucleic acid precur-sors purines and pyrimidines, and vitamins
are tested in turn. If reversal is achieved, this
may point to the reaction or reaction se-quence which is blocked by the inhibitor.
Valuable confirmatory evidence can often be
obtained by the use of genetically engineered
auxotrophic organisms which require a com-pound known to be the next intermediate in a
biosynthetic sequence beyond the reaction
blocked by the antimicrobial agent. Aux-otrophs of this type should be resistant to the
action of the inhibitor. Inhibition in a biosyn-thetic sequence may also be revealed by ac-cumulation of the metabolite immediately
before the blocked reaction. Unfortunately,
the actions of many antimicrobial agents are
not reversed by exogenous compounds. This
especially applies to compounds which inter-fere with the polymerization stages in nucleic
acid and protein biosynthesis, where reversal
is impossible.
4. The ability of an inhibitor to interfere with
the supply and consumption of ATP is usu-ally examined since any disturbance of en-ergy metabolism has profound effects on the
biological activity of the cell. The inhibitor is
tested against the respiratory and glycolytic
activities of the micro-organism, and the ATP
content of the cells is measured. Compounds
which damage biological membranes are
likely to collapse the proton gradient across
the cytoplasmic membrane of bacteria and
thereby block the biosynthesis of ATP.
.5. Useful information can often be gained by
observing the effect of an antimicrobial agent
on the uptake kinetics of a radiolabelled nu-trient, such as glucose, acetate, a fatty acid,
an amino acid, a nucleic acid precursor, or
phosphate. Changes in rate of incorporation
after the addition of the drug are measured
and compared with its effect on growth. A
prompt interference with incorporation of a
particular nutrient may provide a good clue
to the primary site of action.
6. An antimicrobial compound which inhibits
protein or nucleic acid synthesis in cells
without interfering with membrane function
or the biosynthesis of the immediate precur-sors of proteins and nucleic acids, or the gen-eration and utilization of ATP, probably in-hibits macromolecular synthesis directly.
Because of the close interrelationship be-tween protein and nucleic acid synthesis, in-direct effects of the inhibition of one process
on another process must be carefully distin-guished. For example, inhibitors of the
biosynthesis of RNA also block protein
biosynthesis as the supply of messenger
RNA (niRNA) is exhausted. Again, in-hibitors of protein synthesis eventually arrest
DNA synthesis because of the requirement
for continued protein biosynthesis for the ini-tiation of new cycles of DNA replication. A
study of the kinetics of the inhibition of each
mac romolecular biosynthesis in intact cells is
valuable since indirect inhibitions appear
later than direct effects.
7. After the tai^get biochemical system has been
identified in intact cells, more detailed infor-mation is obtained with preparations of en-zymes, nucleic acids and subcellular or-ganelles. The antimicrobial compound is
11
Dewelopment of antimicrobial agents
tested for inhibitory activity against the sus-pected target reaction in vitro. There is a risk,
however, of nonspecific drag effects in vitro,
especially when the drag is added at high
concentrations. Failure to inhibit the sus-pected target reaction in vitro, on the other
hand, even with high drug concentrations,
may not rale out inhibition of the same reac-tion in intact cells for several reasons.
(a) The drug may be metabolized by the
host or the living micro-organism to an
active, inhibitory derivative.
(b) The procedures involved in the purifica-tion of an enzyme may cause desensiti-zation to the inhibitor by altering the
confomiation of the inhibitor binding
site.
(c) The site of inhibition in the intact cell
may be part of a highly integrated struc-ture which is disrupted during the prepa-ration of a cell-free system, again caus-ing a loss of sensitivity to the inhibitor.
Enzyme or organelle preparations from drag-resistant mutants have been successfully used
in identifying the site of attack, and examples
of this approach are described in later chap-ters. Cloning procedures and recombinant
DNA technology greatly facilitate the provi-sion of suspected protein targets for in vitro
evaluation.
Application of microarray expression and
proteomic technologies in analyzing drug
action
The acquisition of microbial genomic sequences and
remark.able technological developments in molecular
biology provide opportunities to profile the effects of
antimicrobial drags by investigating their effects on
the expression of thousands of genes simultaneously.
Although an antimicrobial drug may target a specific
molecular receptor, its consequent effects on microbial
metabolism and gene expression are not only pleio-tropic, i.e. multiple, but may also be characteristic.
The ability to assess the impact of drugs on the expres-sion of many different genes simultaneously enables
investigators to place compounds with closely similar
primary sites of action into related sets. Gene tran-scription profiling of novel agents with unknown sites
of action may therefore provide valuable clues as to
their primary target receptors. One microarray tech-nology involves the synthesis of short oligonu-cleotides in a high-density array directly on a solid sur-face, or ‘chip’. The oligonucleotides are selected using
total genomic DNA from the micro-organism to repre-sent each open reading frame (orf). In this way many
thousands of genes (or at least fragments of genes) can
be arrayed. Messenger RNA extracted from cells cul-tured in the presence and absence of the drag under in-vestigation is then hybridized to the immobilized
oligonucleotides to reveal how the levels of expression
of individual microbial genes are affected by the drag.
There are numerous opportunities for experimental
artefacts in analyzing microarray expression, and var-ious controls are essential to ensure reproducible data.
With this caveat in mind, patterns of gene expression
can be obtained which are characteristic of specific
modes of drug action, i.e. inhibition of protein or nu-cleic acid synthesis. Microarray experiments yield
thousands of data points and the evaluation of such
large amounts of information is a considerable chal-lenge. Several different methods of data analysis are
used, including hierarchical clustering, self-organiz-ing maps, principal components analysis and vector
algebra. It is worth restating that even with all this
methodology, analysis of microarray expression is
concerned with finding patterns of responses to the in-hibitory actions of antimicrobial agents and is not cur-rently capable of precisely defining the primary mo-lecular target of drug action.
Similar comments apply to proteomic analysis of
drag action. This method assesses the effects of drugs
on the patterns of protein expression in microbial cells.
Recently, proteomic analysis was applied to the effects
of 30 antibacterial drugs from all the important types
of agents on protein expression in Bacillus subtilis.
Two-dimensional polyacrylamide electrophoresis was
used to separate radiolabelled cytoplasmic proteins.
Each of the 30 drags produced complex but repro-ducible and characteristic patterns of protein expres-sion. Combinations of microarray RNA expression
and proteomic technologies should eventually offer a
12
1,4, Pharmacological biochemistry
highly refined approach to the characterization of drug
action and expedite the ultimate definition of the pri-mary molecular targets.
1.4.3 The study of the interactions between
antimicrobial agents and their molecular
target
Early mode of action studies concentrated on reveahng
the biochemical processes and pathways inhibited by
antimicrobial drugs. However, scientists are no longer
satisfied with this level of explanation alone and aspire
to define dmg action in molecular temis, i.e. the details
of the specific interactions between drugs and their
target sites. In order to achieve this level of under-standing of drug action, techniques such as X-ray crys-tallography and nuclear magnetic resonance spec-troscopy (NMR) are used to generate visual images of
the molecular interactions between drug and macro-molecule. Recombinant DNA technology enables the
role of specific amino acids or nucleotide residues in
macromolecule^rug interactions to be defined. The
stractural elucidation of supramoleculai” organized
structures, such as membranes and ribosomes, proved
to be a more fomiidable undertaking, largely because
of the difficulty in obtaining diffracting crystals of
these stmctures for X-ray analysis. Nevertheless, in the
past few years even the structure of bacterial ribo-somes has been revealed by X-ray crystallography, to-gether with remarkable details of their interactions
with drugs of major clinical importance which inhibit
protein biosynthesis (Chapter 5).
1.4.4 Pharmacological biochemistry
Effective antimicrobial drugs possess a combination of
advantageous properties: potency and selectivity at the
target site, good absoiption from the site of administra-tion, appropriate distribution within the body of the in-fected host, adequate persistence in the tissues and ab-sence of significant toxicity to the patient. Each of
these attributes may require distinct molecular charac-teristics. For optimum activity, all these characteristics
must be combined in the same molecule. The absorp-tion, distribution, metabolism, excretion and toxicity
of drugs are therefore essential subjects for investiga-tion. Activity requires an inhibitory concentration of
the drug at the target site which must be sustained long
enough to allow the body’s defences to contribute to
the defeat of the infection. The concentration is deter-mined by the rates of absorption and excretion and also
by the metabolism of the dmg in the tissues of the host.
The extent of binding of the drug to host proteins can
also be important. While extensive binding to plasma
proteins can increase drug persistence in the body, it
may also reduce effectiveness because the activity of
drugs depends on the concentration of free (unbound)
compound in the immediate environment of the infect-ing micro-organism. The methods for studying such
factors using modern analytical techniques are well es-tablished. The data can help to explain species differ-ences in the therapeutic activities of drugs and provide
a sound basis for recommendations on the size and fre-quency of doses for treating patients. The study of the
phamiacological biochemistry of drugs is a highly spe-cialized field and is beyond the scope of this book;
only passing mention is therefore made of the pharma-cological factors that influence the activities of antimi-crobial drugs.
1.4.5 Selectivity of action of antimicrobial
agents
Safe and effective antimicrobial drugs are by defini-tion highly selective in their action on the infecting
pathogens. In some cases the molecular targets inhib-ited by drugs are specific to the microbial cell. In other
cases drugs may act on biochemical mechanisms that
are common to both microbe and host. Possible expla-nations for selectivity of action in the latter situation
can include sufficient structural differences between
the microbial and host enzymes catalyzing the same
reaction to permit a selective attack on the microbial
enzyme, specificity due to selective concentration of
the drug within the microbial cell, and finally, differ-ences between the rates of turnover of target molecules
in the pathogen and its host that provide a basis for se-lectivity of action (see eflornithine, Chapter 6).
13
Dewelopment of antimicrobial agents
1.4.6 Biochemistry of microbial resistance
The effectiveness of antimicrobial drugs frequently
declines after sustained use, owing to the emergence of
drag-resistant organisms. This enormously important
problem has been studied in great depth by micro-biological, biochemical and molecular genetic meth-ods. Such studies have revealed the genetic basis for
the emergence of drag-resistant bacteria, fungi and
virases (Chapter 8) and defined the biochemical mech-anisms of resistance (Chapter 9). The mechanism of
some forms of resistance in eukaryotic pathogens,
such as chloroquine resistance in the malarial parasite,
is proving more difficult to define but is nevertheless
the subject of much research attention because of the
re-emergence of malaria across much of the tropical
and subtropical regions of the world.
1.5 Current trends in the discovery of
antimicrobial drugs
The relentless increase in drag resistance among bac-teria, fungi, virases and protozoal pathogens provides
an urgent stimulus for the discovery of new antimicro-bial drags. It is hoped that several developments in re-search technologies during the past decade will facili-tate the discovery process; these are briefly outlined
below.
1.5.1 Bioinformatics and genomics
Many of the antimicrobial drags in current clinical use
were discovered empirically by screening against cul-tures of micro-organisms or directly against model in-fections in experimental animals. Explanations of the
biochemical and molecular basis of drug action often
emerged only after years of use in patients. In contrast,
the modem approach to drug discovery is usually
driven by the perceived molecular target. Now that at
least 70 complete bacterial genomic sequences are
available, in addition to sequences from some viruses,
pathogenic yeasts and the malarial parasite, Plasmod-ium falciparum, the enormous and ever-expanding
wealth of genomic information from both micro-or-ganisms and their mammalian hosts provides many
opportunities to identify potential molecular targets
which are either pathogen-specific or sufficiently dif-ferent in sequence from the mammalian counterparts
to offer the possibility of drug selectivity. The com-puter-based technology of bioinformatics is used to
search micro-organisms for proteins with highly con-served sequences which suggest functions that are es-sential for viability. Validation of such proteins as
drug targets is then obtained by targeted gene knock-out experiments in pathogens grown in vitro and in in-fected animals. In the search for potential broad-spec-tram antibacterial drag activity, target proteins are
sought that have a high degree of sequence identity
across the major pathogens, both Gram-positive and
Gram-negative.
1.5.2 High-throughput screening versus
targeted screening
After an attractive molecular target has been selected
by bioinformatic ‘data mining’ and a microbiological
demonstration of essentiality for viability has been
carried out, sufficient protein is produced by gene
cloning and expression technologies to allow screen-ing to begin. Advances in laboratory robotic proce-dures have made it possible to rapidly screen ex-tremely large numbers of compounds. Whereas 20
years ago screening perhaps 100 or so compounds per
week against an enzyme target might have been con-sidered satisfactory, screening a million compounds
per week is now not unusual. The enoimous quantities
of data generated by such high-throughput screens cre-ated new challenges in evaluating and filtering com-pounds of interest for the succeeding stages of screen-ing cascades. The provision of chemicals in such vast
numbers also places considerable demands on com-pound collections and on synthetic chemistry. Fortu-nately the miniaturized techniques of combinatorial
chemistry are capable of synthesizing prodigious num-bers of compounds.
For some scientists, however, the intellectual ap-peal of high-throughput screening is limited and its
productivity in delivering novel effective drags has
been questioned. An alternative approach is that of tar-geted screening, based upon an appreciation of the
stmcture and function of the target protein. The ability
14
1.6 Scope and layout of the book
of genetic engineering and protein production facili-ties to provide substantial quantities of previously in-accessible proteins has expanded the opportunities for
the determination of three-dimensional structures by
X-ra}’ crystallograph}’ and NMR. Visualization of pro-tein structure on the computer monitor screen enables
chemists to design potential inhibitors in silico which
can then be synthesized and evaluated in targeted bio-chemical screens. At this stage, the numbers of com-pounds may be quite small but they are soon expanded
as promising compounds are further modified to opti-mize potency of inhibition, activity and safety in vivo.
In all likelihood both high-throughput and targeted
screening approaches will be employed for the fore-seeable future in the search for novel, effective antimi-crobial drugs.
In this book we have sought to provide well-estab-lished evidence for the biochemical actions and molec-ular targets of many of the best-known agents used in
medicine. Although much of the content is devoted to
antibacterial drugs, which constitute by far the largest
group of antimicrobial agents in use today, we have
also brought together information on the biochemical
activities of commonly used antifungal, antiprotozoal
and antiviral drugs. One chapter is devoted to the
means by which antimicrobial compounds enter and
leave their target cells and their relevance to the intrin-sic resistance of micro-organisms to dmgs. The last
two chapters consider the genetic and biochemical
basis of acquired drug resistance, respectively.
Wherever possible we have grouped drugs ac-cording to their types of biochemical action rather than
by their therapeutic targets. However, one chapter
brings together several drugs with other and unusual
modes of action.
F8 8 jfi r wi SS& $” $” JOi ^St S’s li SI! ff^ PSlSSCvl! SUCIillSiSSlI
Baudow, .1. E. et al. (2003) Proteomic approacli to under-sttmding antibiotic action. Antimicrob. Agents
Chemother. 47, 948.
Greenwood, D. (1995) Antimicrobial Chemotherapy. 3rd
Edn. Oxford University Press, Oxford.
Le Faun, J. (1999) Tlie Rise and Fall of Modern Medicine.
Little, Brown & Co., New York.
.Sneader, W. (1985) Drug Discovery: The Evolution of Mod-ern Medicines. John Wiley & Sons, New York.
Volker C. and Brown. .1. R. (2002). Bioirrformatics and the
discover)’ of novel anti-microbial agents. Ciirr. Drug
Targets. 2, 279.
15
Chapter two
2 a ff” i i i ^ ff^ w i ^ ^ B^ ^ ff^w sa l fi^ fi*^ O^ i i ^JIJ’S ^ i i ft 9 S $>fi n n %i^ Ir H %i^ B B « ^ %i^ B Sp 9 9 v^ ^^JBl v^ n B W w CS « »
In the search tbr differences between microbial patho-gens and animal cells that could provide the basis for
selective antimicrobial attack, one evident distinction
lies in thek general structure. The animal cell is rela-tively large and has a complex organization; its bio-chemical processes are compartmentalized and differ-ent functions are served by the nucleus with its
surrounding membrane, by the mitochondria and by
varioiis other organelles. The cytoplasmic membrane
is thin and lacks rigidity. The cell exists in an envkon-nient controlled in temperature and osmolariy in mam-mals and birds. It is constantly supplied with nutrients
from the extracellular fluid. Bacteria and fungi live in
variable and often hostile envkomnents and they must
be able to withstand considerable changes in external
osmolality. Some micro-organisms have relatively
high concentrations of low molecular weight solutes in
then cytoplasm. Such cells suspended in water or in
dilute solutions develop a high internal osmotic pres-sure. This would inevitably disrupt the cytoplasmic
membrane unless it were provided with a tough, elas-tic outer coat. This coat is the cell wall, a characteris-tic of bacteria and fungi which is entirely lacking in
animal cells. It has a protective function but at the
same time it is vulnerable to attack, and a number of
antibacterial and antifungal drugs owe thek action to
their ability to disturb the processes by which the walls
are synthesized. Since there is oo parallel biosynthetic
mechanism ui animal cells, substances affecting this
process may be highly selective io their antimicrobial
action.
The term ‘wall’ will be used to describe all the
cell covering which lies outside the cytoplasmic mern-braoe. The structures of the walls of bacteria aod fungi
are very different from each other, as are the biosyn-thetic processes involved in their elaboration. This re-sults in susceptibility to quite distinct antimicrobial
agents.
The structure of the bacterial wall not only differs
markedly from that of fungi but also varies consider-ably from one species to another. It nevertheless fol-lows general patterns which are related to the broad
moiphological classification of bacteria. Classically
this has been based on the responses towards the Gram
stain, but the well-tried division into Gram-positive
and Gram-negative types has a significance far beyond
that of an empirical staining reaction. The most evi-dent differences are worth recalling.
Many Gram-negative bacteria are highly adapt-able organisms which can use inorganic nitrogen
17
The cell walls of bacteria and fungi
compounds, mineral salts and a simple carbon source
for the synthesis of their whole stracture. Their cyto-plasm has a relatively low osmolarity. Typical Gram-positive cocci or bacilli tend to be more exacting in
their nutritional needs. They are usually cultivated on
rich undefined broths or on fairly elaborate synthetic
media. In their cytoplasm, Gram-positive bacteria con-centrate amino acids, nucleotides and other metabo-lites of low molecular weight and consequently have a
high internal osmolarity. However, not all bacteria fit
this neat division. The Gram-negative cocci, the rick-ettsias, the chlamydias and the spirochetes, for exam-ple, are all Gram-negative bacteria with exacting
growth requirements. The mycoplasmas lack a rigid
wall structure and although technically Gram-nega-tive, they are best treated as a separate group lying out-side the usual Gram stain classification.
For many years the bacterial wall was considered
to be a rigid structure, largely because when bacteria
are disrupted, the isolated walls retain the shape of the
intact organisms. More recent evidence, however,
shows that this concept of rigidity must be revised. The
peptidoglycan saccules (see later discussion) of the
bacterial wall can expand or contract in response to
changes in the ionic strength or the pH of the external
environment. This responsive flexibility is a property
of the wall itself and can even be seen by the unaided
eye when salt solutions are added to quantities of walls
pelleted by centrifugation. When intact bacteria are
subjected to osmotic stress, water moves through the
wall and membrane into the cytoplasm. The conse-quent swelling of the cell, bounded by the membrane,
is accommodated to some extent by the limited elastic-ity of the wall, although even stretchable stractures
break when sufficiently stressed. The wall breaks and
the cell then bursts as a result of the turgor pressure on
the thin cytoplasmic membrane, it is important to real-ize that during cell growth and proliferation, bacterial
cell walls are highly dynamic stractures, continually
undergoing biosynthesis, extension and remodeling. It
is this dynamic character which renders bacteria sus-ceptible to antibiotics which attack the biosynthesis
and the integrity of cell walls.
Most of the work on wall structure has been done
with Gram-positive cocci and bacilli and with enteric
bacteria and other Gram-negative rods. The extent to
which the stractural generalizations apply to groups
outside these classes is uncertain.
2.2.1 The Gram-positive wall
The basic stracture of the cell walls of Gram-positive
bacteria is relatively simple (Figure 2.1), although
there are many differences of detail across the species.
The wall which lies outside the cytoplasmic membrane
is usually beween 15 and 50 nm thick. Bacteria can be
broken by shaking with small glass beads and the walls
separated from cytoplasmic material by washing and
differential centrifugation. in electron micrographs
these wall preparations resemble empty envelopes torn
in places where the cytoplasmic contents were re-leased. The major part of the Gram-positive wall is a
large polymer consisting of two covalently linked
components. One of these components, forming at
least 50% of the wall mass, is peptidoglycan (some-times referred to as murein or mucopeptide). Its cross-linked stracture provides a tough, fibrous fabric that
gives strength and shape to the cell and enables it to
withstand a high internal osmotic pressure. The
amount of peptidoglycan in the wall shows that it cov-ers the cell in a multilayered fashion, with cross-link-ing both within and between the layers. Attached to the
peptidoglycan is an acidic polymer, accounting for
30-40% of the wall mass, which differs from species
to species. Often this is a teichoic acid—a substituted
poly(D-ribitol 5-phosphate) (see Figure 2.8)—or a sub-stituted glycerol 3-phosphate (lipoteichoic acid). In
some bacteria teichoic acid is replaced by poly(A’-acetylglucosamine 1-phosphate) or teichuronic acid (a
polymer containing uronic acid and iV-acetylhex-osamine units). Bacteria that normally incoiporate tei-choic acid in their walls can switch to teichuronic acid
under conditions of phosphate limitation. The acidic
character of the polymer attached to the peptidoglycan
ensures that the cell surface is strongly polar and car-ries a negative charge. This may influence the passage
of ions, particularly Mg-+ and possibly ionized drags,
into the cell. The teichoic acid or other acidic polymer
is readily solubilized and released from the insoluble
peptidoglycan by hydrolysis in cold acid or alkali. The
nature of the linkage is described later.
18
2.2 Structure of the bacterial wal
Lipoteichoic
acid  Peptidoglycnii
Cytoplasmic
membrane
FIGURE 2.1 The arrangement of the cell envelope of Gram-positive bacteria. Note that the term ‘cell envelope’ includes
both the cytoplasmic membrane and the outer layers of the cell. The components are not drawn to scale. (This diagram was
kindly provided by Philip Kerkhoff.)
Other components of the Grain-positive wall vary
widely from species to species. Protein is often present
to the extent of 5 10%, and protein A of Staphylococ-cus aureus is apparently linked covalently to peptido-glycan. Proteins and polysacdiarides frequently occur
in the outennost layers and provide the main source of
the antigenic properties of these bacteria. Mycobacte-ria and a few related genera difler from other Gram-positive bacteria in having large amounts of complex
lipids in their wall structure. The unique features of the
mycobacterial cell wall are described later in this chap-ter.
2.2.2 The Gram-negative wall
The Gram-negative wall is far more complex. Wide-ranging studies of its structure have been concentrated
on the Enterobacteriaceae and on Escherichia coli in
panicular. The diagram in Figure 2.2 illustrates the
general arrangement of the components of the Gram-negative cell envelope, which includes the cytoplasmic
membrane as well as the cell wall. When cells of Es-cherichia coli are fixed, stained with suitable metal
salts, sectioned and examined by electron microscopy,
the cytoplasmic membrane is readily identified by its
‘sandwich’ appearance of two electron-dense layers
separated by a lighter space. The cieai^ layer immedi-ately outside the cytoplasmic membrane has been de-scribed as the peiiplasmic space. However, tedmiques
in electron microscopy such as txeeze-etching and
freeze-substitution reveal that a rich, dense periplasm
occupies the peiiplasmic ‘space’, containing a wealth
of biochemicals, including enzymes, transport pro-teins, secreted materials, components of peptidoglycan
and the bacterial outer membrane (see later discus-sion). The electron-dense layer, about 2 nni thick, im-mediately outside the periplasm represents the pepti-doglycan component of the wall. It is much thinner
than in Gram-positive bacteria and may constitute only
5 to 10% of the wall mass. Even so, it contributes sub-stantially to wall strength. Cells rapidly lyse when
treated with lysozyme, an enzyme which specifically
degrades peptidoglycan. In Escherichia coli the pepti-doglycan is covalently linked to a lipoprotein which
probably projects into the outer regions of the wall.
The outer regions of the Gram-negative cell wall have
been the most difficult to characterize. The various
19
The cell walls of bacteria and fungi
Poiln
0-antlgen
side chain
Phospholipids
Outer
membrane
Lipoprotein
Peptidoglyccin
layers
Periplnsmic
space
Cytoplnsmic
membrane
IntecirnI
protein
FIGURE 2.2 The aiTangement of the various layers of the cell envelope of Gram-negative bacteria. The components are
not drawn to scale. (This diagram was kindly provided by Philip Kerhoff.)
components together form a stractm-e 6-10 nm thick,
called the outer membrane. Like the cytoplasmic
membrane, it is basically a lipid bilayer (giving rise to
the two outermost electron-dense bands), hydrophobic
in the interior with hydrophilic groups at the outer sm*-faces. It also has protein components which penetrate
the layer partly or completely and form the membrane
‘mosaic’.
Despite these broad structural similarities, the
outer membrane differs widely in composition and
function from the cytoplasmic membrane. Its main
constituents are a lipopolysaccharide, phospholipids,
fatty acids and proteins. The phospholipids, mainly
phosphatidylethanolamine and phosphatidylglycerol,
resemble those in the cytoplasmic membrane. The
stmcture of the lipopolysaccharide is complex and
varies considerably from one bacterial strain to an-other. The molecule has three parts (Figure 2.3). The
core is built from 3-deoxy-D-/nanno-octulosonic acid
(KDO), hexoses, heptoses, ethanolamine and phos-phoric acid as structural components. The three KDO
residues contribute a structural unit which strongly
binds the divalent ions of magnesium and calcium, an
important feature that stabilizes the membrane. Re-moval of these ions by chelating agents leads to release
of some of the lipopolysaccharide into the medium; at
20
2.2 Structure of the bacterial wal
Mannose -^ Abequose
f
Rhamnose
i Galactose
Mannose -^ Abequose
Rhamnose
Galactose
Glucose •-«”
Heptose
Heptose
N – Acetyl
glucosamine
Galactose
Q)
U
KDO Phosphorylethanolamine
KDO -t KDO
Lipid A
FIGURE 2.3 Structure of the lipopolysaccharide of the cell
envelope of Salmonella typhimurium. The diagram has been
simplified by omitting the configuration of the glycosidic
linkages and omitting the 0-acetyl groups from the abequose
units. KDO: S-deoxy-D-manno-octulosonic acid. Lipid A
consists of a P-l,6-linked diglucosaniine residue to which
lauric, myristic, palmitic and 3-D(-)-liydroxymyristic acids
are bouud. The heptose residues of three lipopolysaccharide
polymers are shown linked by phosphate diester bridges. Al-though there are considerable structural variations in the
antigen side chains among Salmonella species, the core
polysacch£iride and lipid A are probably common to all wild-type salmonellae. The core structure in Escherichia coll is
more variable.
the same time, the membrane becomes pemieable to
compounds that would otherwise be excluded. The
core polysaccharide is linked to the antigenic side
chain, a polysaccharide which can vary greatly from
one strain to another even within the same bacterial
species. Usually it consists of about 30 sugar units, al-though these can vary in both number and structure. It
forms the outermost layer of the cell and is the main
source of its antigenic characteristics. At the opposite
end, the core of the lipopolysaccharide is attached to a
moiety known as lipid A which can be hydrolyzed to
glucosamine, long-chain fatty acids, phosphate and
ethanolamine. The fatty acid chains of lipid A, along
with those of the phospholipids, align themselves to
form the hydrophobic interior of the membrane. The
outer membrane is therefore asymmetric, with lipo-polysaccharide exclusively on the outer surface and
phospholipid mainly on the inner surface.
The most abundant proteins of the outer mem-brane in Escherichia coli are the porin proteins and
lipoprotein. Electron microscopy of spheroplasts lack-ing peptidoglycan reveals triplets of indentations in
the membrane surface, each 2 nm in diameter and 3
nni apart, through which the stain used in the prepara-tion readily penetrates. This is interpreted as showing
that the porin protein molecules stretch across the
membrane in groups of three, enclosing pores through
which water and small molecules can diffuse. The size
of the pores explains the selective pemieability of the
Gram-negative outer membrane; they freely allow the
passage of hydrophilic molecules up to a maximum
molecular weight of 600-700. Larger flexible mole-cules may also diffuse through the pores, although
with more difficulty. Artificial vesicles can be made
with outer membrane lipids. Without protein, these are
impermeable to solutes, but when porins are incoipo-rated, they show pemieability characteristics similar
to those of the outer membrane itself. The role of
porins in influencing the penetration of antibacterial
drugs into Gram-negative bacteria is explored in
Chapter 7.
Lipoprotein is another major component of the
outer membrane proteins. About one-third is linked to
peptidoglycan and the remaining two-thirds are unat-tached but form part of the membrane. The nature of
the attachment of lipoprotein to the side chains of pep-tidoglycan is discussed later. About one in twelve of
the peptide side chains is substituted in this way. This
arrangement anchors the outer membrane to the pepti-doglycan layer. The fatty acid chains of the lipoprotein
presumably align themselves in the hydrophobic inner
layer of the outer membrane and the protein moiety
21
The cell walls of bacteria and fungi
may possibly associate with matrix protein, reinforc-ing the pore structure.
Many other proteins with specialized functions
have been identified in the outer membrane. Some of
these are transport proteins that allow access to mole-cules such as vitamin Bp or nucleosides which are too
large to penetrate the pores of the membrane. Outer
membrane proteins that contribute to the function of
multidrag efflux pumps are described in Chapter 7.
2.3 Structure and biosynthesis of
peptidogiycan
The structure and biosynthesis of peptidogiycan have
special significance relative to the action of a number
of important antibacterials and have been studied ex-tensively. The biosynthesis of peptidogiycan was first
worked out with Staphylococcus aureus. Although
bacteria show many variations in peptidogiycan struc-ture, the biosynthetic sequence in Staphylococcus au-reus illustrates the general features of the process. In
this description the enzymes involved will be referred
to, where appropriate, by their biochemical names and
also by the more recent popular abbreviations derived
from the genetic nomenclature. The biosynthetic se-ciuence may be conveniently divided into four stages.
2.3.1 Stage 1: Synthesis of UDP-A/-acetylmuramic acid
The biosynthesis starts in the cytoplasm with two
products from the normal metabolic pool, iV-acetylglu-cosamine 1-phosphate and UTP (Figure 2.4). UDP-A’~
acetylglucosamine (I) formation is catalyzed by N-acetyl-1-phosphate glucosamine uridyl transferase
(GlmU) with the elimination of pyrophosphate. This
nucleotide reacts with phosphoenol pyruvate catalyzed
by UDP-iV-acetylglucosamine enolpymvyl transferase
(MurA) to give the coiTesponding 3-enolpyravyl ether
(11). The pyruvyl group is then converted to lactyl by a
reductase (MurB) that requires both flavin NAD and
NADPH as cofactors, the product being UDP-A”-acetylmuramic acid (111, UDPMurNAc). Muramic
acid (3-0-D-lactyl-D~glucosamine) is a distinctive
amino sugar derivative found only in the peptidogiy-can of cell walls.
0 0 0
II II II
)-PO-P-C
1 I
OH OH
(HO)2-P-0-PO-P-0-CH2
NH COCH3
/V-Acety!glucosamine l-phosphate Uridine triphosphate
(UTP)
CH^OH
UDP
NH COCH3
HO\j ^ U
^ / MH H I
CH2OH
Pyrophosphate
‘UDP
O’ NH COCH3
CH2 = C-COOH II
UDP
NH COCH3
CH, 0
II II
C-O-P(OH),
COOH
Phosphoenolpyruvate
UDP-W-acetylmuramic acid
(Abbreviation UDPMurNAc)
FIGURE 2.4 Peptidogiycan synthesis in Staphylococcus aureus. Stage 1; fonnation of UDP-Ai’-acetyimuramic acid.
22
2.3 Structure and biosynthesis of peptidoglycan
Successive addition
UDP Mur NAc “—;—:
01 L-aianme,
‘” D-glutamic acid
L-lysine
to carboxyl group of
muramic acid
Racemase
L–Alan!rie ^==::::::::::z± D-Alanin0
Synthetase
2D-Alanine •” o-Alanyl-D-aianine
CH,
1 ^
UDP
O NHCOCH3
I CH, COOH* rCH]NH,
I i 1. j 14
CHg-CH-CONH’CH-CONH-CH-pHpl-CONH-CH-COOH
IV
CH,
I
H^N-CH-CONH-CH-COOH
COOH*  |CH2]4NHj CH3 CH3
I ‘ i L| ‘J I I
UDPlViurNAc-NH-CH-CONH-CHfCHJ -CONH-CH •CONH-CH-CONH-CH-COOH
UDP-W-acetyltnuramyl pentapeptide
FIGURE 2.5 Peptidoglycaii synthesis in Staphylococcus aureus. Stage 2: fonnatioii of UDP-W-acetylmuramyl pentapep-tide. Addition of eachi amino acid and tlie final dipeptide requires ATP and a specific enzyme. L-Lysine is added to tlie y-cai’~
boxyl group of D-gltitamic acid; tlie a~carboxyl group (marked *) is amidated at a later stage in the biosynthesis.
2.3.2 Stage 2: Building the pentapeptide side
chain
Five amino acid residues are next added to the car-boxyl group of the muramic acid nucleotide (Figure
2.5). Each step requires ATP and a specific amino acid
Ugase. L,-Alar]itie is added first by MiirC. The next two
residues added are D-glutamic acid catalyzed by MurD
and then eitiier L-lysine or wiejo-diaminopimelic acid
by amino acid-specific forms of MurE. The incorpora-tion of either L-lysine or w.ejo-diarninopimeIic acid
into the pentapeptide side chain is characteristic of in-dividual bacterial species. Tiiese latter amino acids are
attached via theh a-amino groups to the y-carboxyl
group of the glutamic acid. In StapJiylococus aureus
and Streptococcus pneumoniae, but not in other bacte-ria, the a-carboxyl group of the glutamic acid is ami-dated at a later stage in the biosynthesis; this amino
acid residue is sometimes referred to as D-isoghita-mine. The biosynthesis of the pentapeptide is com-pleted by addition, not of an amino acid, but of a
dipeptide, D-alanyl-D-alaiiine, which is synthesized
separately. A racemase acting on L-alaniiie converts it
to D-alaoine, and a ligase joins two molecules of D-alanine to give the dipeptide. The linkage of i3-alanyl-D-alaoine to the tripeptide chain is catalyzed by MurF.
The completed UDP-i¥-acetylinuramyl Intermediate
(V) with its pendant peptide group will be referred to
as the ‘nucleotide pentapeptide’.
The three-dimensional structures of MurC,
MurD, MurE and MurF have all been solved by X-ray
crystallography and are generally very similar. Since
these enzymes are unique to bacteria, detailed knowl-edge of the structures may eventually lead to the de-sign of highly specific antibacterial drugs.
3: Membrane-bound reactions
leading to a linear peptidoglycan polymer
The biosynthesis up to this point is cytoplasmic, while
the succeeding steps occur oo membrane structures.
The first membrane-associated step involves the
formation of a pyrophosphate link, catalyzed by
23
The cell walls of bacteria and fungi
UDP~A?-acetylmuramyl pentapeptide phosphotrans-ferase (MraY), between the nucleotide pentapeptide
and undecaprenyl phosphate (the phosphate ester of a
C55 isoprenoid alcohol), which is a component of the
cytoplasmic membrane, to form a complex referred to
as lipid I. In this reaction UMP is released and be-comes available for reconversion to UTP, which is
needed in the first step of peptidoglycan biosynthesis
(Figure 2.6). All subseciuent reactions occurring while
the intermediates are linked to undecaprenyl phos-phate take place without release from the membrane.
An essential step in this membrane-bound reaction se-quence is the addition of a second hexosamine residue
through a typical glycosidation by UDP~iV-acetylglu-cosamine catalyzed by a glycosyl transferase (MurG)
(Figure 2.6). The modified disaccharide known as
lipid II is formed by a 1,4-p linkage with liberation of
UDP The involvement of undecaprenyl phosphate is
not unique to peptidoglycan biosynthesis. It is also
concerned in the biosynthesis of the polysaccharide
chain in the 0-antigen produced by Salmonella ty-phimuriurn and in the formation of the polysaccharide
elements of the lipopolysaccharides of Gram-negative
bacteria; in Gram-positive bacteria it fulfils a similar
role in the biosynthesis of teichoic acid or polysaccha-rides of the wall. The structure of MurG has also been
solved and active sites identified for inhibitor design
studies.
At about this point in the biosynthesis oi Staphy-lococcus aureus peptidoglycan and in many other
Gram-positive bacteria, an extending group is added to
the e-amino group of the lysine unit in the nucleotide
pentapeptide. Glycine and a glycine-specific transfer
RNA (tRNA) are involved in this process during which
a pentaglycine group is added. This reaction, which is
not found in Gram-negative bacteria, is unlike the
tRNA reactions in protein biosynthesis because ribo-somes are not involved; the five glycine units are
added successively to the lysine from the nitrogen end
(the reverse direction of protein biosynthesis). The re-sultant product (VUl, Figure 2.6) with 10 amino acid
units is referred to as the disaccharide decapeptide and
retains a free terminal amino group. In the biosynthe-sis of peptidoglycans in certain other bacterial species,
for example in Escherichia coli, in which no extending
group is added, the later reactions involve the e-amino
group of mejo-diaminopimelic acid (or equivalent di-amino acid) instead of the terminal amino group of
glycine. During the membrane-bound stage in the
biosynthesis of Staphylococcus aureus peptidoglycan,
the carboxyl group of D-glutamic acid is amidated by a
reaction with ammonia and ATP.
The disaccharide decapeptide (VIII) is now at-tached to an ‘acceptor’, usually regarded as the grow-ing linear polymer chain. In this reaction the disaccha-ride with its decapeptide side chain forms a p-linkage
from the 1 position of the W-acetylmuramic acid
residue to the 4-hydroxyl group of the terminal A’-acetylglucosamine residue in the growing polysaccha-ride chain. Because this reaction occurs outside the cy-toplasmic membrane, the disaccharide-decapeptide
linked to the undecaprenyl phosphate (lipid 11) first
moves across the membrane to gain access to the ac-ceptor on the external face of the membrane. The re-leased undecaprenyl pyrophosphate is reconverted by
a specific pyTophosphatase to the corresponding phos-phate, ready for another cycle of the membrane-bound
part of the synthesis. The extension of the glycan
chains thus occurs by successive addition of disaccha-ride units catalyzed by glycosyl transferases.
2.3.4 Stage 4. Cross-linking
The linear peptidoglycan (IX, Figure 2.6) formed in
stage 3 contains many polar groups which make it sol-uble in water. It lacks mechanical strength and tough-ness. These attributes are introduced in the final stage
of biosynthesis by cross-linking, a process well known
in the plastics industry for producing similar results in
synthetic linear polymers. The mechanism involved in
cross-linking peptidoglycan is a transpeptidation reac-tion requiring no external supply of ATP or similar
compounds. In Staphylococcus aureus, the transpepti-dation occurs between the temiinal amino group of the
pentaglycine, side chain and the peptide amino group
of the terminal D-alanine residue of another peptide
side chain; D-alanine is eliminated and a peptide bond
formed (Figure 2.7). In Staphylococcus aureus pep-tidoglycan, the cross-linking is quite extensive and up
to 10 peptide side chains may be bound together by
bridging groups. Since the linear polymers themselves
are very large, it is likely that the whole of the peptido-glycan in a Gram-positive bacterium is made up of
24
2.3 Structure and biosynthesis of peptidoglycan
UDPMurNAo-Pentapeptide  Membrane bound
O
n
0 O
II I
H 4’cH„-C=CH-CH,l 0-P-OH
CH;, OH
Translocase
CH„ M M r I 1
OP-0-P-0-[CH2-CH=CH-CH2J-_ H
I I
OH OH (Membrane)  + UMP-O NHCOCHj
I
CHjCH-CO • pentapeptide
• Uridine diphosplio-W-acetyl
qlucosamine
CiH^OiH  CHjOH
O
NHCOCH3
V . O O
Ao-P-O-P-0-[cH2-CH=CH-CH2] – H
I OH OH
VII
CH^OH
J O
0 NHCOCH3
CHaCH-CO-pentapeptide
5 Glycine + tRNAQfy
CHjOH p 0
+ UDP
CH.,
I
NHCOCH3
!l 11 r I n
O, ^0-P-O-P- O tCH,-C=CH–CH, + H VIII
CHsj-NH-f COCHJNHI-^ H
I 1
OH OH
NHC0CH3
CH3  CONH,
1
CH,
I ‘
CH3
CHj-CHCONH-CH-CONH-CH-iCH,! -CONH-CH-CONH-CH-CONH-CH-COOH
Pyrophosphatase
CHjOH
•O
+ acceptor
(growing polysacclnaride ohain^
Reused
Membrane bound
+ P: + HfcH,-C=GH-CH,] ^ 0-P
1… • I – J 11 I I
CH 3
OH
OH
NHCOCH, ‘ NHCOCH,
‘ O ‘
I
CH3CCO – Decapeptide iv
Linear peptide-polysaccharide
FIGURE 2.6 Peptidoglycan synthesis in Staphylococcus aureus. Stage 3: formation of the linear peptidoglycan. Tlie struc-ture of the decapeptide side chain is shown in VIII.
The cell walls of bacteria and fungi
GlcNAc
CH,
I
CO
[NHCHjCOj,
NH
I CHj CONH. [CHjJ, CH3
MurNAc-NHCHCONHCH[CHJ , CONHCHCONHCHCONHCHCOOH
CH3
GlcNAc
GlcNAc
(X
NH ,
i ‘
CH j
CO
[COCH^NH].
NH
CH3  CH3
I  [CH,].  CH,
I
MurNAc
GlcNAc
HOOCCHNHCOCHNHCOCHNHCOrCH,],CHNHCOCHNH-MurNAc
NH2
I
CH,
I
CO
rNHCHjCO]
GlcNAc
NH
^1
I
CONH,
Transpeptidase
CH3
GicNAc
CH
!
CONH2 [CHj] CH3 + NHjCHCOOH
MurNAc-NHCOCHCONHGHrCH] CONHGHCONHCHOO
! L 32 I
GlcNAc
MurNAc
NH
!
CHj
CO
!
[COCH^NH],
– I
NH
i
MurNAc
CH,
GlcNAo
HOOCGHNHCOGHNHCOCHNHCO [CHjjCHNHCOCHNHMurNAc
CONH2  GlcNAc
FIGURE 2.7 Peptidoglycan
synthesis in Staphylococcus au-reus. Stage 4: cross-linljing of
two linear peptidoglycan chains.
The linear polymers have the
structure IX (Figure 2.6) GlcNac:
A’-acetylglucosaminyl residue.
The dashed arrows show points at
which further cross-links may be
formed with other polymer
chains. MurNAc; A’-acetylmu-raniyl residue.
units covalently bound together. This gigantic bag-shaped molecule has been called a sacculus. There is
also a mechanism for constantly breaking it down and
reforming it to allow cell growth and division. Peptido-glycan hydrolases, which hydrolyze the polysaccha-ride chains of peptidoglycan and others attacking the
peptide cross-links, exert this essential catabolic activ-ity during cell growth.
2.3.5 Penicillin-binding proteins
The membrane-bound enzymes involved in linking the
disaccharide deca- or pentapeptide to the growing lin-ear peptidoglycan and the subsequent cross-linking re-action are referred to as penicillin-binding proteins or
PBPs (Table 2.1). The PBPs are regarded as the spe-cific targets for penicillin and the other p-lactam an-2i
2.3 Structure and biosynthesis of peptidoglycan
TABLE 2.1 Properties of penicillin-binding proteins of Escherichia coli
Protein no.
Molecular mass
(Kilodaltons)  Enzyme activities  Function
la
lb
0
3
4
5
6
91
91
66
60
49
41
40
Transpeptidase
Traiisglycosylase
Transpeptidase
Traiisglycosylase
Transpeptidase
Transpeptidase
DD–carboxypeptidase
DD–carboxypeptidase
DD-carboxypeptidase
Peptidoglycau cross-linking
Peptidoglycau cross-linking
Peptidoglycan cross-linking
Peptidoglycan cross-linking
Limitation of peptidoglycan
cross-linking
Limitation of peptidoglycan
cross-linking
Limitation of peptidoglycan
cross-linking
tibiotics. As we shall see, the covalent reaction be-tween p-lactam antibiotics and the PBPs, which inac-tivates their transpeptidase function but not the trans-glycosylase activity, is central to the antibacterial
activity of these drugs. PBPs vary from species to
species in number, size, amount and affinity for p~lac-tains antibiotics. The PBPs fall into two major groups
of high (> 60 kDa) and low (< 49 kDa) molecular
mass, respectively. The PBPs with a high molecular
mass are essentially two-domain proteins classed as A
or B. In both classes the C-terminal domain is respon-sible for transpeptidation and is the target for penicillin
binding and p-lactam action. Class A proteins also cat-alyze the transglycosylation reactions at the N-termi-nal domains. PBPs la and lb of Eschericliia coli ex-emplify this bifunctional type. The monofunctional
class B proteins lack transglycolase activity. Mono-functional glycosyl transferases have been identified
in both Gram-positive and Gram-negative bacteria, al-though not all glycosyl transferases appear to be essen-tial to bacterial viability. In Escherichia coli, PBPs la
and lb provide the key enzyme activities involved in
peptidoglycan synthesis. The synthetic role of PBP2 is
specifically involved in cellular elongation and that of
PBP3 with the formation of the cell septum during cell
division. The low molecular mass PBPs, which include
PBPs 4, 5 and 6 in Escherichia coli, are monofunc-tional DD~carbox3’peptidases that catalyze transfer re-actions from D-alanyl-D-alanine temiinated peptides.
Although these PBPs are also inactivated by p-lac-tams, this may not be central to their antibiotic action.
Nevertheless, carboxypeptidases of this type are con-venient to purify and have been widely used as models
for the nature of the interaction between PBPs and
penicillin. The most widely studied enzymes are the
extracellular DD-carboxypeptidases produced by
Streptomyces species and carboxypeptidases solubi-lized from the membranes of Escherichia coli and
Bacillus stearothermophilus. The Streptomyces en-zymes display some transpeptidase activity besides
their high carboxypeptidase activity.
In addition to the seven ‘classic’ PBPs listed in
Table 2.1, a further five have been added to the collec-tion: PBPlc, PBP7, DacD, AmpC and AmpH. An ex-tensive study of deletion mutants reveals that only
PBPs 2 and 3 plus PBPla or lb are essential for the
growth and division of rod-shaped bacteria under lab-oratory conditions. However, combinations of the ac-tivities of the other PBPs may be necessary for growth
and viability in more demanding conditions, for exam-ple, in an infected host. The possible significance of
the ‘new’ five PBPs in relation to the antibacterial ac-tion of p-lactams remains to be explored.
2.3.6 Variations in peptidoglycan structure
Many variations are found in peptidoglycan stracture
between one species of bacteria and another or even
between strains of the same species and only a general
27
The cell walls of bacteria and fungi
account is possible here. All peptidoglycans have the
same glycan chain as in Staphylococcus aureus except
that the glucosamine residues are sometimes iV-acy-lated with a group other than acetyl. 0~Acetylation of
glucosamine residues is also found in some organisms.
The peptide side chains always have four amino acid
units alternating L-, D~, L~, D- in configuration. The sec-ond residue is always D-glutamic acid, linked through
its Y~carboxyl group, and the fourth is invariably D-ala-nine. The peptidoglycan from Staphylococcus aureus
(type A2) is characteristic of many Gram-positive
cocci. Peptidoglycans of this group, and the related
types A3 and A4, have similar tetrapeptide side chains
but vary in their bridging groups. The amino acids in
the bridge are usually glycine, alanine, serine or threo-nine, and the number of residues can vary from one to
five. In type Al peptidoglycans, the L-lysine of the type
11 peptide side chain is usually replaced by meso~2,6,~
diaminopimelic acid, and there is no bridging group.
Cross-linking occurs between the D-alanine of one side
chain and the 6-amino group of the diaminopimelic
acid of another. This peptidoglycan type is character-istic of many rod-shaped bacteria, both the large fam-ily of Gram-negative rods and the Gram-positive
bacilli. In the less common type B peptidoglycans,
cross-linkage occurs between the a-carboxyl group of
the D-glutamic acid of one peptide side chain and the
D~alanine of another through a bridge containing a
basic amino acid.
2.3.7 Cross-linking in Gram-negative bacteria
In contrast to the multiple random cross-linkage of
peptidoglycan which is found in the Gram-positive
cocci, the peptidoglycan of Escherichia coli and simi-lar Gram-negative rods has on average only a single
cross-link between one peptide side chain and another.
These bacteria contain, besides the transpeptidases
concerned in cross-linkage, other enzymes known as
DD-carboxypeptidases which specifically remove D-alanine from a pentapeptide side chain. Carboxypepti-dase 1 is specific for the terminal D-alanine of the pen-tapeptide side chain, whilst carboxypeptidase 11 acts
on the D-alanine at position 4 after the terminal D-ala-nine has been removed. DD-Carboxypeptidase 1 there-fore limits the extent of cross-linking.
The peptidoglycan sacculus determines the over-all shape of the cell, and the peptidoglycan is laid down
with a definite orientation in which the polysaccharide
chains ran peipendicular to the main axis of rod-shaped organisms such as Escherichia coli.
2.3.8 Attacliments to peptidoglycans
Within the cell wall, the polymeric peptidoglycan is
usually only part of a larger polymer. In Gram-positive
cocci it is linked to an acidic polymer, often a teichoic
acid (Figure 2.8). The point of attachment is through
Teschoic
acid
Tri (glycerol)
phosphate
Glycan
chain
Mi 1/
l-Phospho-W-acetyl-glucosamine \ NHCOCH3
HgCCH-CO-Peptlde
W-Acetylmuramic acid
Linkage groups
FIGURE 2.8 Teichoic acid and
its linkage to peptidoglycan in the
wall of Staphylococcus aureus.
28
2.4 Antibiotics tliat inhibit peptidoglycan biosynthesis
the 6-hydroxyl group of muramic acid in the glycan
chain. Only a small fraction of the muramic acid
residues is thus substituted. In Staphylococcus aureus
cell walls, teichoic acid is joined to peptidoglycan by a
linking unit consisting of three glycerol l~phosphate
units attached to the 4 position of j^-acetylglucosarnine
which engages through a phosphodiester group at po-sition 1 with the 6~hydroxyl group of muramic acid.
This type of linkage seems to occur with polymers
other than teichoic acid, e.g. with polylA’-acetylglu-cosaniine l~phosphate) in a Micrococcus species. The
acid-labile /V-acetylglucosarnine 1-phosphate linkage
and the alkali-labile phosphodiester linkage at position
4 explain the ease with which teichoic acid can be split
off from peptidoglycan. Within the cell wall, the syn-thesis of teichoic acid is closely associated with that of
peptidoglycan.
In the Gram-positive mycobacteria, the peptido-glycan carries quite a different polymeric attachment.
Arabinogalactan is attached to the 6 position of some
of the iV-glycolylmuraniic acid residues of the glycan
chain through a phosphate ester group. Mycolic acids
(complex, very long-chain fatty acids) are attached by
ester links to the C-5 position of arabinose residues of
the arabinogalactan.The mycobacterial cell wall thus
has a high lipid content.
In Escherichia coli and related bacteria, the pep-tidoglycan canies a lipoprotein as a substituent (Figure
2.9). The lipoprotein consists of a polypeptide chain of
58 amino acid units of known sequence with lysine at
the C-terminal and cysteine at the N-tenxiinal. This is
attached to the 2-carboxyl group of meso~2.(3-di~
aminopimelic acid in a peptide side chain of Els-cherichja coli peptidoglycan which has lost both D-ala-nine groups. Attachment is by an amide link with the
e-amino group in the terminal lysine of the polypep-tide. At the opposite end of the polypeptide chain, the
cysteine amino group canies a long-chain fatty acid
joined as an amide, and its sulfur atom forms a thio-ether link with a long-chain diacylglycerol.
Lipoprotein occurs in enteric bacteria other than
Elscherichia coli, but it may not be common to all
Gram-negative bacteria, although small amounts have
been detected in Proteus mirabilis.
2.4 Antibiotics thst inhibit pcptidooivcsn
b io”‘i¥iithp”ii”4
The conclusion that a pailicular antibiotic owes its an-tibacterial activity to interference with peptidoglycan
biosynthesis rests on several lines of evidence:
I. Bacteria suspended in a medium of high os-motic pressure are protected from concentra-tions of the antibiotic that would cause lysis
and death in a normal medium. Under these
conditions the cells lose the shape-determin-ing action of the peptidoglycan and become
spherical; they are then known as sphero-plasts. These retain an undamaged cytoplas-mic membrane, but their wall is deficient or
considerably modified. Spheroplasts are in
CHjOR’
CHOR^
R^’NHCHCO—
Glycan
chain -MurNAc-GlcNAc-t
Ala
I
D-Glu
• “V
NH  COOH
56 amino acid units
COOH
I
-NHC H-fcHgl-(L) GH-[CH 3 GHNH,
I L J3
NHCO (D)
Cysteine (1)  Lysine (58)
Lipoprotein
FIGURE 2.9 Lipoprotein and
its linkage to peptidoglycan in the
envelope iyf Escherichia coli.
^u
The cell walls of bacteria and fungi
principle viable and if the antibiotic is re-moved, they can divide and produce progeny
with noimal walls.
2. Several species of bacteria have wails con-taining no peptidoglycan. These include the
mycoplasmas, the halophilic bacteria tolerant
of high salt concentrations and bacteria in the
L-phase where the nomial wall structure is
greatly modified. If a compound inhibits the
growth of common bacteria but fails to affect
bacteria of these special types, it probably
owes its activity to interference with peptido-glycan synthesis.
3. Subinhibitory concentrations of these antibi-otics often cause accumulation in the bacter-ial cytoplasm of uridine nucleotides of A’-acetylmuramic acid, with varying numbers of
amino acid residues attached which represent
intemiediates in the early stages of peptido-glycan biosynthesis. When an antibiotic
causes a block at an early point in the reac-tion sequence, it is not surprising to find an
accumulation of the intemiediates immedi-ately preceding the block. However, quanti-ties of muramic acid nucleotides are also
found in bacteria treated with antibiotics
known to affect later stages in peptidoglycan
biosynthesis. It seems that all the biosyn-thetic steps associated with the membrane are
closely interlocked, and inhibition of any one
of them leads to accumulation of the last
water-soluble precursor, UDP-A/^-acetylmu-ramyl pentapeptide (V, Figure 2.5).
2.4.1 Bacitracin
Bacitracin is a polypeptide antibiotic (Figure 2.10)
which is too toxic for systemic administration but is
sometimes used topically to kill Gram-positive bacte-ria by interfering with cell wall biosynthesis. The an-tibiotic is ineffective against Gram-negative bacteria,
probably because its large molecular size hinders pen-etration through the outer membrane to its target site.
Bacitracin inhibits peptidoglycan biosynthesis by
binding specifically to the long-chain C^j-isoprenol
pyrophosphate in the presence of divalent metal ions.
CO
Leu
D-Glu
GH CH
D-Phe
lie .
lie
t D-Orn
V Lys
His Bacitracin A
D-Asp
L-Asn
H., H
‘”C C,.
Fosfomycin
FIGURE 2.10 Antibiotics which inhibit the biosynthesis of
the precursors of peptidoglycan.
In the formation of the linear peptidoglycan (IX, Fig-ure 2.7), the membrane-bound isoprenol pyrophos-phate is released. Normally this is converted by a py-rophosphatase to the corresponding phosphate which
thus becomes available for reaction with another mol-ecule of UDPMur-iV-Ac-pentapeptide (V, Figure 2.6).
Interaction between the lipid pyrophosphate and a
metal ion-bacitracin coordination complex blocks this
process and eventually halts the synthesis of peptido-glycan. The identity of the divalent metal ion bound to
the antibiotic in bacterial cells is uncertain but could
well be either Mg'”*” or Zn^^. Bacitracin forms 1:1 com-plexes with several divalent metal ions, and investiga-tions employing nuclear magnetic resonance and opti-cal rotary dispersion (ORD) indicate the involvement
of the imidazole ring of the histidine residue of the an-tibiotic in metal ion binding. Additional likely sites of
metal ion interaction include the thiazoline moiety and
the carboxyl groups of the D-aspartate and D-glutamate
residue.
2.4.2 Fosfomycin (phosphonomycin)
This antibiotic has the very simple structure shown in
Figure 2.10. It acts on infections caused by both
30
2.4 Antibiotics tliat inhibit peptidoglycan biosynthesis
Gram-positive and Gram-negative bacteria but al-though its toxicity is low, until recently it achieved
only limited use in clinical practice. However, there is
a resurgence of interest in fosfomycin for the treat-ment of serious infections resistant to other antibi-otics. Fosfomycin inhibits the first step of peptido-glycan biosynthesis, nameh’, the condensation of
UDP-7V~acetylglucosamine (I) with phosphoenol py-ravate (PEP) catalyzed by UDP-A’-acetylglucosamine
enolpyruvyi transferase (MurA), giving the intermedi-ate (11) that subsequently yields UDP-A’-acetyimu-rarnic acid (111) on reduction (Figure 2.4). Fosfomycin
inactivates MurA by reacting covalently with an es-sential cysteine residue (Cys-115) at the active center
of the enzyme to form the thioester illustrated in Fig-ure 2.11. This reaction is time-dependent and is facil-itated by lJDP-,’V-acetylglucosamine, which appeai^s to
‘chase’ the other substrate (PFP) from the active site
and promotes a conformational change in the enzyme.
Both these effects are believed to expose the nucle-ophilic Cys-115 for reaction with the epoxide moiety
of fosfomycin. The three-dimensional structure of
MurA (from Escherichia coli) complexed with UDP-A’-acetylglucosamine and fosfomycin has been deter-mined by X-ray crystallograph}’. The analysis con-finned the covalent interaction of the antibiotic with
Cys-115 and also revealed that there are hydrogen
bonds between the antibiotic and the enzyme and
lJDP-/V”-acetyl glucosamine.
This antibiotic also has a simple structure (Figure
2.10). Cycloserine is active against several bacterial
species, but because of the central nerirous system dis-HgC
SH
Cys~115  /
Cys-115
OH
‘P0,2-FIGURE 2.11 Fosfomycin inactivates UDP-zV-acetylghi-cosamiiie enolpyruvoyl transferase (MurA) by reacting with
the essential cysteine residue (Cys-li5) at the active center
of the enzyme to fomi a thioester.
turbances which ai^e experienced by some patients,
clinically it is limited to occasional use in individuals
with tuberculosis that is resistant to the more com-monly used drugs. Cycloserine produces effects in
bacteria that are typical of compounds acting on pepti-doglycan biosynthesis. Thus when cultures of Staphy-lococcus aureus are grown with subinhibitory concen-trations of cycloserine, the peptidoglycan precursor
(IV, Figure 2.5) accumulates in the medium, suggest-ing a blockage in the biosynthesis immediately beyond
this point.
In fact, cycloserine inhibits alanine r and
D-alanyi-D-alanine ligase, the two enzymes concerned
in making the dipeptide for completion of the pen-tapeptide side chain. Molecular models reveal that cy-closerine is structurally related to one possible confor-mation of D-alanine, so that its inhibitory action on
these enzymes appears to be a classic example of isos-teric interference. The observation that the action of
cycloserine is specifically antagonized by the addition
of D-alanine to the growth medium also supports the
postulated site of action. The affinity of cycloserine
for the ligase is much greater than that of the natural
substrate, the ratio of K^^ to K^ being about 100. In a
compound acting purely as a competitive enzyme in-hibitor, this sort of KJK. ratio is probably essential for
useful antibacterial activity. The greater affinity of cy-closerine for the enzyme may be connected with its
rigid stmcture. This could permit a particularly accu-rate fit to the active center of the enzyme, either in the
state existing when the enzyme is uncombined with its
substrate or in a modified conformation which is as-sumed during the normal enzymic reaction. Rigid
structures of narrow molecular specificity are com-mon among antimicrobial agents and similar consider-ations may apply to other types of action; this theme
will recur in later sections. The three-dimensional
structures of both alanine racemase and D-alanyl-D-alanine ligase are available and it will be interesting to
see whether cycloserine does indeed interact with the
active sites of these enzymes according to this concept
of inhibition.
Cycloserine enters the bacterial cell by active
transport (see Chapter 7). This allows the antibiotic to
reach higher concentrations in the cell than in the
medium and adds considerably to its antibacterial
efficacy.
31
The cell walls of bacteria and fungi
2.4.4 Glycopeptide antibiotics
Vancomycin (Figure 2.12), which is a member of a
group of complex glycopeptide antibiotics, was first
isolated in the 1950s, but its real clinical importance
only emerged with the inexorable spread of methi-cillin-resistant staphylococci (MRSA; see Chapters 9
and 10). The use of vancomycin and structurally re-lated glycopeptides has markedly increased because of
their value in treating serious infections caused by
MRSA and other Gram-positive bacteria. Because of
their relatively large molecular size, the glycopeptides
are essentially inactive against the more impermeable
Gram-negative bacteria. The antibacterial action of
glycopeptide antibiotics depends on their ability to
bind specifically to the terminal D-alanyl~D-alanine
group on the peptide side chain of the membrane-bound intemiediates in peptidoglycan synthesis (com-pounds VI-IX in Figure 2.6). It is important to note
that this interaction occurs on the outer face of the cy-toplasmic membrane. The glycopeptide antibiotics
probably do not enter the bacterial cytoplasm, again
because of their molecular size. The complex which is
formed between vancomycin and D-alanyl~D-alanine
Oritawancin
NHj
«^^ -Vancomycin
“?NH, H
Teicoplanin
OH
FIGURE 2.12 Glycopeptide antibiotic inhibitors of peptidoglycan synthesis that are increasingly important in the treatment
of infections caused by drug-resistant staphylococci.
32
2.4 Antibiotics tliat inhibit peptidoglycan biosynthesis
has been studied in considerable detail. The complex
blocks the transglycosylase involved in the iiicoipora-tion of the disaccharide-peptide into the growing pep-tidoglycan chain and the DD~transpeptidases and DD~
caitoxypeptidases for which the i:)-alanyl-D-alanine
moiety is a substrate. Both peptidoglycaii chain exten-sion and cross-linlcing are therefore inhibited by gly-copeptide antibiotics. This is, in fact, a most unusual
mode of inhibition in that the antibiotic prevents the
utilization of the substrate rather than directly interact-ing with the target enzymes.
The side chains of the amino acids of the hep-tapeptide backbone of vancomycin are extensively
cross-linked to fomi a relatively concave carboxylate
cleft into which the D-akmyl-D-alanine entity binds
noncovalently via hydrogen bonds and hydrophobic
interactions. Furthemtore, NMR and X-ray crystallo-graphic studies show that vancomycin spontaneously
forms a dimeric structure which enables the antibiotic
to bind to two D-alanyl-D-alanine peptide units at-tached either to the disaccharide-peptide precursor or
to adjacent growing peptidoglycan strands. Another
glycopeptide antibiotic, teichoplanin (Fig 2.12), is
considerably more potent than vancomycin against
some important Gram-positive pathogens. It is thought
that the j^-substituted fatty acyl side chain that distin-guishes teichoplanin from vancomycin serves to an-chor teichoplanin in the cytoplasmic membrane. This
localization may facilitate the interaction of the drug
with the D-alanyl-D-alanine target site. In contrast with
vancomycin, teichoplanin does not form dimers. Thus
although the dimerization of vancomycin may in prin-ciple facilitate its antibacterial action, the dimerizing
potential is relatively weak and it is unclear whether
the dimer is indeed a significant contributor to the an-tibiotic activity of vancomycin in vivo. The semisyn-thetic glycopeptide, oritavancin (Fig 2.12), is strongly
dimerized and this may be a factor in the highly potent
antibacterial activity of this promising drug.
2.4.5 Penicillins, cephalosporins and other
p”lactam antibiotics
Penicillin was the first naturally occuning antibiotic to
be used for the treatment of bacterial infections, and
the story of its discovery and development is one of the
most inspiring in the history of medicine. Penicillin is
one of a group of compounds known as p-lactam an-tibiotics which are unrivalled in the treatment of bacte-rial infections. Their only serious defects include an
ability to cause immunologic sensitization in a small
proportion of patients, a side effect which prevents
their use in those affected, and the frequency of emer-gence of bacteria resistant to p-lactams. The original
penicillins isolated directly from mold fermentations
were mixtures of compounds having different side
chains. The addition of phenylacetic acid to the fer-mentation medium improved the yield of penicillin
and ensured that the product was substantially a single
compound known as penicillin G or benzylpenicillin
(Figure 2.13). The first successful variant was obtained
by replacing phenylacetic acid by phenoxyacetic acid
as the added precursor. This gave phenoxymethyipeni-cillin or penicillin V (Figure 2.13). The main advan-tage of this change was an improvement in the stabil-ity of the penicillin towards acid. The ready
inactivation of penicillin G at low pH limited its use-fulness when it was given by mouth smce a variable
and often considerable fraction of the antibacterial ac-tivity was destroyed in the acidic environment of the
stomach. Penicillin V thus improved the reliability of
oral doses. These early penicillins, produced directly
by fermentation, were intensely active against Gram-positive infections and gave excellent results in strep-tococcal and staphylococcal infections and in pneumo-nia. They were also very active against Gram-negative
infections caused by gonococci and meningococci, but
were much less active against the more typical Gram-negative bacilli.
A further advance in the versatility of the peni-cillins was achieved by workers at the original
Beecham company (now part of GlaxoSmithKline)
with the development of a method for the chemical
modification of the penicillin molecule. Bacterial en-zymes were found that remove the benzyl side chain
from penicillin G, leaving 6-aminopenicillanic acid,
which could be isolated and then acylated by chemical
means. This discovery opened the way to the produc-tion of an almost unlimited number of penicillin deriv-atives, some of which have shown important changes
in properties compared with the parent penicillin. The
value of increased stability has already been men-tioned, and some semisynthetic penicillins show this
33
The cell walls of bacteria and fungi
(H)
.C C. ^
CH3
! /-•CH 3
‘•COOH
Genera! penicillin structure
Side chain
(R)
^^^^CHjCO “
/V-OCHjCO- -H–OCH3
OCH3
,ci co -^= ^ N- O
( V-CHCO -HO- f VCHCO -NHg
CHCO-Vg/COON a
Name
Benyl penicillin
Penicillin G
Phenoxymetliyl
penicillin
Penicillin V
e-Aminopanicilianic
acid
IMethicillin
Cloxaciilin
Armpiciilin
Amoxycillin
IVIecillinam
Ticarcillin
H
r— — N —”^ V Of” * F
«^ COOH
0
General cephalosporin structure
Substituents
OOCCH-|CH2|jC0- CH3COOCH2 H
NH3
H- CH3COOCHJ H
(y~CH,CO~- (Q-CH , H
/~~VcHCO – CH-, H
r^CGO – HjNGOO- H
NOCH3
li >-CCQ – CH3COO- H
NOCH3
Kj y”°V.COO H
H,<JC0 – -CH=CH, H
s
N-‘°-wCOO H
., K.^’^”7r \ CH, CH,
H,.<JC0~ _^^^^_^r^ ,
N-OCH3 ®^’= ^
0
Name
Cephalosporin C
7-Aminocephalosporanic acid
Cephaloridine
Ceptialexin
CafuroKJme
HR756
Ceflxime
Ceftazidime
Ceftriaxone
OOCH-[CHJ CO-i+ t 2J3 HjNCOO- CH3O- CephamycinC
M,iMUU ^ y CO- .CH s<? ‘ N CH3O Cefotetan
i
\_J CH3OCOCH3
C = NOCH3
Cefotaxime
FIGURE 2.13 Representative penicillins and cephalosporins.
34
2.4 Antibiotics tliat inhibit peptidoglycan biosynthesis
property. Other modified penicillins (e.g. methiciiiin
and cloxacillin, Figure 2.13) are much less susceptible
to attack by p-lactaniase, an enzyme which converts
penicillin to the antibacterially inactive peniciiloic
acid and gives rise to the commonest fomi of resist-ance to penicillin (Chapter 9).
The discovery of the p-lactamase inhibitor, clavii-lanic acid (Figure 2.14), which is a p~lactam itself but
without useful antibacterial activity, provided an op-portunity to coadminister this agent with p-lactamase~
o o [i n
HOOCCHCHjCHjCNHCHCNH. OCH3
NHj  CH3
o Suifazecin (a monobactam)
SOjNa
“OOC
“;CHCHjS[CHJ,. CH = C-^N H
3 nn
DOC CH -I CHT H O—{‘ V—
NH,
C-CONH^v H
N-OH I ‘
{syn) J N^
Nocardicin A
OH
COOH
H3C 1 J—i^V
I J> S CH^CH^NHj. I \^c
° \ O J”,, H
COOH * ^rrtrtiA
CHjOH
> S CHjCHjINHj
‘COOH
Thienamycin
COOH
Ciavyianic acid
COOH
Meropenem
FIGURE 2.14 Additional P-lactam compounds and ci-iastatin, an inhibitor of mammalian metabolism of thien-amycin. Clavulanic acid is an intiibitor of serine-active-site
P-lactamases.
sensitive compounds such as amoxycillin (Figure
2.13) in mixtures such as augmentin (a 1:1 mixture of
amoxycillin and clavulanic acid) and timentin (a 1:1
mixture of ticarcillin and clavulanic acid).
Another striking change brought about by chem-ical modification of the penicillin side chain was an in-crease in activity against Gram-negative bacteria, a
property found in several derivatives, including ampi-cillin, amoxycillin, carbenicillin and ticarcillin (Figure
2.13). This increase in Gram-negative activity is ac-companied by a lessening of activity towards Gram-positive bacteria. Ampicillin is one of the most widely
used antibacterial agents. In mecillinam (Figure 2.13),
where the side chain is attached by an azomethine link
rather than the usual amide bond, the activity spectrum
of the original penicillin molecule has been completely
reversed. This compound is highly active against
Gram-negative bacteria but reciuires 50 times the con-centration for an equal effect on Gram-positive organ-isms. It can be used in the treatment of typhoid fever,
which is caused by the Gram-negative bacterium Sal-monella typhi.
Cephalosporin C (Figure 2.13), originally iso-lated from a different organism than that used to pro-duce penicillin, has a structure in its nucleus similar to
that in the penicillins. The biogenesis of the nuclei in
these two classes of antibiotics is now known to be
identical except that in cephalosporin biosynthesis the
thiazolidine ring of the penicillin nucleus undergoes a
specific ring expansion to form the dihydrothiazine
ring of the cephalosporin nucleus. Besides this similar-ity in structure and biogenesis, cephalosporin C and its
derivatives act on peptidoglycan cross-linking in the
same way as the penicillins. Cephalosporin C itself is
not a useful antibacterial drug, but like the penicillins,
it is amenable to chemical modification. Enzymic re-moval of the side chain gives 7-aminocephalosporanic
acid, which can be chemically acylated to give new de-rivatives. A second change in the molecule can also be
made by a chemical modification of the acetoxy group
of cephalosporin C. The first successful semisynthetic
cephalosporin was cephaloridine. Many others have
followed; a selection of some the best known is shown
in Figure 2.13. Most are only effective when given by
injection, but cephalexin and cefixime can be given by
mouth. Cefuroxime is unaffected by many of the com-mon P-lactamases and can be used against bacterial
The cell walls of bacteria and fungi
strains which are resistant to other P-lactam anti-biotics; it can be useful in infections that are due to
Neisseria or Haemophilus. The related compound, ce-fotaxime, has enjoyed considerable success. Other
agents such as ceftazidime and ceftriaxone are useful
because of the fonner’s improved antipseudomonal ac-tivity and the latter’s enhanced half-life in the body,
which pennits a more convenient dosing schedule, for
example, once or twice daily.
The cephamycins resemble the cephalosporins,
but have a methoxy group in place of hydrogen at po-sition 7. Cefotetan (Figure 2.13) is a semisynthetic de-rivative of cephamycin C. The cephamycin derivatives
are not readily attacked by P-lactamases and have ad-vantages over the cephalosporin derivatives, with ac-tivity against Proteus and Serratia species.
The enormous success of the penicillins and
cephalosporins stimulated a seairh for other naturally
occurring P-lactam compounds. These have been
found in a variety of micro-organisms. Some of the
most interesting are shown in Figure 2.14. In the cai’-bapenem, thienamycin, the sulfur atom is not part of
the ring, but is found in the side chain. This compound
is remarkable for its liigh potency, broad antibacterial
spectrum and resistance to P-lactamase attack, but it is
both chemically unstable and susceptible to degrada-tion by a dehydropeptidase found in the kidneys. The
iV-fonnimidoyl derivative of thienamycin is chemi-cally more stable but must administered as a 1:1 mix-ture with cilastatin (Figure 2.14), an inhibitor of the
renal peptidase. A further development in the cai’-bapenem series has been the appearance of the syn-thetic compound meropenem (Figure 2.14). This drug
is not readily degraded by renal peptidase and can
therefore be administered as a single agent. Merope-nem is active against Gram-positive and Gram-nega-tive pathogens, including many which are resistant to
other P-lactams.
Other P-lactam antibiotics include the monobac-tams (e.g. sulfazecin. Figure 2.14); the name comes
from monocyclic bacterial P-lactams) which are de-rived from bacteria and represent the simplest P-lactam structures with antibacterial activity so far dis-covered. Many semisynthetic derivatives have been
made and exhibit excellent anti-Gram-negative activ-ity, with much weaker activity against Gram-positive
bacteria. In contrast, the monocyclic nocardicins (Fig-ure 2.14) appear to offer less activity and are of more
historic than clinical interest. Interest in the p-lactam
family remains intense and novel drugs with improved
properties continue to be developed.
2.4.6 Mode of action of penicillins and
cephalosporins
As with many other antibiotics, early attempts to dis-cover the biochemical action of penicillin led to con-flicting hypotheses. Gradually it became accepted that
the primary site of action lay in the production of cell
wall material, and more specifically in the biosynthe-sis of peptidoglycan.
Evidence for this site of action rests on several
difterent types of experiment. Staphylococcus aureus
cells were pulse-labelled with [^”^Clglycine, and pepti-doglycan was isolated from their walls after a further
period of growth in unlabelled medium. The labelled
glycine entered the pentaglycyl ‘extending group’.
The polysaccharide backbone of the peptidoglycan
was then broken down by an A’-acetylmuramidase,
leaving the individual muramyl peptide units linked
only by their pentaglycine peptide chains. After the
products were separated by gel chromatography, ra-dioactivity was found in a series of peaks of increasing
molecular weight representing the distribution of the
pulse of [^”^Clglycine among peptide-linked oligomers
of varying size. A parallel experiment done in the pres-ence of penicillin showed the radioactivity to be as-sociated largely with a single peak of low molecular
weight, presumably the un-cross-linked muramyl pep-tide unit, with much less radiolabel in the oligomers.
The penicillin had thus inhibited the peptide cross-linking.
In another experiment, ‘nucleotide pentapeptide’
was prepared with [^”^CJalanine. This was used as a
substrate for an enzyme preparation fi-om Escherichia
coll in the presence of UDP-A?~acetylglucosamine.
This system carried out the entire biosynthesis of pep-tidoglycan, including the final stage of cross-linking.
Peptidoglycan was obtained as an insoluble product
containing [^"'"C] from the penultimate D-alanine of the
substrate; the terminal D-[^'*C]alanine was released
into the medium, partly from the transpeptidase cross-linking reaction and partly from a carboxypeptidase
3i
2.4 Antibiotics tliat inhibit peptidoglycan biosynthesis
that removed temiinal D~alanine residues from cross-linked products. In a parallel experiment, penicillin
was added at a concentration that would inhibit growth
of Escherkiiia coli. Biosynthesis of peptidoglycan
then proceeded only to the stage of the linear polymer
(IX, Figure 2.6), which was isolated as a water-soluble
product of high molecular weight labelled with [^'''C].
No D-[''''C]alanine was liberated because the penicillin
suppressed both the cross-linking transpeptidase reac-tion and the action of DD-carboxypeptidase.
The understanding of the mechanism of p-lactam
action was considerably advanced by the discovery of
the penicillin-binding proteins refeiTed to in Section
2.3.5. Of the PBPs in Escherichia coli and many other
bacteria, PBPla and FBPlb are the key enzymes in-volved in peptidoglycan biosynthesis. PBP2 and FBP3
are concerned, respective!}’, with remodelling of the
peptidoglycan sacculus during septation and cell divi-sion. All these PBPs are targets of P-lactam antibiotics.
Different p-lactams exhibit different affinities for the
various PBPs and these can in turn be con-elated with
different morphological effects. Drugs which bind
most strongly to PBPs la and lb cause cell lysis at the
lowest antibacterial concentration. Compounds such
as the cephalosporin, cephalexin, bind more strongly
to PBP3 and inhibit septation, leading to the fomiation
of filaments, which are greatly elongated cells. An-other variation is found with mecillinam, which binds
almost exclusively to PBP2 and causes cells to assume
an abnomial ovoid shape. Cells overproducing PBP2
have enhanced amounts of cross-linked peptidoglycan
and are very sensitive to mecillinam.
The interaction of a penicillin or cephalosporin
(1) with the enzyme (E) can be represented as:
E + I ^ EI ^ EI* ^ E + degraded inhibitor.
The first step is reversible binding to the enzyme.
The second stage, involving chemical modification of
the inhibitor with covalent binding to the enzyme, is ir-reversible, as is the final stage of enzyme release. For
high antibacterial activity, k-^ should be rapid, prevent-ing release of inhibitor through reversal of the initial
binding, and k^ should be slow to maintain the enzyme
in the inactive EI* form and to avoid significant reac-tivation. Measurements show that the widely used P~
lactam antibiotics have just such characteristics, and
this scheme goes far to explain their outstanding effec-tiveness. There is good reason to suppose that the inac-tivation mechanism is the same with cross-linking
transpeptidases as with DD-carboxypeptidases. The na-ture of the end products of penicillin degradation de-pends on the enzyme involved. It may be a simple
opening of the P-lactam ring to give the penicilloate or
there may be more extensive breakdown leading to the
production, from benzylpenicillin, of phenylacetyl
glycine. Those enzymes which yield penicilloate ai^e
equivalent to slow-acting P~lactamases. There is evi-dence to suggest that active P-lactamases are relatives
of carboxypeptidases and transpeptidases in which re-action k^ is rapid instead of very slow.
The mechanism of action of DD~carboxypepti~
dases and cross-linking transpeptidases resembles that
of certain esterases and amidases. These enzymes pos-sess reactive groups associated with their active cen-tres, which undergo transient acylation in the course of
enzymic action. z\ntibiotics containing a P-lactam ring
behave chemically as acylating agents. The action of
penicillin on the PBPs thus involves acylation of the
enzymically active site in the second reaction to form
the inactive complex EI*. This explanation was sup-ported by experiments with purified DD~carboxypepti~
dases from Bacillus stearothermopMlus and Bacillus
subtilis. The enzyme was allowed to react briefly with
|”*C|benzylpemcillin or with a substrate analogue,
[''*C]Ac,L~Lys-D”Ala~D~lactate; D-lactic acid is the
exact hydroxyl analogue of D-alanine, and use of this
derivative enabled the transient enzyme reaction inter-mediate to be trapped. In peptide fragments from the
Bacillus stearothermophilus enzyme, radioactivity
was found in a peptide with 40 amino acid residues
and the label was shown to be associated with the same
specific serine residue, whether the reactant was ben-zylpenicillin or the substrate analogue. Similar results
were found with the Bacillus subtilis enzyme from
which a labelled 14-unit peptide was isolated. This
peptide showed extensive homology with 14 residues
of the Bacillus stearothermophilus peptide and the
label was associated with the corresponding serine
residue. It was concluded that penicillin binds to the
active site and acylates the same serine as the sub-strate. Unlike the substrate, the degraded penicillin
37
The cell walls of bacteria and fungi
was released very slowly (reaction k^ in the scheme
shown) and thus blocked further access of substrate to
the site.
How can this action of penicillin be related to its
stracture? The most widely quoted explanation de-pends on the similarity of the spatial orientation of the
principal atoms and polar groups in the p-lactam nu-cleus to one particular orientation of the D~alanyl-D~
alanine end group of the pentapeptide side chain of
peptidoglycan precursors (see Figure 2.15). When the
two structures are compared, the peptide bond between
the alanine units is seen to correspond in position to the
C—N bond in the p-lactam ring which is believed to
be responsible for the acylating activity. Such a group
bound to the cross-linking transpeptidase close to its
active centre could well usurp the acylating function
implicit in the normal reaction of the substrate with the
enzyme. When the stractures (Figure 2.15) are com-pared more critically, it becomes apparent that the
agreement between them is imperfect but can be much
improved if the peptide bond of the D-alanyl-D-alanine
end group is represented, not in its normal planar form,
but twisted nearly 45° out of plane. This may imply
that the confomiation of the penicillin molecule re-sembles the transition state of the substrate rather than
its resting fomi. During the enzymic transpeptidation,
the peptide bond quite possibly undergoes this sort of
distortion.
An altemative model is based on a comparison of
certain electrostatic potentials of benzylpenicillin and
synthetic iV-acyl-D-alanyl-D-alanine peptides. Calcula-tion of these potentials reveals a significant similarity
in the coplanarity of key electrostatic negative wells of
both benzylpenicillin and the dipeptide terminal. The
coplanarity of these wells may facilitate the attack of
an electrophilic centre in the catalytically active serine
of the target PBP on the p-lactam C—N bond. With
some modifications this model may be applicable to all
Penicillin
H
Labile bond
Alanyl-alanine
end group  Bond brol<en during the
transpeptidation reaction
associated with cross-linking
FIGURE 2.15 Comparison of the
structures of penicillin with that of the
D-alanyl-D-alanine end group of the pep-tidoglycan precursor. [Reproduced by
permission of the Federation of Ameri-can wSocieties for Experimental Biology
from i. L. Strominger et al. Fed. Proc.
26, 18(1967).]
38
2.5 Drugs that interfere with the biosynthesis of tlie ceil wall of mycobacteria
types of P”lactam drag. However, the precise details of
tlie interactions between P-lactam and PBPs will have
to await data from the various on-going
X-ray crystallographic studies of p-iactam-PBP
complexes.
wjf ffiujf i BJ i&M ^fe & i i £3 & 111% . Etf i i Ctf i S * W W i & i i & i i Cff
b iOS¥iifliP’*&i^ of ttip CPI I WI^I I of
mft #tf°ft #% &% ^ f^a*O b i**! ^
Mycobacteria are responsible for two devastating dis-eases’ tuberculosis {Mycobacterium tuberculosis) and
leprosy, or Hansen’s disease {Mycobacterium leprae).
The ceil wall of mycobacteria is remarkably complex
and underlies many of the characteristic properties of
these organisms, including then extremely low perme-ability and intrinsic resistance to commonly used an-tibiotics. The reader is referred to a review provided in
‘Further reading’ at the end of this chapter for detailed
infonnation on^he cell wall of mycobacteria. A key
feature of the mycobacterial cell envelope that distin-guishes it from most other bacteria is the mycolyl-arabinogalactan-peptidoglycan complex. Arabino-galactan is linked to the peptidoglycan through a
phosphodiester link between the C-6 of 1()~14% of the
muramic acid residues and a disacchaiide linker unit
attached to the galactan. Arabinogalactan itself is a
unique polysaccharide consisting of linear galactan
chains composed of alternating 5- and 6-linked P~D-galactofuranose units which in turn £ire linked though
C-5 of some of the 6~linked galactofuranose units to
extensively branched chains of D~arabinofuranose
(arabinan). Approximately two-thirds of the nonreduc-ing terminals of arabinan are esterified to long-chain
mycolic acids. There are other lipids in the mycobac-terial cell outer envelope in addition to the mycolic
acids, including a range of complex glyco- and pepti-dolipids. The lipoidal nature of this complex wall is a
significant contributor to the impemieability of my-cobacteria to many solutes, including some antibiotics.
The characteristically slow growth rate of mycobacte-ria also presents a considerable challenge to the suc-cessful chemotherapy of infections caused by these
bacteria, which usually requires several months of
continuous drag treatment.
Zl.D. ! ioUnidZi u
Isoniazid (Figure 2.16) provides one of the founda-tions of combination therapy for tuberculosis. The rel-ative ease with which Mycobacterium tuberculosis be-comes resistant to individual drugs led to the concept
of combining several chemically distinct drugs with,
as it later turned out, different modes of action. In com-bination variously with rifampicin, ethambutol, pyraz-inamide and occasionally streptomycin, isoniazid is an
effective antitubercular drug which has been in use
since 1952. However, it is only since the 199()s that
biochemical and genetic data have revealed the molec-ulai” mechanisms underlying the antimycobacterial ac-tion of isoniazid.
NHNH,,  N=N H  CHpOH CH,>OH
CH3CH2CHNHCH2CH2NHCHCH2CH3
Isonicotinic acid  Ethambutol
NH 2
Ethionainid©  Pyrazinamide
FIGURE 2.16 Structures of synthetic
compounds used iu combination therapy of
tuberculosis. The structure of the microbial
metabolite isonicotinic acid can be seen to
resemble that of isoniazid.
•^ Q
The cell walls of bacteria and fungi
In the mycobacteria, the inhA gene encodes an
enzyme that has been identified as a major molecular
target for isoniazid and the structurally related drug,
ethionamide (Figure 2.16). This enzyme, abbreviated
to InhA, catalyzes the NADH-dependent reduction of
the 2-franj~enoyl-acyl canier protein (ACP), an essen-tial reaction in the elongation of fatty acids. Long-chain substrates containing between 16 and 18 carbon
atoms are preferentially used by InhA, an observation
which implicates the reductase in the biosynthesis of
the mycolic acids. Inhibition of the biosynthesis of
mycolic acids therefore disrupts the assembly of the
mycolyl-arabinogalactan-peptidoglycan complex and
causes the loss of cell viability. While mutations in
inhA confer resistance to isoniazid, studies with re-combinant InhA show that isoniazid itself is only a
weak inhibitor of the enzyme. The drug is in fact first
converted by oxidative cellular metabolism to a reac-tive metabolite which is beheved to bind to and inhibit
the reductase in the presence of NADH bound to the
enzyme. Isoniazid is metabolically unstable in my-cobacteria, owing to the activity of a unique mycobac-terial catalase-peroxidase encoded by the katG gene.
Studies with the recombinant fomi of this enzyme
show that it converts isoniazid to several chemically
reactive derivatives, isonicotinic acid (Fig 2.16) being
the major product. The electrophilic nature of these
compounds would enable them to acylate or oxidize
vulnerable amino acid residues in the target reductase,
although direct evidence for this is lacking. The two-stage concept of the mechanism of action of isoniazid
is strengthened by the existence of two fomis of my-cobacterial resistance to the drag. One type of mutant
has a defective katG gene that precludes the conver-sion of the prodrug to its active form. The second re-sistant phenotype depends on an isoniazid-resistant
variant of InhA that is characterized by a markedly
lower affinity for NADH, which minimizes the attack
of the isoniazid metabolite on the enzyme.
Recently, the product of another gene, kasA, has
been proposed as an alternative primary site of action
for isoniazid. The kasA gene encodes the enzyme p-ketoacyl synthase (KasA), which may be involved in
the biosynthesis of C, g~~Cj^ fatty acids required for the
elaboration of mycolic acids. The case for the KasA
enzyme as a primary target for isonaizid rests largely
on isoniazid-resistant clinical isolates of mycobacteria
with mutations solely in the kasA gene, i.e. with no
mutations in either inhA or katG genes. However, there
is contrary evidence of clinical isolates with mutations
in kasA which retain sensitivity to isoniazid. In sum-mary, the weight of experimental and observational ev-idence supports the concept of the InhA enzyme as the
primary target for isoniazid (and also ethionamide), al-though a possible contribution from KasA cannot be
ruled out at this stage.
2.5.2 Ethambutol
The antibacterial activity of isoniazid is confined to
Mycobacterium tuberculosis. Ethambutol (Figure
2.16), which has been in clinical use against tuberculo-sis since 1961, has a broader spectrum of action, in-cluding Mycobacterium avium, a serious opportunist
pathogen in patients with AIDS. Despite many years
of use, the molecular basis of the bacteriostatic action
of ethambutol was identified only recently. It had long
been known that the drag in some way blocked the
biosynthesis of the polysaccharide arabinan, but the
actual mechanism was not known. The target for
ethambutol was eventually established by cloning the
genetic elements responsible for resistance to this drag
in Mycobacterium avium. The stractural genes embA,
embB and embC all encode arabinosyl transferases
which appear to have similar functions in polymeriz-ing arabinose into arabinan. In vitro evidence obtained
with a cmde broken cell preparation from Mycobac-terium smegmatis indicates that ethambutol inhibits
the transfer of a hexa-arabinosfuranosyl unit, from the
phospho-decaprenol canier complex, to arabinan.
Most clinical isolates of Mycobacterium tuberculosis
that are resistant to ethambutol have mutations in
embB. The molecular target of ethambutol therefore
seems to be arabinosyl transferase, with the product of
the embB gene being the most important. The mecha-nism of inhibition of the arabinosyl transferases by
ethambutol remains to be established and will proba-bly await the purification of the enzymes, which are
predicted to be integral membrane proteins with mul-tiple anchoring, transmembrane domains and an exter-nal domain.
Disruption of the biosynthesis of the arabino-galactan component of the mycobacterial cell enve-40
2.6 The fungal cell wall as a target for antifungal drugs
lope may increase cellular permeability to other drugs.
This could account for the valuable clinical synergism
that is achieved when ethambutol is combined with a
drug of large molecular size such as rifampicin.
2.5.3 Pyrazinamide
Although first recognized for its substantial antimy-cobacterial activity in the 1950s, this synthetic drug
was not introduced into the combination therapy for
tuberculosis until the mid-1980s. Pyrazinamide (Fig-ure 2.16) is a bacteriostatic agent which is especially
useful against semidormant populations of My-cobacterium tuberculosis located in acidic intracel-lular compartments such as the phagolysosomes of
macrophages.
The active form of pyrazinamide is believed to be
P3’razinoic acid (Figure 2.17), formed by the action of
an intracellular bacterial amidase, referred to as pyraz-inamidase. Some pyrazinamide-resistant strains of M.
tuberculosis lack pyrazinarnidase activity. Genetic and
biochemical evidence strongly suggests that the anti-bacterial activity of pyrazinamide rests upon the inhi-bition by p3’razinoic acid of a multifunctional fatty
acid synthase (type I) encoded by xhefasi gene, which
results in suppression of mycolic acid biosynthesis. By
inhibiting the type 1 fatty acid synthase (FAS 1), pyriz-inamide blocks the provision of fatty acid precursors
for another fatty acid synthase (FAS II) which has an
essential role in the elongation of mycolic acids.
2 ^% i B S £% ffi 1 1 S^ tf^ SS i tf^ £ ^ i i \SiiS^& i i ^& C? foil ‘sil ^ ff^fi^ O&’s’ ffl’S’l& fi** « %^ il l ^ ^ i %iH i i \M ^S i \^ ^ ^ i i ^w %^ S i %^ %3^ ^S %p%^ i %M ^ ^ % i vji i
a i ^ B*! B*! i W^ ^% «»& i ^ ^1 1 £°1 ^
Fungal infections (mycoses) pose an ever-increasing
threat to health across the world. Immunocompro-mised individuals, including AIDS patients, those on
immunosuppressive drugs following organ transplan-tation, cancer patients undergoing chemotherapy, peo-ple recovering from major surgery and patients receiv-ing prolonged antibacterial treatment, are all at risk
from infections caused by a variety of fungal patho-gens. Compared with the wealth of drugs available to
treat bacterial infections, the current therapeutic op-tions for fungal infections are much more limited.
NH ,
Pyrazinamide
pyrazinamidas© “‘M ‘
Pyrazinoic acid
FIGURE 2.17 Conversion of the prodrug pyrazinamide to
the active molecule bv bacterial amidase.
Although it serves functions analogous to those
of the bacterial cell wall, the structure of the fungal
wall is very different from that of its bacterial counter-part. Critically, fungal walls do not contain peptidogly-can, so neither p~lactam nor glycopeptide antibiotics
have any effect on the viability of fungi. The fungal
wall is a multilayered structure whose major macro-molecular components include chitin, glucan and
mannoproteins. Neither chitin nor glucan occurs in
mammalian or bacterial cell walls, so the biosynthesis
of these materials provides potential targets for spe-cific antifungal drug action. Because glycosylated pro-teins are found in all eukaryotes, the biosynthesis of
fungal mannoproteins may be rather less attractive as
a target for chemotherapy. However, the sugar residues
of glycosylated proteins are very different in fungi and
humans and could conceivably offer opportunities for
drug design. The composition and organization of the
cell wall vary significantly among the various fungal
species and define the identity of the organisms. Chitin
is a linear l,4~p-linked homopolymer of/V-acetylglu-cosamine. In yeasts chitin contributes as little as 2% to
the cell wall mass, while there can be as much as 60%
chitin in some mycelial fungi. Nevertheless, chitin is
essential for fungal growth, even in species with very
small amounts of the polymer. Cflucan is a p-1,3-linked linear glucose homopolymer with varying
amounts of p-l,6~ and p~l,4-glucose side chains, de-pending on the species. The mannoproteins make up
complex chains of rnannose linearly bonded by 1,6-links to which oligomannoside side branches are at-tached by 1,2- and 1,2-a bonds. The polysaccharide
structures are covalently linked to protein via a l,4~p-disaccharide of A’-acetylglucosamine residues by ei-ther A~glycosyiation of asparagine or 0~glycosylation
at the free hydroxyl groups of threonine or serine
residues. Some idea of the diversity of mannoproteins
41
The cell walls of bacteria and fungi
may be gauged from the fact that between 40 and 60
differem mannoproteins can be isolated from yeast
cell walls.
The arrangement of these various polymers in the
wall of the important fungal pathogen Candida albi-cans is illustrated in Figure 2.18. The insoluble poly-mers chitin and glucan confer mechanical strength on
the wall. The function of the mannoproteins is less
clear but appears to be essential because inhibitors of
N- and 0~glycosylation are lethal, although it should
be remembered that the effects of inhibition of these
reactions are not confined to the biosynthesis of
mannoproteins.
2.6.1 Inhibitors of chitin biosynthesis
The enzyme chitin synthase catalyzes a reaction in
which an A’~acetylglucosamine residue is transferred
from the donor molecule, UDP~A?-acetyl glucosamine,
to the nonreducing end of the growing chitin chain,
with the concomitant release of UDP. Chitin synthase
exists in several foims, none of which has been puri-fied so far. Two related groups of antibiotics inhibit
chitin synthase, the polyoxins and nikkomycins (Fig-ure 2.19). Both types are analogues of UDP-A’-acetyl-glucosamine and presumably inhibit the enzyme by
competition with this substrate. In the yeast Saccha-romyces cerevisae, the gene encoding chitin synthase 1
is essential for repairing damage to the intercellular
septum incurred during the separation of daughter
cells. The product of the chitin synthase 2 gene is
specifically involved in the biosynthesis of the septum
itself while that of the chitin synthase 3 gene produces
•— Fibrils
“—IVIannoprotein
•«—p-Glucan
-<—p-Glucan + Chitin
•”—IVIannoprotein
•*—Cytoplasmic membrane
FIGURE 2.18 The general arrangement of layers in the
fiinga! cell envelope. The components are not drawn to scale.
It should be remembered that the precise structure of the fun-gal cell envelope is markedly species-specific.
most of the chitin in the bud scar and lateral cell wall.
No single synthase appears to be essential for cell via-bility, but the loss of all three in mutants of Saccha-romyces cerevisiae is lethal. Chitin synthase exists in
multuple isozymic forms in Candida albicans and pos-sibly in other pathogenic fungi.
The susceptibility of fungi to polyoxins and
nikkomycins varies considerably and may be due to
difterences in the distribution and sensitivities of the
chitin synthase isoforms to these antibiotics. Another
factor which determines susceptibility to polyoxins is
their transport into fungal cells by a peixnease that nor-mally carries dipeptides. Candida albicans is intrinsi-cally resistant to polyoxins because of the low activity
of this permease. Despite these potential problems, it
is hoped that the best of the currently available in-hibitors of chitin synthase, nikkomycin Z, may eventu-ally find a place in clinical medicine. The essential role
of chitin in fungi has encouraged a search for other,
more effective inhibitors of the chitin synthases, so far
O / “^p/O-^l/O ^ / UDP-N-acetyl-D-g!ucosamin
THO Me^o HO OH
HO
NH
HN”’^COO H
COOH / Polyoxin D
H \ J
HO OH
Nikkomycin Z
OH NH2
HO OH
FIGURE 2.19 Antifungal agents that inhibit eel! wall
chitin synthesis, together with the substrate UDP-A’-acetyl-glucosamine.
2.6 The fungal cell wall as a target for antifungal drugs
it must be said, with little success. Purification of the
enzymes and the provision of adequate amounts for
screening purposes are likely to be prerequisites for
further progress.
2.6.2 Inhibitors of glucan biosynthesis
In glucan biosynthesis, the enzyme L3-P~glucan syn-thase catalyzes the transfer of glucose from UDP-glu-cose to the insoluble, growing glucan polymer. Unlike
chitin synthase, glucan synthase has been purified to
homogeneity. The enzyme consists of two subunits,
one of which is an integral membrane protein, molec-ular mass 215 kDa, with multiple transmembrane he-lices. The other subunit is a much smaller protein (20
kDa) that interacts with GTP-binding proteins and is
only loosely associated with the cell membrane. The
function of the smaller subunit is apparently to activate
the catalytic activity of the membrane-bound protein
through interaction with the GTP-binding protein
complexes. Two closely homologous fomis of glucan
synthase have been identified in Saccharoinyces cere-visiae, designated as FKSl and FKS2. FKSl is domi-nant during vegetative growth whereas FKS2 has an
essential role in sporulation. Genomic analysis of 5’ac-charomyces cerevisiae predicts a third possible glucan
synthase, FKS3, although it remains uncharacterized
at present. Sequence-related glucan S3’nthases have
been found in other yeasts and in filamentous fungi.
Five genes apparently encoding glucan synthases have
been identified in the genome sequence of Candida
albicans.
Echinocandin B (Figure 2.20) is a member of a
large family of naturally occurring and semisyntheti-cally modified lipopeptide antibiotics which have
HO OH
Caspofungin
FIGUR E 2.20 Inhibitors of the bio-synthesis of the glucan polymer in
fungal cell waUs.
43
The cell walls of bacteria and fungi
potent activity in vitro against Candida spp. and
against the filamentous Aspergillus spp. These com-pounds are powerful non-competitive inhibitors of 1,3-P~glucan synthase. This specificity may explain the
lack of activity against fungi where glucan is not
mainly 1,3-p-linked. hi Cryptococcus spp., for exam-ple, a dangerous pathogen affecting the respiratory
tract, the glucan is mostly 1,3-a-linked and the organ-isms are resistant to the cyclic lipopeptide antibiotics.
Studies with an echinocandin-resistant mutant of Sac-charomyces cerevisiae identified the membrane-bound
component of l,3~p-D-glucan synthase as the likely
target of the drag. Unfortunately the details of the in-hibitory mechanism, including the site of interaction
between the antibiotic and the enzyme, are not known
at present. The clinical usefulness of echinocandin B is
limited by its propensity to cause lysis of red blood
cells, which is thought to be due to its extended
lipophilic side chain. Another member of the
echinocandin group, Caspofungin (Figure 2.20), re-cently entered clinical practice for the treatment of As-pergillus infections unresponsive to other drugs and
disseminated Candida infections. Caspofungin is
likely to have the same mode of action as echinocandin
B. Another semisynthetic cyclic lipopeptide, Micafun-gin, has recently enetered clinical practice in Japan.
2.6.3 Disruption of the function of
mannoproteins
Pradimicin A (Figure 2.21) belongs to a unique group
of antibiotics originally isolated from Actinomadura
hibisca and is active against Candida spp., Cryptococ-cus spp. and Aspergillus spp. The antifungal action in-volves a change in the permeability of the cell mem-brane, which may result fom the ability of pradimicin
to form an insoluble complex with mannan in the pres-ence of calcium ions. Although this points to some
fomi of interference with mannoprotein function, the
biochemistry of the antifungal action of pradimicin re-quires further investigation. As yet, this drag has not
been used to treat fungal infections in human patients
although it has shown promise in the treatment of ex-perimental infections.
CH3O
./ -~OH
OH O  ° ^ \ NHCH3
HO
H
FIGURE 2.21 Pradimicin A, an experimental antibiotic ac-tive against several species of yeast pathogens. Its mode of
action may depend upon interference with the function of
cell wall mamioproteins.
Further reading
Allen, N. Fi. and Nicas, T. I. (2003). Mechanisms of action of
oritavancin and related glycopeptide antibiotics. FEM.S
Microbiol. Rev. 26,511.
Bremian, P. J. (1995). The envelope of mycobacteria. Annu.
Rev. Biochem. 64, 29.
Denome, S.A. et al. (1999). Escherichia coli mutants lacking
all possible combination of eight penicillin binding
proteins: viability, chai’acteristics and implications for
peptidoglycan synthesis. / . Bact. 181, 3981.
Doyle, R. J. and Marquis, R. E. (1994). Elastic, flexible pep-tidoglycan and bacterial cell wall properties. Trends
Microbiol. 2, 57.
Ghuysen, J.-M. et al. (1996). Penicillin and beyond: evolu-tion, protein fold, multimodular polypeptides and mul-tiprotein complexes. Microbial Drug Resistance 2,
163.
Goffm, C. and Ghuysen, J.-M., (2002). Biochemistr\’ and
comparative genomics of SxxK superfamily acyltrans-ferases. Microbiol. Molec. Biol. Rev. 66, 702.
Green, D. W. (2002). The bacterial cell wall as a source of
antibacterial targets. Expert Opin. Then Targets 6, 1.
Liu, J. and Balasubramanian, M. K. (2001). l,3P-Glucan
synthase: a useful drug for tmtifungal drugs. Cun:
Drug Targets-Infect. Disord. 1, 159.
Ming, L.-J. and Epperson J. D. (2002). Metal binding and
structure-activity relationship of the nietallo-antibiotic
peptide bacitracin. / . Inorg. Biochem. 91, 46.
44
2.6 The fungal cell wall as a target for antifungal drugs
Odds F. C , Brown, A. J, P. and Gow, A. R, (2003). Antifun- Skarzyiiski, T. et al. (1996). Structure of UDP-W-acetyl glu-gal agents: mechanisnis of action. Trends Microbiol. cosamine enol pyruvyl transferase: an enzyme essen-11, 272. tial for the synthesis of bacterial peptldoglycan, com-Prescott, L. M., Harle)’, J. P. and Klein D. A. (1996). Micro- plexed with substrate UDP-;V~acetyl glucosamine and
biology. Wm. C. Brown, Dubuque, lA. the drug fosfomycin. Structure 4, 146,5.
ScliroederE. K. etal. (2002). Drugs that inliibitmycolic acid Van Hiejenoort, J. (2001). Formation of giycan chains in the
biosynthesis in Mycobacterium tuberculasis. Curr. synthesis of bacterial peptidoglycan. Glycobiol. 11,
Pharm. Biotechnol. 3, 197. 25R.
C hanfpr thrpp
3 1 RHiprohp killpi””?’ antispntirt anri
d” ” ^ J A J A
3 ^ 3 S^ W £!5& tf^ W €!ft S^W ^ 3 ^&B 5 5 i ^^i» ^ i,C i 5 5 i, ^
The major interest throughout this book lies in the
mechanism of action of drugs that can be used against
microbial infections. For this purpose the compound
must normally be absorbed and cuxulate in the blood.
However, there is also a requirement in medicine, in
industry and in the home for substances that kill bac-teria and other micro-organisms on the surface of the
body or in other places. Such products ai^e known as
disinfectants, sterilants, antiseptics or biocides, the
choice of temi depending on the circumstances in
which they are used. ‘Disinfectant’ describes products
intended for use in the presence of dut and dense bac-terial populations, for example, in cleaning animal
quarters or drains. ‘Biocide’ is used more pailiculaily
for preservatives that prevent bacterial and fungal at-tack on wood, paper, textiles and other kinds of or-ganic material and in pharmaceutical preparations.
‘Antiseptic’ is a term usually reserved for a substance
that can be safely applied to the skin and mucosal
surfaces to reduce the chances of infection by killing
the surface bacteria. ‘Sterilants’ are substances used to
sterilize an enclosed space; since penetration is
paramount in this application, sterilants are usually
gaseous.
The requirements for a compound having disin-fectant or antiseptic action differ markedly from those
needed in a systemic drug. Many compounds used suc-cessfully against microbial infections do not actually
kill micro-organisms, but ooly prevent their multipli-cation, and most are inactive against nongrowing or-ganisms. A cessation of microbial growth is often all
that is needed in treating an infection, provided that the
immune defences of the body can be mobilized to re-move pathogens present in relatively small numbers.
Furthermore, systemic antimicrobial agents often have
a fairly limited spectrum of action. This is acceptable
since the compound can be selected according to the
nature of the infection that is being treated. Antiseptics
and disinfectants, in contrast, are usually required to
have a broad-spectrum killing effect. Antiseptics and
preservatives used in ointments, creams, eyedrops and
multidose injections must obviously be free from tox-icity against the host tissues.
A distinction is oifen made between ‘static’ and
‘cidal’ compounds, but the division is by no means
clear-cut. There is no certain way of detemiining
whether a micro-organism is dead. The usual method
of assessing the killing effect of an antiseptic is by
measuring the ‘viable count’ of a previously treated
bacterial or fungal suspension. The antiseptic is first
inactivated and dilutions of the suspension are added
to a rich medium. The organisms are deemed to be
alive if they give rise to colonies. Many compounds are
static at low concentrations and cidal at higher concen-trations, and the effect may also depend on the condi-tions of culture. However, for antiseptics and disinfec-tants, a cidal effect is required under all normal
conditions of application. Such compounds must be
able to kill micro-organisms whether they are growing
47
Antimicrobial agents and cell membranes
or resting; they must be able to deal with most of the
common bacteria likely to be found in the environment
and, ideally, fungi and viruses as well. Bacterial and
fungal spores are usually much more difficult to kill.
Many of the older disinfectants are compounds of
considerable chemical reactivity. Their antimicrobial
action presumably depends on their ability to react
chemically with various groups on or in the organism,
thus killing them. Such compounds include hydrogen
peroxide, the halogens and hypochlorites, the gaseous
sterilants ethylene oxide, ozone, etc. Salts and other
derivatives of the heavy metals, particularly of mer-cury, probably owe their antimicrobial effect to reac-tion with vital thiol groups. In disinfection, their high
reactivity and toxicity limit their scope and they are not
generally acceptable for the more delicate uses as anti-septics. For this purpose three main groups of com-pounds are widely used: alcohols, phenols and cationic
antiseptics. The ether compound triclosan is also a
popular and effective antiseptic. The main emphasis
with these agents has been their efficacy against bacte-ria. Increasingly, however, there is concern that they
should have useful activity against fungi and viruses.
Although there are differences among the actions of
the various types of antiseptics, they have several com-mon features:
1. Antiseptics interact readily with bacteria, the
amount adsorbed increasing with an increas-ing concentration in solution. The adsorption
isotheiTH sometimes shows a point of inflec-tion which corresponds to the minimum bac-tericidal concentration; higher concentra-tions lead to a much greater adsorption of the
compound.
2. The extent to which micro-organisms ai^e
killed is governed by three principal factors:
concentration of the antiseptic, cell density,
and time of contact.The adsorption of a given
amount of the compound per cell leads to the
killing of a definite fraction of the microbial
population in a chosen time intei-val.
3. The lowest concentration of the antiseptic
that causes the death of micro-organisms also
brings about leakage of cytoplasmic con-stituents of low molecular weight. The most
immediately observed effect is a loss of
potassium ions. Leakage of nucleotides is
often detected by the appearance in the
medium of material having an optical absorp-tion maximum at 260 nm. Some loss of cyto-plasmic solutes is not in itself lethal, and cells
that have been rendered leaky by low concen-trations of an antiseptic will often grow nor-mally if they are immediately washed and
placed in a nutrient medium. The increased
permeability is a sign of changes in the mem-brane which may be initially reversible but
become irreversible on prolonged treatment.
4. The necessary characteristic of antiseptics is
their biocidal action, but there is often a low
and rather narrow concentration range in
which their effect is biostatic. At these low
concentrations, certain biochemical func-tions associated with the microbial mem-brane may be inhibited but may not necessar-ily lead to cell death.
5. In the presence of higher concentrations of
antiseptic and after prolonged treatment, the
compound usually penetrates the cell and
brings about extensive disruption of normal
cellular functions, including inhibition of
macromolecular biosynthesis and eventually
precipitation of intracellular proteins and nu-cleic acids.
The primary effect of these antiseptics on the cy-toplasmic membrane is thus established beyond doubt,
but secondary actions on cytoplasmic processes are
less well defined and may vary from one compound to
another. Examples of evidence of action for particular-compounds will be given as illustrations.
3.1.1 Phenols
Crude mixtures of cresols solubilized by soap or alkali
and originally introduced as Lysol are still used as
rough disinfectants. They need to be applied at high
concentrations and are mitant and toxic, but they kill
bacteria, fungi and some viruses. For more refined ap-plications as antiseptics, chlorinated cresols or
xylenols (Figure 3.1) are commonly used since they
are less toxic than the simpler phenols. In general, the
48
3.1 Microbe killers: antiseptics and disinfectants
Chlorocresol
Br
I
HO H C—C—CH^OH
NO,
Bronopol
CHglCHp] N—CH,
CH3
Cetrimide
// \  NH.C.NH.C.NH[CH2].NH.C.NH.C.NH
NH
Chlorohexidine
-CI
NH NH
FIGURE; 3.1 Some commouly used
antiseptics. The tbrmula tor cetrimide
shows the main components in the
preparations nomially sold. Homo-logues with other chain lengths, espe-cially Cjg, are also present.
primary action of the phenolic disinfectants and anti-septics is to cause the denaturatioo of microbial pro-teins, the first target being the proteins of the ceU enve-lope, leading to lethal changes in membrane
penneability.
3.1.2 Alcohols
Alcohols are widely used as inexpensive antiseptics,
disinfectants and preservatives. Ethanoi, for example,
is a reasonably effective skin antiseptic as a 60 70%
solution which kills both bacteria and viruses. Iso-propanol (propan-2-ol) (at least 70%), which is
slightly more effective as a bactericide than ethanoi but
is more toxic, can be used to sterilize instruments such
as clinical thermometers. The more complex coni-poiind known as bronopol (2-bronio-2-nitropropatie-1,3-diol, Figure 3.1) is an effective preservative for cer-tain pharmaceutical products and toiletries, although
there are concerns about the potential toxicity of some
of the decomposition products of this compound, in-cluding formaldehyde and possibly nitrites, upon its
exposure to light.
The antibacterial effects of the alcohols can be
traced to a disrtrption of membrane function. The ac-tion of short-chain alcohols such as ethanoi is probably
dominated by the polar function of the hydroxyl group,
which may fonn a hydrogen bond with the ester
groups of membrane fatty acid residues. In contrast to
ethanoi, longer-chain alcohols gain access to the hy-drophobic regions of membranes and this probably ac-counts for the increasing potency of antimicrobial ac-tion up to a maximum chain length of 10 carbon atoms.
The interaction of alcohols with cell membranes pro-duces a generalized increase in permeability which is
lethal at higher concentrations. Bronopol may exert an
additional effect by interacting with thiol groups in
membrane proteins.
0 . i.O UoliOni G ctniioupliG o
This classification covers a number of compounds dif-fering considerably in chemical type. Their common
features are the presence of strongly basic groups at-tached to a fairly massive lipophilic molecule. Al-though antiseptic action is found quite widely in com-pounds having these characteristics, the degree of
activity is sharply dependent on stnictui^e within any
particular group. For instance, in cetrimide, a quater-nary alkylammonium compound (Figure 3.1), the
length of the main alkyl chain is 14 carbon atoms and
the activity of other compounds in the same series falls
49
Antimicrobial agents and cell membranes
off markedly with longer or shorter chains. Cetrimide
combines excellent detergent properties and minimal
toxicity with a useful antiseptic action. However, it is
not very potent against Proteus and Pseudomonas
species and has little antiviral activity, except against
viruses with a lipid envelope. Experiments with & -cherichia coli labelled with -‘^P show that with in-creasing concentrations of cetrimide the loss of cell
viability closely parallels the degree of leakage of ra-dioactivity from the bacteria. An effect on bacterial
growth, however, is noticeable at concentrations that
affect neither viability nor permeability. At bacter-icidal concentrations, the bacterial membrane is
ruptured.
One of the best and most widely used of the
cationic antiseptics is chlorhexidine (Figure 3.1). This
compound has two strongly basic groups, both
biguanides; it is often formulated as the digluconate,
which has good solubility in water. Chlorhexidine is
much less surface active than cetrimide and has little
detergent action. However, it acts against a wide range
of bacteria at concentrations between 10 and 50 (ig
ml^^ and it also has useful activity against Candida al-bicans. Its toxicity is low and it has so little irritancy
that it can be used on the most sensitive mucosal sur-faces. For example, it is a useful aid to oral hygiene.
Periodic rinsing of the mouth with chlorhexidine solu-tion greatly reduces the population of Streptococcus
mutans on the teeth. This minimizes the production of
dental plaque and reduces periodontal infections that
give rise to gingivitis. It also decreases the incidence of
some types of caries. An important feature of this ac-tion is the strong binding of chlorhexidine to the tis-sues in the mouth, including the teeth, with subsequent
slow release which maintains an antibacterial action
over an extended period.
Chlorhexidine exerts effects on the cytoplasmic
membrane that are characteristic of cationic antisep-tics. At concentrations that just prevent the growth of
Streptococcus faecalis, it inhibits the adenosine tri-phosphatase of the membrane. The effect can be
shown in isolated membranes and on the solubilized
enzyme derived from them. The proton motive force
produced by the proton gradient across the cytoplas-mic membrane, which drives ATP synthesis, is also
dissipated. A similar concentration of chlorhexidine
inhibits the net uptake of potassium ions by intact cells
and suppresses macromolecular biosynthesis. The in-teraction between chlorhexidine and the cell mem-brane probably involves electrostatic binding between
the cationic groups of the antiseptic and the anionic
groups of phospholipids in the membrane. Hydropho-bic interactions between the hexamethylene chains of
chlorhexidine and the aliphatic chains of the phospho-lipids also contribute to the stability of the complex.
When bacteria are treated with a range of concentra-tions of chlorhexidine and then examined for leakage
of cytoplasmic solutes, the degree of leakage increases
with concentration up to a maximum and then declines
at higher concentrations. Low concentrations of
chlorhexidine provoke the release of K”*” ions, nu-cleotides and sugars. Electron microscopy shows that
the cells from higher levels of chlorhexidine treatment
are grossly altered. The increased membrane pemie-ability apparently allows the antiseptic to enter the cy-toplasm and cause precipitation of the nucleic acids
and proteins, resulting in the death of the cells. Under
these circumstances, leakage is probably prevented by
simple mechanical blockage. With Gram-negative
bacteria, chlorhexidine damages the outer membrane
as well as the cytoplasmic membrane. This can be seen
as ‘blistering’ in electron micrographs (Figure 3.2).
This phenomenon will be discussed further in connec-tion with the action of polymyxin.
3.1.4 Triclosan: an antiseptic in a class of
its own
Triclosan (2,4,4′-trichloro-2′-hydroxyphenyl ether.
Figure 3.1) has been used for over 30 years as an anti-septic in medical handwashes and in many household
products, including toothpaste, mouthwash, soaps, and
deodorants, and in kitchen cutting boards. With broad-spectrum antimicrobial activity, although relatively
poor against pseudomonads, and minimal toxicity (at
least as a topical agent), triclosan has therefore en-joyed considerable success. For much of its history the
antimicrobial activity of triclosan was thought to be
caused by a direct membrane-damaging effect similar
to that of the other common antiseptics. More recent
research, however, reveals that the primary target of
triclosan is lipid biosynthesis, the inhibition of which
in turn leads to a loss of membrane integrity and func-50
3.1 Microbe killers: antiseptics and disinfectants
i^.^~
FIGURE 3.2 Electron micrograph of a cross-section of an Escherichia coli cell after treatment with cMorhexidine (30 iig
ml-‘), showing’blistering’of the cell wall.
tion. The first clue came with the isolation of a strain
of Escherichia coli resistant to the antiseptic. Gene
mapping located resistance in the fabi gene that en-codes the enzyme enoyl-acyl carrier protein reductase,
or FaM. This enzyme plays a key role in the biosynthe-sis of short-chain fatty acids and is a homologue of the
InhA enzyme involved in mycolic acid biosynthesis in
mycobacteria described in Chapter 2. Purified Fabl
from triclosan-resistant Escherichia coli was unaf-fected by the antiseptic, whereas the enzyme from the
triclosan-sensitive bacterium was strongly inhibited.
The essential cofactor for Fabl is NAD”” and, remark-ably, triclosan increases the affinity of the enzyme for
NAD’*’ by some four orders of magnitude. A tight ter-nary complex is formed that involves Fabl, NAD’*’ and
triclosan with a dissociation constant for triclosan of
20-40 pM. X”Ray analysis of the complex reveals that
the 2-hydroxy-3-chlorophenyl ring of triclosan inter-acts with enyzme-bound NAD”” and with the enzyme
itself via hydrogen bonding to the phenolic hydroxy!
group of tyrosine-156. The 2,4~dichlorophenyl ring of
the antiseptic, which is rotated 90° out of the plane of
51
Antimicrobial agents and cell membranes
the hydroxychlorophenyl ring, forms a hydrogen bond
between the 4-CI atom and alanine-95. There are also
hydrophobic contacts between this ring and the side
chain of methionine-159.
It is intriguing that the InhA enzyme of Mycobac-terium tuberculosis, which is the molecular target of
the active metabolite of the antitubercular drug isoni-azid (Chapter 2), is also inhibited by triclosan. How-ever, /Mycobacterium tuberculosis is not susceptible to
triclosan and there is evidence that the bacterium har-bors another form of enoyl-aCP reductase which is not
inhibited by triclosan and therefore assumes the func-tion of InhA when that enzyme is inhibited. These ob-sen’ations, along with the recent finding that systemic
doses of triclosan are effective against experimental
infections in mice, suggest the possibility that the
stracture of triclosan could conceivably provide a
starting point for the design of novel antitubercular
drags.
3.2 Cationic peptide antibiotics
Several classes of polypeptide antibiotics are known,
all of which have their origins in natural sources. Com-pounds with cyclized peptide regions include the tyro-cidins, gramicidins and polymyxins (Figure 3.3). The
tyrocidins and gramicidin S are cyclic decapeptides.
These contain one or sometimes two free amino
groups. They are more active against Gram-positive
than against Gram-negative bacteria. The polymyxins
have a smaller polypeptide ring attached to a polypep-tide chain terminating with a branched 8- or 9-carbon
fatty acid residue. They have five free amino groups
associated with the diaminobutyric acid units. All the
cyclized peptide antibiotics carry a positive charge.
The antibacterial action of polymyxins is directed par-ticularly against Gram-negative organisms although
the selectivity can be altered dramatically by chemical
modification. For example, the penta-iV-benzyl deriva-tive of polymyxin is highly active against Gram-posi-tive bacteria.
In addition to the cyclized peptide antibiotics,
there are several hundred linear peptides with antimi-crobial activity which have been isolated from many
different species of prokaryotes and eukaryotes. These
linear peptides, e.g. magainin 11 (Figure 3.3), have an
enormous variety of sequences and structures but re-tain certain features in common. They are generally
12-50 amino acids in length and, like the cyclized pep-tides, carry an overall net positive charge owing to an
excess of basic lysine and arginine residues over acidic
amino acids.
Polypeptide antibiotics have only a minor place
in medicine because they also damage mammalian cell
membranes. The polymyxins may be used systemi-cally in severe Pseudomonas infections, although
there is considerable risk of kidney damage. Polymy-xin is bactericidal and acts on nongrowing as well as
growing cells. At low concentrations its bactericidal
action parallels the degree of release of cytoplasmic
solutes. It is strongly and rapidly bound to bacteria.
Val
f
Tyr
\  n
Asn*
/ Phe
Tyrocidin A
Orn’
/
Pro
Leu-Phe
A^Bu
(D)
PUe  Orn
Asn-Gramicidin S
GIGKFLHSAKKFGKAFVGEIMNS
Magainin I
– AjBu
-AjBu -A,Bu
!
Thr
A,Bu
Thr
AjBu-C0[CH2|-CH^
Polymyxin B,
‘C2H5
~CH3
FIGURE 3.3 Peptide antibi-otics that damage bacterial cell
membranes. A2BU: 2,4-dianiino-butyric acid. The arrows show the
direction of the peptide bonds.
Except where shown, all peptide
linkages involve a-amino and a-carboxyl groups. Configurations
are L unless otherwise indicated.
The formula for magainin 11 uses
the conventional abbreviations
for amino acids.
52
3.2 Cationic peptide antibiotics
With Salmonella typhimurium, the binding of 2 x 10″‘
molecules of polymyxin per cell is known to be bacle–ricidal. In Gram-negative bacteria, antibiotics of the
polymyxin group apparently bind first to the outer
membrane, affecting mainly the lipopolysacchaiide.
The gross effects of polymyxin on the outer membrane
are sometimes revealed in electron micrographs as
blisters, similar to those caused by chlorhexidine (Fig-ure 3.2). The swellings may be due to an increase in
the surface area of the outer leaf of the outer mem–braiie. The parallels between the action of polymyxin
and chlorhexidine are quite striking. In both, the bind-ing and antibacterial effects are antagonized by excess
of calcium or magnesium ions, indicating that the dis-placement of divalent ions is an impoitaiit feature of
their action. The disorganization of the outer mem-brane by polymyxin enables the antibiotic to gain ac-cess to the cytoplasmic membrane, which in turn is
damaged by the antibiotic. The disruption of normal
membrane function brought about by polymyxin re-sults in a generalized increase in membrane penneabil-ity and the loss of essential cellular nutrients and ions,
such as K’^.
Physical measurements of various kinds all sup-port the conclusion that the antibacterial action of
polymyxin is caused primarily by its binding to mem-branes. The positively char-ged peptide ring is thought
to bind electrostatically to the anionic phosphate head
groups of the membrane phospholipid, displacing
magnesium ions which normally contribute to mem-brane stability. At the same time, the fetty acid side
chain is inserted into the hydrophobic inner region of
the membrane. The effect is to disturb the normal or-ganization of the membrane and to alter its permeabil-ity characteristics.
The tyrocidins (Figure 3.3) are also bactericidal
and promote leakage of cytoplasmic solutes. Their ac-tion on the bacterial membrane permits passage into
the cell of ions that are normally excluded, and under
some conditions this causes uncoupling of oxidative
phosphorylation as a secondary effect. Gramicidin S, a
closely related compound (Figure 3.3), acts similarly.
It lyses protoplasts from Micrococcus lysodeikticus
but not those from Bacillus brevis. Since it is bacteri-cidal towards the fomier organism but not the latter, it
is reasonable to suppose that both its action and speci-ficity depend upon its effect on the cytoplasmic mem-brane, but a detailed explanation is lacking. The tyro-cidins act not only on bacteria but also on the fungus
Neurospora crassa. In this organism, concentrations of
the antibiotic that stop growth and cause leakage of
cell contents also cause an immediate fall in membrane
potential, a consequence of the destruction of the per-meability baiTier.
In both the tyrocidin group and in the polymyx-ins, the cyclic molecular stmcture is important for an-tibacterial activity. The presence of basic groups is also
essential, but in other respects the molecules can be
varied considerably without losing activity. The simple
symmetrical structure of gramicidin S has been sub-jected to many modifications. Activity is preserved
when the ornithine units are replaced by arginine or ly-sine groups but is lost by modifications that destroy the
basic character of the terminal groups. The compound
in which glycine replaces L-proline is fully active.
Moreover one L-proline residue together with the ad-jacent D-phenylalanine can be replaced by a 8-aminopentanoic acid group without losing antibacter-ial activity. The resulting compound has only nine
peptide groups, but retains the same ring size. Acyclic
compounds having the same sequence of amino acids
as gramicidin S show only slight antibacterial action.
The importance of the cyclic structure lies in the
maintenance of a well-defined, compact conformation
in solution. This has been shown by nuclear magnetic
resonance, optical rotatory dispersion and other physi-cal measurements. In tyrocidin A and gramicidin S the
conformation is determined by lipophilic association
between the nonpolar side chains of the amino acids,
particularly leucine, valine, proline and phenylalanine,
and by hydrogen bonding between the peptide groups.
Three regions have been defined in the molecular to-pography of tyrocidin A: a hydrophobic surface; a flat
hydrophilic opposite surface consisting of the peptide
groups of most of the amino acids in equatorial posi-tions; and a helical hydrophilic region, accommodat-ing the amide groups of asparagine and glutamine and
the tyrosine hydroxyl group. Gramicidin S shows a
similar arrangement, based on a pleated-sheet struc-ture. In both antibiotics the ornithine amino groups,
which are essential for antibacterial activity, stand out
from the hydrophilic surface.
Out of the enormous variety of linear cationic
peptides with antibiotic activity, only a handful of
53
Antimicrobial agents and cell membranes
compounds have reached the stage of clinical evalua-tion. The most interesting to date appear to be the ma~
gainins (Figure 3.3), which were originally isolated
from frog skin. The action of the linear cationic pep-tides on micro-organisms is broadly similar to that of
the cyclized compounds. That is, in the case of Gram-negative bacteria, there is an initial interaction with the
negatively charged lipopolysaccharides on the the sur-face of the outer membrane. This enables the peptides
to insert into and cross the outer membrane, in the case
of Gram-positive bacteria and fungi, the thick cell wall
provides only a partial barrier to the cationic peptides.
The cytoplasmic membrane is then open to attack. The
linear peptides again interact electrostatically with the
anionic outer surface of the membrane, allowing the
peptides to insert into the lipid bilayer. Precisely what
happens to the membrane at this point is unclear; one
suggestion is that the peptides may orient themselves
to fomi channels across the membrane in the manner
of the staves of a barrel, the so-called barrel-stave
model. Alternatively, the peptides may bind so effec-tively and in such quantity to the outer surface of the
cytoplasmic membrane that they wreck the intergrity
of the membrane entirely. This is known as the caipet
model. Whatever the exact details, the fundamental
permeability characteristics of the cytoplasmic mem-brane are compromised, allowing the loss of essential
solutes from the cytoplasm and the possible ingress of
peptide antibiotics into the cytoplasm, with subsequent
disruptive interactions with vital macromolecular enti-ties such as enzymes and other proteins.
3.3 lonophoric antibiotics
Several classes of antibiotics may be grouped together
because of their common property of facilitating the
passage of inorganic cations across membranes by the
formation of hydrophobic complexes with the ions or
by forming ion-pemieable pores across the mem-branes. Although these compounds were discovered
through their antibacterial activity, they are not used in
human bacterial infections because of their lack of
specificity. They act equally effectively on the mem-branes of animal cells and may therefore be toxic.
However, some ionophores have applications in veteri-nary medicine and animal husbandry which are dis-cussed later. lonophoric compounds are also of consid-erable biochemical interest and are widely used as ex-perimental tools. As antimicrobial agents, they are
active against Gram-positive bacteria whereas Gram-negative bacteria are relatively insensitive because
their outer membranes are impermeable to hydropho-bic compounds of the molecular size of the iono-phores. Monensin and lasalocid have useful activity
against the protozoal parasite Eirneria tenella, the or-ganism that causes coccidiosis in poultry and against
Clostridium perfringens, another pathogen of eco-nomic importance in the broiler chicken industry.
There is also some evidence for the selective toxicity
of certain ionophores against the malarial parasite (see
below).
3.3.1 Valinomycin
This was the first member of a group of related com-pounds to be discovered and is among the most widely
studied. It is a cyclic depsipeptide in which amino
acids alternate with hydroxy acids in a ring that con-tains both peptide and ester groups (Figure 3.4). An
important feature, which is common to all the cyclic
ionophores, is the alternation of D- and L~configura-tions in pairs around the 12 components of the ring
stracture.
Valinomycin forms a well-defined complex with
potassium ions. X~Ray analysis of this complex re-veals a highly ordered structure (Figure 3.5) in which
the potassium atom is suirounded by six oxygen
atoms. The ring structure is puckered and held in a
cylindrical or bracelet-like form by hydrogen bonds
roughly parallel to its axis. The ability to achieve such
a conformation depends entirely on the alternation of
D- and L-centres. The dimensions are such that the
potassium atom is exactly accommodated. The ion en-tering the complex must shed its normal hydration
shell; the complex retains the positive charge carried
by the ion. The stracture observed in the crystal is sub-stantially maintained in solution. Although valino-mycin will also form a complex with sodium, the
smaller sodium atom fits much less exactly into the
stmcture, and this complex has a stability constant
1000 times smaller than that of the potassium
complex.
54
3.3 lonophoric antibiotics
– (D)
Val •, ^va i
Lac
/
(D) Val
{D>Hiv
Val  L-C^LK
,Val
(D)
Lac
\
Val
Hiv (D)
Valinomycin
Nonactin
FIGURE 3,4 Antibiotics enhancing tlie permeability of membranes to potassium ions. In tiie valinomycin structure: Val
represents valine; Lac, lactic acid; and Hiv, 2-hydroxyisovaleric acid. The arrows indicate the direction of peptide or ester
bonds. Configurations are L unless otherwise indicated. Dotted lines separate the repeating units.
The high specificity of valinomycin towards the
potassium ion and the physical properties of the com-plex are consistent with its postulated action on biolog–ical membranes. The binding of the potassium ion in
the structure of valinomycin increases the lipophihcity
of the antibiotic and thereby promotes its diffusion into
the hydrophobic regions of the membrane. The
lipophilic molecule moves physically through the
membrane lipids, carrying potassium, and returns in
the protonated form. In a passive membrane the flow is
determined solely by the concentration of potassium
ions on each side of the membrane, but in mitochon-I’lGURE 3..5 Stereophotographs of a model of the potassium complex of valinomycin. To obtain a three-dimensional ef-fect, the diagram should be held about 50 cm from the eyes and attention concentrated on the space between the two pictures.
With practice, three pictures can be seen, the middle one showing a full stereoscopic elTect. The central metal ion is seen co-ordinated to six oxygen atoms. Nitrogen atoms are labelled N and the methyl groups of the lactyl residues are labeled M, Hy-drogen bonds are shown by thin lines. The solitary hexagonal ring is a liexane of crystallization, (We ai’e grateful to Mciry
Pinkertou and L. K. .Steinrauf for allowing us to reproduce this picture.)
Antimicrobial agents and cell membranes
dria supplied with an energy source, potassium is
taken in by an energy-coupled process against the con-centration gradient. Ttie process is highly effective,
one vahnomycin molecule being able to transport lO”’
ions per second, a turnover rate higher than that of
many enzymes. The transport of potassium by valino-mycin and similar ionophores shows saturation kinet-ics with respect to the cation; sodium ions inhibit
potassium transport although they undergo little trans-port themselves. The kinetic results are well explained
by a model in which the ionophore at the membrane
surface first fomis a hydrophilic cation complex. This
is transformed into a hydrophobic complex which can
then cross the membrane. The rate of the transfomia-tion from one type of complex to the other determines
the turnover number.
Vahnomycin specifically drains Gram-positive
bacteria of potassium and growth ceases because of the
requirement for potassium in cellular metabolism. If
the potassium content of the medium is raised to that
normally present in the cytoplasm, the inhibitory action
of vahnomycin is prevented. A characteristic second-ary effect of vahnomycin in growing aerobic bacteria is
to disturb oxidative phosphorylation. In analogous
fashion, vahnomycin disrupts oxidative phosphoryla-tion in the mitochondria of eukaryotic ceUs.
3.3.2 Nonactin
Another series of antibiotics known as the macrotetro-lides, exemplified by nonactin (Figure 3.4), have a
cyclic stmcture which also permits the enclosure of a
potassium ion in a cage of eight oxygen atoms (the car-bonyl and tetrahydrofuran oxygens), with the rest of
the molecule fomiing an outer lipophilic shell. To pro-duce this structure, the ligand is folded in a form re-sembling the seam of a tennis ball and is held in shape
by hydrogen bonding. The action of the macrotetro-lides closely resembles that of vahnomycin.
3.3.3 Monensin
lonophoric antibiotics of another broad group, typified
by monensin (Figure 3.6), carry a carboxyl group. In
these compounds the molecule itself is not cyclic, but
as with vahnomycin, a metal complex is formed in
which the ion is surrounded by ether oxygen atoms and
the outer surface is lipophilic. At physiological pH the
carboxyiate group of monensin is likely to be ionized
and located in the external environment of the cell. The
lipophilic character of the rest of the molecule enables
it to insert into the lipid bilayer of the cell membrane.
Positively charged, monovalent metal ions in the aque-ous medium associate initially with the negatively
charged carboxyl ions of monensin molecules, con-verting them into neutral species. Water molecules of
solvation bound to free metal ions are lost during the
interaction with the antibiotic. NMR spectroscopy
shows that following the initial interaction of a metal
ion with the carboxyl group, the monensin molecule
wraps itself around the metal, with five oxygen atoms
binding strongly and a sixth oxygen interacting more
weakly with the enclosed metal atom. This folding of
the molecule brings the carboxyl group at one end into
a position where it can form strong hydrogen bonds
with the alcohol groups at the other end; the structure
is thus stabilized into an effectively cyclic fomi.
Sodium ions are bound by monensin in preference to
potassium ions. The presence of the carboxyl group
promotes an electrically neutral cation-proton ex-change across the membrane by moving as an undisso-ciated acid in one direction and as a cation-anion com-plex with no net charge in the other direction. This
distinguishes monensin and stucturally similar
ionophores from vahnomycin and nonactin, in which
the metal complex carries a positive charge. The mech-anism by which the sodium ion is disgorged from the
monensin complex at the opposite face of the mem-brane seems likely to involve a sequential reversal of
the association process as the complex encounters the
internal aqueous environment.
Monensin is a compound of considerable com-mercial importance. It was first introduced as a treat-ment for the protozoal infection cocciodiosis in chick-ens, and proved to be of exceptional utility. It has
shown few signs of the development of resistance
which usually limits the effective life of drugs sold for
treating coccidiosis. Monensin also improves the uti-lization of feedstuffs in ruminants. Its action depends
on altering the balance of free fatty acid production by
rumen bacteria in favour of propionate at the expense
of acetate. Propionate is energetically more useful to
5i
3.3 luiiophciric siitibicitios
MrjLjl
en’s
HO^ CH^OH 1^ 0. ^ /
i ,v H HOOC
Monensin
OCH  ‘^r CH3 -‘
,0 -H3C
H i _H y”7y'”‘NHCH 3
I O*
^ O”‘ / \f ‘
H
OH
A 23187
.NH
COOH
HO-HjC-CH3 I – H ^H3
H S”^i ^H% / / -H OH 0  H
X~537A
CHjCH^
\- ?
0 -OH
CH3
FIGURE 3.6 Three more ionophoric antibiotics. Monensin preferentially complexes sodium ions by coordination with the
oxygen atoms marked by asterisks. The other two compounds complex with calcium ions. All three compounds have coc-cidiostatic activity.
the animal than acetate. There is also a lessening of the
metabolically wasteful production of methane. The
molecular basis of these effects is not certain, but the
shifts in nimen metabolism can probably be attributed
to differential antimicrobial actions on the complex
population of micro-organisms in the rumen. The ac-tion of monensin on cell membranes is not species-specific. Its lack of toxicity when given orally to farm
animals probably depends upon its limited absoiption
from the gastrointestinal tract.
3.3.4 Ionophoric antibiotics specific for
divalent cations
The ionophores considered so far form complexes only
with monovalent metal ions. A few ionophores are
known that fomi complexes with divalent ions. One of
the best known of these is the antibiotic calcimycin,
otherwise known as A23187 (Figure 3.6). This forms a
2:1 complex with calcium or magnesium ions, the cal-cium complex having the higher stability; it binds
monovalent ions only weakly. As with monensin, it is
not a cyclic molecule but is able to fold into an effec-tively cyclic conformation by the fomiation of a hydro-gen bond between a carboxyi oxygen and the NH
group of the pyrrole ring. The divalent metal ion is
held in octahedral co-ordination between the polar
faces of tVi’o ligand molecules. This gives an electri-cally neutral complex with a hydrophobic outer sur-face. It acts as a freely mobile carrier of these ions and
causes progressive release of magnesium, uncoupling
of oxidative phosphorylation and inhibition of adeno-sine triphosphatase in mitochondria suspended in a
magnesium-free medium. Like monensin, divalent
cationophoric antibiotics have coccidiostatic activity,
for example, the antibiotic lasalocid, or X-537A (Fig-ure 3.6).
57
Antimicrobial agents and cell membranes
Gramicidin A
Gramicidin A (Figure 3.7) (which is quite unrelated to
gramicidin S) has many biochemical properties reseni-bhng those of valinomycin. It shows a specificity to-wards potassium ions and promotes their passage
across hpid membranes. However, studies have shown
that its mechanism of action is different. The most sig-nificant demonstration of this distinction depends
upon measurements of the electrical conductivity of
artificial membranes separating aqueous layers con-taining potassium ions. Conditions can be chosen
where addition of valinomycin, nonactin or gramicidin
A at 0.1 uM concentration lowers the resistance of the
FIGURE 3.7 Gramicidin A. One possible helical structure having 6.3 residues per turn. Bonds drawn inwards are directed
down the helix; those drawn outwards are directed up. D-Amino acid residues are circled.
58
3.4 Antifungal agents that interfere with the function and biosynthesis of membrane sterols
membrane at least 1 OOO-fold. If the temperature is then
lowered gradually, the membrane reaches a transition
point at which its lipid la3’er effectively changes phase
from liquid to solid. In the presence of valinoniycin or
nonactin, a 2° C fall in temperature at the transition
point causes a dramatic rise in membrane resistance,
but in a similar experiment with gramicidin A, resist-ance rises only slowly as the temperature falls. The ef-fect with compounds of the valinomycin type is under-standable since they require a liquid membrane for
mobility and movement. Gramicidin A acts by the for-mation of a pore that permits the flow of ions through
a rigid membrane. Gramicidin A is a linear polypep-tide in which alternating amino acid residues have the
L-configuration. The remaining residues are either D-amino acids or glycine. The C~terminal is amidated
with ethanolamine and the N~terminal canies a formyl
group. The configuration allows the molecule to form
an open helical structure held together by hydrogen
bonds lying almost parallel to the axis of the cylinder.
One possible helical fomi is shown in Figure 3.7. The
inside of the helix is lined with polar groups and there
is a central hole about 0.4 nm in diameter. The fatty
side chains of the amino acids form a lipophilic shell
on the outside. One such molecule is not long enough
to form a pore across a membrane, but head-to-head
dimerization is believed to occur by bonds between the
forrnyl groups. The existence of dimerization is sup-ported by measurements in artificial membranes which
show that conductJince is proportional to the square of
the concentration of gramicidin A. The length of the
dimer is calculated to be 2.5-3.0 nm, which is some-what less than the thickness of the fatty acid layer in
many membranes, so some distortion probably occurs
during pore formation.
Conductivity measurements suggest that these
pores have a transient existence, a small fraction of the
antibiotic being in the fomi of pores at any given time.
The life of a channel measured in a phosphatidyl-ethanolamine artificial membrane was only 0.35 s.
However, while a pore is in existence, its transporting
capacity is high. One channel is estimated to convey
3 X 10” K+ ions s”^ under a potential gradient of 100
mV. Thus a low concentration of gramicidin A is a
very effective canier of potassium ions. Divalent
cations are too large to traverse the gramicidin pores
but block the free passage of monovalent ions.
3.3.5 Antiprotozoal activity of ionopliores
With the exception of monensin and lasalocid, the
practical applications of ionophores are very limited.
However, the pore-forming ionophores gramicidin D,
a linear peptide structurally related to gramicidin A,
and lasalocid show a surprising degree of selective
toxicity for the malarial protozoal parasite Plasmod-ium falciparum. The sensitivity of the organism was
studied during its intraerythrocytic stage. The normal
permeability characteristics of the red cell membrane
are drastically altered by the intracellular presence of
the parasite; permeability to Na’^ and Ca-^”*” ions is in-creased whereas permeability to K’*’ is decreased. The
selective toxicity of the ionophores may be due to
preferential partitioning into the cell membranes of
infected red cells, leading to abnormal ion fluxes into
the intraerythrocytic environment of the malarial pro-tozoan. It remains to be seen whether the interesting
in vitro activities of the ionophores can be translated
into safe and effective treatments for malaiial
infection.
3 0 , «\ §^ ffi i ffi i i S^ i”^ 5^ i 5^ i”^ S3fi ff^ a ^ “HH^ ^% B* i S^H*fiS ^”B fiS ^0& n^ ^ J^°%5 5 ^ n i Qpffi 5 5 \J9 C^ n C« ^»« %^ n n % ^ Q^i i C^ ^ 5 a a 0^%^ i a %^ i %^
with the function and biosynthesis of
3.4.1 Polyene antibiotics
The polyenes constitute a large group with varied mo-lecular structures which interact with membranes in an
especially interesting way. There are about 200 poly-enes, all produced by Streptomyces spp. Of these, only
a few ai”e sufficiently nontoxic for clinical use and only
one is used to treat systemic infections in man—am-photericin B (Figure 3.8). Polyenes ai^e active against
fungi, including yeasts, but not against bacteria. Sys-temic fungal infections are potentially dangerous, and
intravenous amphotericin B, which is usually specially
formulated with lipids, can halt infections which might
otherwise be fatal. Nystatin (Figure 3.8) is useful as a
topical agent to treat localized candidal infections on
mucosal surfaces. The polyenes are not absorbed from
the gastrointestinal tract but can be given by mouth to
combat fungal growth in the oral cavity and gut. The
relatively low toxicity of pimaricin (Figure 3.8)
S«Q
Antimicrobial agents and cell membranes
OH O OH OH OH OH 0
Amphotericin B
OH 0 OH OH OH OH O
Nystatin
H,C
Pimaricin
FIGURE 3.8 Polyene antifungal agents.
pemiits its use in food as a preservative against fungal
contamination.
The primary site of interaction of polyenes is the
fungal sterol, ergosteroi. The sterol composition of the
fungal membrane is important in determining the sen-sitivity to the polyene antibiotics, and the difference in
their relative affinities for ergosteroi (the major sterol
of fungal membranes) and the predominant cholesterol
of mammalian membranes allows limited clinical
applications of the polyenes. The ergosteroi molecule
adopts a cylindrical three-dimensional stmcture which
may favour its interaction with polyenes in preference
to the sigmoidal structure of cholesterol. Although the
molecular details of the interactions between polyenes
and ergosteroi and cholesterol remain uncertain, the
similarity between the sterols means that the therapeu-tic safety margins of polyenes are low. The compounds
are therefore inherently toxic to mammals, and the side
effects, such as kidney and nei-ve damage, of even the
best compound, amphotericin B, restrict their clinical
use. This toxicity can be reduced to some extent by ad-ministering the drug as a complex with cholesterol and
phosphatidylglycerol.
Bacteria are not affected by amphotericin B, or
other polyenes since their membranes do not contain
sterols. The mycoplasma Acholeplasma laidlawii does
not need sterols in its membrane, but can incorporate
either cholesterol or ergosteroi when they are added to
the growth medium. Ergosterol-containing organisms
are sensitive to amphotericin B, whereas cells grown
without sterols are not affected. The addition of ergos-teroi or digitonin to yeast cell cultures prevents ampho-tericin from being toxic; this results from the complex-ing of amphotericin by these agents. Mutant yeasts
which have a block at some stage of ergosteroi synthe-sis are resistant to amphotericin because there is no
longer a target for the polyene in the cell. Fortunately
this type of resistance is not clinically important.
The general action of polyenes is to increase the
permeability of fungal membranes, but the specific ef-fects of individual polyenes show considerable differ-ences. Filipin, for example, causes gross disruption of
membranes, with release of K”*” ions, solutes of low
molecular weight and small proteins, whereas A’-suc-cinylperimycin more specifically induces the release
of intracellular K+ ions. Living cells cannot survive a
catastrophic loss of intracellular potassium, so the in-teraction of polyenes with cell membranes soon results
in cell death. The ion peiTneability-enhancing effects
of the polyenes are probably caused primarily by the
drag molecules creating pores in the membranes. Mol-ecular models of amphotericin B and nystatin show a
rod-like structure held rigid by the all-trans extended
conjugated system, which is equal in length to an er-gosteroi molecule. The cross-section of the polyene
stmcture is roughly rectangular. One surface of the rod
is hydrophobic and the opposite surface, studded with
axial hydroxyl groups, is hydrophilic. At one end of
the rod the mycosamine sugar group and the carboxyl
group form a zwitterionic assembly with strongly
polar properties.
A computer-based, ‘virtual’ model of a possible
pore or channel involves eight amphotericin B and
eight cholesterol molecules (the latter molecule was
chosen because of the investigators’interest in polyene
toxicity). The computer also simulated the environ-ment of the membrane with layers of phospholipid sur-rounding the channel. The length of channel was less
60
3.4 Antifungal agents that interfere with the function and biosynthesis of membrane sterols
than the thickness of typical cytoplasmic membranes
and it is possible that in reality two such channels end-to-end may actually bridge the membrane. It is also
thought possible that the lipids around the channel
may accommodate themselves so that the bilayer is
‘pinched in’ somewhat, with the bilayer now approxi-mating the channel length. The stability of the com-puter model of the channel is largely dependent upon
hydrogen bonding between the hydroxyl groups of
neighbouring amphotericin B molecules and the
amino and carboxyl groups of adjacent drug mole-cules. The hydroxyl groups line the internal surface of
the channel and provide the necessary hydrophilic en-vironment for the passage of K”” and other water-solu-ble ionized species. It is suiprising that the computer
simulation did not reveal any structurally specific in-teractions between the sterol and amphotericin B, al-though the cholesterol molecules are essential to the
formation of the channel. Further details of this fasci-nating, though speculative, approach to the puzzle of
the amphotericin B membrane channels are available
in a research paper included under T-^’urther reading’.
Considerably more research will be needed before the
precise details of these channels are revealed. Further-more, the formation of pores does not satisfactorily ex-plain the gross changes in permeability brought about
by polyenes such as filipin. In this case it seems likely
that the insertion of the antibiotic into membranes
causes a more general disraption of their organization
and function.
3.4.2 Inhibition of ergosterol biosynthesis
Whereas the polyenes disnipt membrane function
through direct interaction with membrane ergosterol,
there are several groups of antifungal compounds that
act by inhibiting the biosynthesis of this sterol. An out-line of the biosynthetic pathway from squalene to er-gosterol is shown in Figure 3.9 and indicates the points
of inhibition of several types of antifungal agents that
are useful in either medicine or agriculture.
The azoles are among the most important compounds
cuiTently in use against fungal infections. They are
subdivided into imidazoles or triazoles, according to
whether they have two or three nitrogen atoms in their
five-membered azole ring (Figure 3.10). Unlike the
polyenes, most azoles do not kill fungi but rather act as
fungistatic agents. In their favour is that they are rela-tively nontoxic, with the exception of some of the older
compounds, such as ketoconazole, which has caused
fatal liver damage on rare occasions.
The antifungal action of the azoles depends on
inhibition of the C~14 demethylation reaction in the
biosynthesis of ergosterol. The enzyme involved, 14a-sterol demethylase, is a P^j^ cytochrome protein, oth-erwise known as P45()-ErgIlP or CypSlp, according
to different gene-based nomenclatures. The azoles in-hibit the enzyme by forming a stoichiometric complex
with the iron of the heme component of the enzyme.
The foixnation of the complex is detected by the red
shift of the Soret band of heme from 417 to 447 imi.
The heme iron interacts with the lone pair of electrons
on one of the ring nitrogens, and the complex is fur-ther stabilized by interactions between hydrophobic
moieties in the azole ligand and the enzyme. The con-formation of the active center of 14cx-sterol de-methylase varies among fungal species and the many
mammalian P^j^j, mono-oxygenases. The precise de-tails of the interaction between each of the azole drugs
and the various P^^,, enzymes are therefore likely to in-fluence the inhibitory potency across different fungal
species as well as the potential toxicity in patients (see
later discussion). The azole-mediated inhibition of the
demethylase is noncompetitive for the sterol substrate
and leads to a greater net reduction in flow through the
metabolic pathway than competitive inhibition. The
result is an accumulation of methylated sterols in the
cell and a reduction in the ergosterol content. Methy-lated sterols are more bulky than ergosterol and do not
fit easily into a noixnal membrane structure. This in-terference in the membrane structure is thought to
have adverse effects on membrane-bound enzymes,
such as those concerned with chitin synthesis and nu-trient uptake, either directly on their activity or on
their control. The depletion of ergosterol may also re-sult in interference with its hormone-like actions on
cell growth.
Typical examples of azole antifungals include the
topically active agent miconazole (Figure 3.10), which
is effective against thrush and demiatophyte infec-61
Antimicrobial agents and cell membranes
Allylamines
HO’ X, ^
Lanosterol
HO’
Ergosteroi
FIGURE 3.9 An outline of the biosynthesis of ergosteroi from squaiene, showing the points of inhibition of several types
of antifungal agent.
tions, and ketoconazole (Figure 3.10), which is orally
active and has been used to treat a wide range of fun-gal infections, particularly deep-seated, potentially
life-threatening mycoses. However, ketoconazole has
now largely been replaced by the triazole fluconazole
(Figure 3.10), which is particularly effective in treating
the candidal infections common in immunosuppressed
patients. Chemical substitution of the fluconazole mol-ecule, as in voriconazole (Figure 3.10), has further ex-tended the spectrum of antifungal action from patho-genic yeasts such as Candida albicans to include fila-mentous pathogens like Aspergillus spp. and
Fusarium. Because of the affinity of azoles for some
mammalian P^^o-dependent enzymes, including those
involved with steroid hormone synthesis, problems
can arise during therapy, owing to depletion of testos-62
3.4 Antifungal agents that interfere with the function and biosynthesis of membrane sterols
OH N
I ^N-CHjC-CH ^ i  ll N 1
CHo
O 1 i
CI
\ J “0 ^
” ^
-c-o-H,
–CI
/ =
A.
= \
J
Ketoconazole
CI—<( )>—CH2CHCH-(CH3)^
^ -^ N , OH
W //
Il^{cona:i:ol0  Diclobutrazole
CI
r=( OH o ”  CI
Fenarimol  Vorlcor!a;^of0
FIGURE 3.10 Azole antifungal
agents used in medicine and agri-culture. Note tlie presence of ei-ther two or tliree nitrogen atoms
in the lieterocyclic rings. Al-though fenaiimol has a biochem-ical action similar to that of the
azoles, it is in fact a pyrimidine
derivative.
lerone and glucocorticoids. Nevertheless, the azoles in
clmical use are several hundred times more potent
against lanosterol demelhylation than the correspon-ding reaction in mammals.
The azole diclobutrazole and the pyrimidine de-rivative fenarimol (Figure 3.10), which inhibits ergos-lerol biosynthesis by the same mechanism as the
azoles, have been used in agriculture to treat fungal in-festations of plants.
Allylamines
These compounds (Figure 3.11) also inhibit ergosterol
biosynthesis, but at an earlier stage. Naftifme and toi-naftate are only safe for topical use, while terbinafme
is both orally and topically active. The allylamines are
used to treat dermatophyte infections in humans and
domesticated animals. These agents share the s citric
mode of action by inhibiting squalene epoxidase (Fig-ure 3.9). Squalene accumulates in the cell to a concen-tration at which it becomes toxic, resuhing in cell
death. The reduction in cellular ergosterol caused by
the inhibition of squalene epoxidase is thought to be
less significant for the antifungal action of the ally-lamines than the accumulation of toxic levels of squa-lene. Allylamines are much less active against patho-genic yeasts such as Candida albicans and they have
little effect on mammalian squalene epoxidase. The
niammalian toxicity of naftifine and tolnaftate there-fore probably rests on other properties of these
molecules.
Morpholines
These compounds are too toxic for systemic use in
medicine because of major interference with host
sterol biosynthesis; they therefore are used as agricul-tural fungicides. The morpholines inhibit two stages
63
Antimicrobial agents and cell membranes
Naftiflne
C=CC(CH3)3
Terbinaflne
/ / ^
CH ,
I ^
NCS O
CH ,
Tolnaftate
FIGURE 3.11 AUylamine antifun-gal agents used to treat deniiato-phytic infections.
of the ergosterol biosynthetic pathway. The first tar-get is the enzyme that catalyzes the reduction of the
double bond at the 14-15 position formed after the re-moval of the C-14 methyl group. The second target is
the isomerization of the double bond between C-8
and C-9 of fecosterol to a position between C-8 and
C-7 (A’^-A^ isomerase). The balance between these
two inhibitory activities varies from fungus to fungus
and probably reflects subtle differences in the en-zymes involved. Tridemorph (Figure 3.12) inhibits
Ustilago maydis mainly at the C-14 reduction step,
whereas Botrytis cinerea is inhibited mainly at the C-8 to C-7 isomerization. However, in general the more
important target is likely to be the A'”* reduction since
this enzyme is essential for fungal viability whereas
the A’^-A*^ isomerase is not. There are serious con-cerns over the safety of tridemorph, specifically in re-lation to an apparent link with birth defects. As a re-sult, the use of the compound in agriculture and
horticulture is now prohibited in countries of the Eu-ropean Union.
Further reading
CH3(CH2),;
CH3
CH3
FIGURE 3.12 Tridemorph, a morpholine antifungal. Al-though it was previously used in agriculture, tridemorph is
now banned in many countries because of concern over its
toxicity to humans and domestic animals.
Anderson, O. S. (1984). Gramicidin channels. Ann. Rev.
Physiol, m, 5^1.
Baginski M. et al. (1997). Molecular properties of ampho-tericin B membrane channel: a molecukir dynamics
simulation. Molec. Pharmacol. 52, 560.
Balkis, M. M. (2002). Mechanisms of fungal resistance.
Drugs 62, 1025.
Dobler, M. (1981). lonophores and Their Structures. John
Wiley & Sons, New York.
Gumila, C. et al. (1999). lonophore-phospholipid interac-tions in Langmuir films in relation to ionophore selec-tivity towards P/ai-mod/Mm-infected er\’tlirocytes. ./.
Coll. Interface Sci. 15, 377.
Hancock, R. E. W. (2001). Cationic peptides: effectors in in-nate immunity and novel antimicrobials. Lancet, In-fect. Disord. 1, 156.
Ingram, L. O. and Buttke, T. M. (1984). Effects of alcohols
on micro-organisms. Adv. Microb. Physiol. 25, 254.
Maillard, J.-Y. (2002). Bacterial target sites forbiocide action.
/ . Appl. Microbiol. Symposium Suppl. 92, i.e. 16S.
Odds, R C , Brown, A. .1. P. and Gow, N. A. R. (2003). Anti-fungal agents: mechanisms of action. Trends Micro-biol. 11, 272.
Ridell, E. G. (2002). Structure, conformation and mechanism
in the membrane transport of alkali metal ions by
ionophoric antibiotics. Chiraiity 14, 121.
Russell, A. D. (1986). Chlorhexidine, antibacterial action
and bacterial resistance. Infection 14, 212.
Schroeder, E. K. et al. (2002). Drugs that inhibit mycolic
acid biosynthesis in Mycobacterium tuberculosis.
Curr. Pharm. Biotechnol. 3,197 (includes review of tri-closan action).
64
napterfour
^ 88^ ^
Many antimicrobial substances, both synthetic chemi-cals and natural products, inhibit the biosynthesis of
nucleic acids. However, relatively few of these in-hibitors are clinically useliil as antimicrobial drugs be-cause most do not distinguish between nucleic acid
synthesis in the infecting micro-organism and in the
host. Many inhibitors of nucleic acid synthesis are
therelbre too toxic to the host for safe use as antimicro-bial agents. However, there are important exceptions
which are described in this chapter.
The synthesis of DNA and the various types of
RNA is an essential function of dividing and growing
cells. Inhibition of DNA synthesis rapidly results in in-hibition of cell division. The biosynthesis, recombuia-tion and intercellular exchange of extrachromosomal
elements of DNA in bacteria are also critical ui main-taining the flexible responses of bacteria to changes in
the environment (Chapter 8). Inhibition of RNA syn-thesis is followed by cessation of protein synthesis.
The time elapsing between the inhibition of RNA syn-thesis and the resulting failure of protein biosynthesis
can be used to indicate the stability of messenger RNA
in intact cells.
Substances that interfere with nucleic acid bio-synthesis fall into several categories. The first group
includes several effective antibacterial drugs that inter-fere with the synthesis and metabolism of the ‘building
blocks’ of nucleic acids, i.e. the purine and pyrimidine
nucleotides. Interruption of the supply of any of the
nucleoside triphosphates required for nucleic acid syn-thesis blocks further macromolecular synthesis when
the nonnial nucleotide precursor pool is exhausted.
Structural analogues of purines and pyrimidines and
their respective nucleosides disrupt the supply of cor-rect nucleotides for nucleic acid synthesis and may
also directly inhibit nucleic acid polymerization fol-lowing conversion to the corresponding triphosphates,
either by inhibiting DNA- and RNA-dependent poly-merase activity or by causing premature chain temii-nation. Few such compounds are useful as antibacter-ial drugs because of their lack of specificity, but
several purine and pyrimidine nucleoside analogues
have achieved success as antiviral agents, and a pyrim-idine analogue finds application as an antifungal drug.
Compounds that interfere with the supply of folic acid
also inhibit nucleotide biosynthesis. Interruption of the
supply of tetrahydrofolate soon brings nucleotide and
nucleic acid synthesis to a halt, and inhibitors of dihy-drofolate reductase are useful in antibacterial and anti-malarial therapy.
Although DNA-dependent RNA polymerases are
common to both prokaryotic and eukaryotic cells, sev-eral naturally occurring and semisynthetic antibiotics
specifically inhibit the bacterial forms of these en-zymes. Another group of inhibitors blocks nucleic acid
synthesis by binding to the DNA template. This type of
interaction can prevent both DNA replication and tran-scription into RNA, but, except in the special case of
the antibacterial inhibitors of topoisomerases, this is
too nonspecific to permit broad therapeutic application.
Finally, several series of compounds, known as
topoisomerase inhibitors, block topological changes in
65
Inhibitors of nucleic acid biosynthesis
bacterial DNA that are essential for the organization
and functioning of DNA in cells. These compounds in-clude some of the most valuable antibacterial drugs in
current use.
4.1 Compounds affecting the biosynthesis
and utilization of nucleotide precursors
4.1.1 The sulfonamide antibacterials
The sulfonamides were the first successful antibacter-ial drugs. The original observation was made with the
dyestuff prontosil rabrum, which is metabolized in the
liver to the active drag sulfanilamide. A more effective
derivative of sulfanilamide was sulfapyridine, which
was in turn superseded by compounds with less toxic
side effects. Several of these early compounds are still
in use and their structures are shown in Figure 4.1.
Many other sulfonamide antibacterials have been
developed since. Most of these are probably no more
intrinsically antibacterial than the earlier compounds,
although some are much more persistent in the body
and can therefore be administered less frequently. The
sulfonamides act against a wide range of bacteria, but
their main success immediately following their discov-ery was in the treatment of streptococcal infections and
pneumococcal pneumonia. Gradually the sulfon-amides were displaced by naturally occurring antibi-otics and their derivatives, largely because of the
greater antibacterial potency of antibiotics. However,
sulfonamides have retained a place in the treatment of
certain bacterial and protozoal infections, especially in
combination with inhibitors of dihydrofolate reduc-tase. The stractural requirements for antibacterial ac-tivity in the sulfonamide series are relatively simple.
Starting from sulfanilamide, the modifications have
generally been variations in substitution on the nitro-gen of the sulfonamide group. Substitution on the aro-matic amino group causes loss of activity.
Among the many sulfonamides synthesized is
dapsone (Figure 4.1) which, although it has no useful
action against common infections, has an excellent ef-fect on leprosy and is still a mainstay in the treatment
of this disease, in combination with other drags such
as rifampicin to minimize the risk of development of
resistant mycobacteria. Dapsone is thought to act by
the same biochemical mechanism as the sulfonamides,
but the reason for its specificity in leprosy is not
known.
A few years after the discovery of the antibacter-ial activity of the sulfonamides it was shown that some
/ / WN= N HjN— V I—IN—IN—\A /r
Prontosil rubrum
S-NH ,
O
I? R:H,N ^ ^S-NH -Sulfanilamide
COOH w //
p-Aminobenzoic acid
^^^A\ //  <r\ \ //^NH ,
Diamlnodlphienylsuifone
(Dapsone)
Sulfapyridine
N- ‘
Sulfadiazine
CH3
Sulfadimidine  FIGURE 4.1 Examples of sulfona-mide aiitibacteria! drugs.
4.1 Compounds affecting the biosynthesis and utilization of nucleotide precursors
bacteria have a nutritional requirement for p-aminobenzoic acid, which is involved in the bio-synthesis of folic acid (Figure 4.2). The structural
resemblance between /7~aminobenzoic acid and the
sulfonamides underlies the ability of these dmgs to an-tagonize the stimulatory effect of/j-aminobenzoic acid
on the growth of bacterial cells. Later, the structure of
folic acid was found to contain a/J-aminobenzyl group
and its biosynthesis was shown to be inhibited by the
sulfonamides. The biosynthesis proceeds via the dihy-dropteridine pyrophosphate derivative shown in Figure
4.2, which then reacts with p-aminobenzoic acid with
loss of the p3’rophosphate group to give dihydropteroic
acid. The sulfonamides inhibit the enzyme dihy-dropteroate synthase (DHPS), which catalyzes this lat-ter reaction in an apparently competitive manner.
While DHI^S is essential for the de novo synthesis of
folate in bacteria, yeasts and protozoa, it is absent from
mammals, which acquire folate from the diet. DHPS is
therefore an excellent target offering high specificity
for antimicrobial agents. Almost 60 years after the dis-covery of competitive antagonism between sulfanil-amide and /7~aminobenzoic acid, the structure of
I3HPS from Escherichia coli and Staphylococus au-reus was determined. The structure of the enzyme is
that of an eight-stranded cx/p barrel. The more complex
substrate, 7,8~dihydropteridine, binds in a deep cleft in
the barrel while sulfanilamide (and presumably p~
aminobenzoic acid, although this remains to be deter-mined) binds closer to the surface. The structure of
DFIPS from Mycobacterium tuberculosis has also re-cently been determined in the hope that this may assist
in the design of novel antitubercular drugs.
The sulfonamides can also act as alternative sub-strates, giving rise to reaction products that are ana-logues of dihydropteroate. However, these products
probably do not play a major role in antibacter ial ac-tion since they inhibit the downstream enzymes only at
concentrations higher than those achievable in the cell.
The striking success of the sulfonamides as an-tibacterials, coupled with the early knowledge of their
point of action, led to an extraordinary flun-y of chem-HjN^ N
HjN
OH OH
I I
CHp-P-O-P-O H + HjN—<\ /)—COOH
0 0
HjN ^ ^N ^ ^N
N^^^^^’^N CH^H-( \ />—COOH
HjN^N^N .
OH
Dihydropteroic acid
* • y N XH^H-( x /)—CO-NHCHCOO H
Glutamate + ATP Q H ^ ( CH 1
\ I 2 J
H
OH ^ ^
Tetrahydropteroic acid
Dihydrofolic acid
Dihydrofolate reductase
H
12
:ooH
COOH
OH ^
Tetrahydrofoiic acid
-CO-NHCHCOO H
(CH,) ,
COOH
FIGURE 4.2 The final stages of folic acid biosynthesis. The first reaction in the sequence is catalyzed by dihydropteroate
synthase, which is competitively inhibited by sulfonamides.
67
Inhibitors of nucleic acid biosynthesis
ical research. Every conceivable bacterial growth fac-tor became the model for the synthesis of analogues in
the hope of repeating the success of the sulfonamides
as antibacterial agents. Unfortunately this early effort
was largely fruitless because the apparently simple
model provided by the antagonism of p-aminobenzoic
acid by sulfanilamide was not easily repeated. The sul-fonamides owe their effect to a fortunate set of circum-stances: (a) As we have seen, DHPS and p-aminoben-zoate are absent from animal cells, which acquire their
folic acid from the diet, (b) The inhibition of bacterial
growth is not reversed by folic acid because of its poor
diffusion into the cells. In contrast, the sulfonamides,
like /7-aminobenzoic acid, enter bacterial cells freely,
(c) Many biosynthetic intermediates carry phosphoric
acid groups which tend to prevent their diffusion into
bacteria, and potential inhibitors based on analogous
stmctures share the same difficulty of access. This
problem was not readily appreciated during the early
days of antibacterial drug research.
4.1.2 Inhibitors of dihydrofolate reductase
When the structure of folic acid became known and its
relationship to p-aminobenzoic acid and the sulfon-amides was accepted, a search was made for antago-nists among structural analogues of folic acid itself.
Many were synthesized but proved to be highly toxic
to mammals because folic acid derivatives, in contrast
to p-aminobenzoic acid, play an important part in the
metabolism of animal cells. The toxicity of some of
these compounds towards animal cells is actually
much greater than towards bacteria since bacterial
membranes are almost completely impermeable to
them. The cytotoxic action of the antifolic compound
methotrexate (Figure 4.3), is useful in the treatment of
certain malignancies, rheumatoid arthritis and psoria-sis, although great care must be taken to minimize the
risk of serious side effects.
Although the direct analogues of folic acid were
of no value as antimicrobial agents, other compounds
more distantly related to folic acid have considerable
importance. The potential of this type of compound
was first realized in two drags developed as antimalar-ials, proguanil and pyrimethamine (Figure 4.3). Pro-guanil is a prodrag that is metabolized in the liver to
the active agent, cycloguanil (Figure 4.3), which
closely resembles pyrimethamine.
The exact point of attack of these so-called an-tifolic compounds became apparent when the details
of folic acid biosynthesis were fully worked out. The
H,l
CH ^
CH,
Methotrexate
NHCHCOO H
(CH,)2
COO H
CH P H,N
CH P
H2 \= N
Trimethoprim
CI
// \ H I ‘
^ ‘ V—N-C-N-C-N-C H •
\ — / H II II H I
NH NH CH
Proguanil
Metabolism
in body
CI
H,N
N
H3C CH3
Cycioguani l
NH2  FIGURE 4.3 Drugs that inhibit di-hydrofolate reductase. In the case of
the antimalarial proguanil, its meta-bolite, cyclogiiani!, is the active
inhibitor.
4.1 Compounds affecting the biosynthesis and utilization of nucleotide precursors
step leading to the production of dih3’dropteroic acid
has already been discussed. At this point glutamic acid
is added to give dihydrofolic acid, which must be re-duced to the tetrahydro state (Figure 4.2) by the en-zyme dihydrofolate reductase (DHFR) before it can
participate as a cofactor for one-carbon transfer reac-tions. C3’totoxic analogues of folic acid, such as
methotrexate and the antimalarial drugs mentioned
eaiiier, inhibit DHFR. Although most living cells con-tain DHFR, the enzyme evidently differs in structural
details amongst major groups of organisms, and a use-ful degree of species specificity in the action of in-hibitors is possible. For example, pyrimethamine is
poorly active against the mammalian and bacterial en-zymes, but has an exceptionally strong affinity for the
enzyme from the malarial parasite, which accounts for
its specific antimalarial action. The antimalarial meta-bolite of proguanil has analogous specificity for the
protozoal DHFR. The stmcture of DHFR from several
species, including Escherichia coli, Lactobacillus
casei, and Plasmodium falciparum in chickens and hu-mans, has been revealed by X-ray analysis. Unlike the
DHFR of bacteria and the higher eukaryotes, the en-zyme of the malarial parasite is bifunctional, having
DHFR and thymidylate synthase domains which are
linked by a junctional region. This arrangement may
allow the channelling of the product of thymidylate
synthase, dihydrofolate, directly to the active site of
DHFR for further enzymic processing. Although the
DHFR region has amino acid sequence similarities
with the DHFR enz3’mes of other species, there is
enough difference to allow preferential inhibition by
the antimalaiial compounds pyrimethamine and cy-cloguanil. Full details of the crystallographic analysis
of the interaction between p3’rimethamine and the
DHFR of Plasmodium falciparum are available in a
reference listed in ‘Further reading.’ Pyrimethamine is
used in combination with the sulfa drug sulfadoxine in
the treatment of malaria whereas proguanil is com-bined with chloroquine (Chapter 6).
A highly selective compound against the bacter-ial DHFR is the pyrimidine derivative trimethoprim
(Figure 4.3). Reduction of the activity of bacterial di-hydrofolate reductase by 50% requires a trimethoprim
concentration of 0.01 LlM, whereas the same inhibition
of the human enzyme requires 300 |JM. A recent com-parison of a bacterial DHFR (from Mycobacterium tu-berculosis) with its human counteipart revealed that
the bacterial enzyme contains 159 amino acid residues
compai^ed with 187 in the human protein. F>en though
the human enzyme is significantly longer, the two pro-teins exhibit the same general folding of their three-dimensional structures. Flowever, there is overall only
26% sequence identity, and the two enzymes have
many local regions of difference in their three-dimen-sional structures, including around the active sites. X-Ray analysis shows that trimethoprim binds to the bac-terial enzyme at the active site, with its p3’rimidine ring
held in a deep cleft by hydrogen bonds and van der
Waals interactions with neighbouring amino acids.
The trimethoxybenzyl side chain of the drug extends
out towards the entrance of the cleft and fomis van der
Waals interactions with the amino acids of two sepa-rate helical regions of the enzyme. Presumably be-cause trimethoprim binds so weakly with the human
I3HFR, it has not been possible to study the interaction
by X-ray analysis.
Trimethoprim is effective on its own as an anti-bacterial drug but is more generally used as a combi-nation (cotrimoxazole) with the sulfonamide deriva-tive sulfamethoxazole. The combination is claimed to
have a wider field of antibacterial activity than either
compound alone and is prescribed as a broad-spectrum
alternative to ampicillin. Because the sulfonamide and
trimethoprim block the folic acid biosynthetic path-way at different points, the twofold inhibition is espe-cially effective in depriving bacteria of tetrahydrofo-late. The principle of twofold blockade is also
exploited in the antimalarial combination of pyrimeth-amine with sulfadoxine mentioned earlier.
The reduction in tetraliydrofolate levels in bacte-ria, fungi and protozoa caused by sulfonamides and the
dihydrofolate reductase inhibitors has widespread ef-fects on cells, although the antifolate approach has not
yet been successfully exploited in the treatment of fun-gal infections. Tetrahydrofolate is required as a one-carbon unit donor in the biosynthesis of methionine,
glycine and the formyl group of fMet-tRNA. Tetrahy-drofolate deprivation therefore depresses protein syn-thesis. The major effects, however, are on the biosyn-thesis of purines and pyrimidines, which involve
one-caiton transfer reactions at several stages. The
synthesis of thymine is particularly sensitive to in-hibitors of dihydrofolate reductase because of the
Inhibitors of nucleic acid biosynthesis
requirement for tetrahydrofolate in the transformation
of dUMP to dTMP (Figure 4.4). When cultures of bac-teria are grown in media containing amino acids and
inosine, antagonists of folic acid synthesis cause the
phenomenon known as thymineless death, which can
be prevented by the addition of excess thymine or
thymidine.
4.2 Nucleoside analogues
4.2.1 5-Fluorocytosine (flucytosine, 5-FC)
This pyrimidine analogue (Figure 4.5) was originally
synthesized as an anticancer agent but is now used
mainly to treat certain serious pathogenic yeast infec-tions, including cryptococcosis and candidiasis. Ex-cept in the latter infection, 5-FC is usually adminis-tered in combination with amphotericin B (Figure 3.8).
It is not itself active and must be metabolized to com-pounds that are the effective inhibitors. The uptake of
5-FC into fungal cells is facilitated by the transporter
protein cytosine permease. Subsequently, the com-pound is converted to 5-fluorouracil by cytosine deam-inase. Fortunately this enzyme is absent from human
cells since 5-fluorouracil is highly toxic to all dividing
dihydrofolate reductase
O ^ N
FIGURE 4.5 The antifungal drag 5-fliiorocytosine.
mammalian cells. 5-Fluorouracil enters a complex net-work of fungal nucleotide metabolism. An important
end product is 5-fluorodeoxyuridine-5′-monophos-phate, which inhibits thymidylate synthase and there-fore DNA synthesis. The other major route of metabo-lism that contributes to the antifungal activity of 5-FC
is via the conversion to 5-lluorouridine-5′-monophos-phate, catalyzed by UMP phosphoribosyl transferase,
leading to the incorporation of 5-fluorouridine-5′-triphosphate into RNA. The antifungal action of 5-FC
therefore results from a combination of the inhibition
of DNA synthesis and the generation of abeiTant RNA
transcripts. Filamentous fungi generally lack the suite
of enzymes necessary for the uptake and metablism of
5-FC and are therefore unaffected by the drug.
During therapy with 5-FC, careful monitoring of
blood levels is important to ensure that concentrations
toxic to the kidneys and bone maiTow are not achieved,
while levels are still high enough to suppress the infec-tion and minimize the risk of the emergence of 5-FC-resistant mutants.
A/gA/^o-methylene FH4 ^^2
O
> XJ 0^’^N thymidylate synthase O N
Ribose-PO.
dUMP
Ribose-PO.
dTMP
FIGURFi 4.4 A major consequence of the inhibition of di-hydrofolate reductase is the suppression of thymine biosyn-thesis. Bacteria cultured with trimethoprim undergo ‘thy-mineless death’ when not supplemented with thymine or
thymidine.
4.2.2 Antiviral nucleoside analogues
Several nucleoside analogues have achieved consider-able success as antivkal drugs. Like 5-FC, all these
compounds ai’e prodrugs that ai’e metabolized by host
or viral enzymes to the active inhibitors.
Ribavirin
This nucleoside analogue (l-P-D-ribofuranosyl-I,2,4-triazole-3-carboxamide; Figure 4.6) is an antiviral
drug effective against certain DNA and RNA vkuses.
It has long been used to treat serious lung infections in
young children caused by the respiratory syncytial
virus. In combination with interferon-a, ribavirin also
has an important application in the treatment of hepa-titis C. Ribavirin has direct actions against viruses with
70
4,2 Nucleoside analogues
HjN N
jOCHjCH^OH
H N N Y I I ^O
C JHpCHCHpH
Ganciciclovir
OH H
5-lododeoxyuridine
O CH^OH HOH2
H OH
Arabinosyladenosine (AraA)
CONH
Ribavirin  Azidothymidine (AZT)  FIGURE 4.6 Examples of nucleo-side analogues as antiviral drugs.
multiple sites of interference in vii’us replication and
indirect effects in modulating the host’s immune de-fences against viral infection.
Direct antiviral action. Ribavirin is successively
phosphoryiated by enzymes of the host cells to the cor-responding mono-, di- and triphosphates. The triphos-phate is recognized as a substrate mimic for viral poly-merases, resulting in rnisincorporation into the viral
nucleic acids. Among other effects, misincoi-poration
causes premature termination of the RNA primer chain
essential for DNA synthesis. Misincoiporation of rib-avirin nucleotides into viral RNA may also result in
viral mutagenesis. There is some evidence for this ef-fect in the polio RNA virus, which markedly reduces
the infectivity of the virus. White ribavirin is not used
to treat infection by the polio virus, the mutagenic ac-tion of the drag could be relevant to its clinical efficacy
against hepatitis C, although there is no direct evi-dence for this.
In some viruses the viral messenger RNA is mod-ified at the .5′ end by a ‘cap’ which protects the mRNA
from degradation by host nucleases. Capping involves
the addition of GTP to the 5′-hydroxyl group of RNA
to form a 5′- to 5′ triphosphate link catalyzed by
guanyl transferase. Subsequent methyiation of gua-nine at the 7 position completes the capping process.
Ribavirin triphosphate may block the capping process
in some vinises, e.g. vaccinia, by competing with GTP
at the active site of the guanyl transferase.
The initial conversion of ribavhin to the mono-phosphate by host cell adenosine kinase provides an-other opportunity for interference with viral replica-tion. Ribavirin monophosphate is a weak competitive
inhibitor of another host enzyme, inosine 5′-mono-phosphate dehydrogenase (IMPDH), which catalyzes
a rate-limiting reaction in the biosynthesis of guanine
nucleotides:
inosine XTAT^+ auanosine ,I,AT^II+ , , , , + NAD <-> 5, , , + NADH . 5 -monophospnate 5 -monophospnate
Limitation of the intracellular availability of gua-nine nucleotides by even partial inhibition of the above
reaction is likely to exeit multiple negative effects on
viral replication, including the capping process and
nucleic acid polymerization.
Indirect antiviral action. There is evidence that
ribavirin enhances the host’s immune defences against
the progression of viral infection. A detailed discus-sion of this aspect of ribavirin action is outside the
scope of this book, but in summary, the drug promotes
T-lymphocyte immunity by inducing the production of
several antiviral type I cytoldnes, including interferon-y, tumor necrosis factor-a and interleukin-2. At the
71
Inhibitors of nucleic acid biosynthesis
same time, ribavirin treatmem suppresses the proviral
type 2 cytokines, such as interleukin~4 and interleukin-10. The inhibition of lymphocyte IMPDH and the con-sequent depression in cellular GTP content is thought
to underlie these complex effects.
The value of ribavirin as an antiviral drug has
slowly emerged over many years and its site of action
has often been the subject of controversy. We can now
begin to see that the clinical benefits of the drug owe
much to a combination of the multiplicity of its effects
on viral infection, both direct and indirect.
Acyclovir and ganciclovir
These stmcturally similar compounds (Figure 4.6) are
analogues of guanosine but without the cyclic ribose
group. Despite their similarity, the two drugs have dif-ferent clinical applications. Acyclovir is used to treat
herpes simplex and varicella-zoster infections. While
ganciclovir is also active against heipes virases, its
clinical application is limited to the treatment of cy-tomegalovirus infections, which are particularly trou-blesome in AIDS patients.
Herpes viruses code for a virus-specific form of
thymidine kinase which converts acyclovir and ganci-clovir to their monophosphate derivatives. It is signifi-cant that the thymidine kinase of the host cells has a
much lower substrate affinity for these compounds, so
that uninfected cells do not generate the phosphory-lated derivatives. The drug monophosphates in virus-infected cells are then successively phosphorylated by
host cell kinases to the triphosphate level. The triphos-phates of both drugs are good substrates for the DNA
polymerases encoded by heipes viruses but are only
poorly recognized by the host polymerases. The result-ing preferential incoiporation of the drug triphos-phates into viral DNA causes premature chain teimi-nation because the antiviral nucleotides lack a 3′-0H
group on their sugar residues, thus preventing forma-tion of the 3′-5′-phosphodiester bonds necessary for
chain extension.
The action of ganciclovir against cytomegalo-viras is rather different. In cytomegalovirus-infected
cells the drug is again first converted to the monophos-phate, though not by thymidine kinase, which the cy-tomegalovirus lacks. Instead, it is believed that another
viral kinase, encoded by the UL97 gene, may be re-sponsible for the first-stage phosphorylation. The sub-sequent stages in the metabolism of ganciclovir
monophosphate and its antiviral action resemble those
in herpes-infected cells.
Vidarabine
This drag (9-p~D-arabinosyladenine, AraA; Figure
4.6) has a fairly broad antiviral spectrum, including ac-tivity against heipes viruses, cytomegalovirus and the
Epstein-Barr viras. Its triphosphate metabolite inhibits
viral DNA polymerase, and viral ribonucleotide reduc-tase and is also incorporated into viral DNA. Because
these effects occur at concentrations of vidarabine
below those needed to inhibit host DNA synthesis, the
compound can be used topically against herpes infec-tions of the eye and brain.
5-lododeoxyuridine
This is another nucleoside (Figure 4.6) which has been
used against herpes infections, especially of the
cornea, but is less specifically antiviral than the com-pounds described earlier. The similar van der Waals
radii of the iodine atom (0.215 nm) and the methyl
group (0.2 nm) of thymidine enable the drug to replace
thymidine in DNA with considerable efficiency since
its triphosphate is readily accepted as a substrate by
DNA polymerase. The incorporation of 5-iododeoxy-uridine into viral DNA leads to errors of replication
and transcription and the eventual termination of viral
proliferation. Unfortunately the compound is also in-corporated into host DNA and the resultant toxicity
limits its use to topical application to the eye and skin.
4.3 Inhibitors of the reverse transcriptase of
the human immunodeficiency virus
The search for effective treatments against the virus
that causes AIDS has been, and continues to be, one of
the greatest therapeutic challenges of modem medi-cine. The progress of the infection and its associated
pathology are insidious and iiTeversible damage to the
immune system may occur if the disease is not diag-nosed sufficiently early. Nevertheless, considerable
progress has been achieved in developing drags that
72
4,3 Inhibitors of the rewerse transcriptase of the human immunodeficiency wirus
limit viral proliferation and confer significant clinical
benefits. Currently, 16 drugs are approved for the treat-ment of HIV infection, ‘len of these are inhibitors of
the characteristic reverse transcriptase (RT) of HIV
which transcribes the single-stranded RNA genome
into a single-standed DNA and subsequently synthe-sizes a complementary strand of DNA. The resulting
double helical DNA is then capable of integration into
the chromosomes of the host cell. The other six drags
inhibit the viral protease and are discussed in Chapter
6. The inhibitors of reverse transcriptase include six
nucleoside analogues, one nucleotide analogue and
three non-nucleoside compounds (Figures 4.6, 4.7,
4.8). The rapid acciuisition by HIV of resistance to any
single drug (see Chapter 7), and the consequent and
ominous likelihood of therapeutic failure, now man-dates the exclusive use of the multidrag regime known
as highly active antiretroviral therapy or HAART,
combining inhibitors of both RT and the viral protease.
Nevertheless, the search for new and improved anti-HIV drags continues apace and many new inhibitors of
RT and other agents are currently under investigation.
4.3.1 Azidothymidine
This drug (3′-azido-3′-deoxythymidine; AZT, Figure
4.6) was the first effective anti~HIV drag to be discov-ered. The compound was originally developed as a po-tential anticancer drag but was then found to be effec-tive against the replication of HIV in AIDS patients.
The compound is first efficiently converted by the host
O
HNA^CH 3
NH,
HO—i°Q N
Zidovudine (AZT)  Zaicitabine (ddC)
NH,
Didanosine (ddl)
O
HO
O.
HNA ^
Tenofovir disoproxil fumarate
NH,  A
H^N
HO
NH
o, “
Stavudine
o
Lamivudine  Abacavir
HO—P—COOH
HO
Foscarnet
FIGURE 4.7 Inliibitors of the reverse
transcriptase of HIV used in the treat-ment of HIV infection and AIDS. These
agents act at the active center of the
enzyme.
73
Inhibitors of nucleic acid biosynthesis
Nevirapine
CH3SO2NI
Efavirenz
HN
\  N N
N- ‘
H O
Delavirdine
FIGURE 4.8 Non-nucleoside inhibitors of ttie reverse tran-scriptase of HIV used in tlie treatment of HIV infection and
AIDS. Ttiese agents bind in a pocltet sited away from the ac-tive center of the enzyme.
cell thymidine kinase to the monophosphate deriva-tive, AZT-MP. The subsequent phosphorylations to the
di~ and triphosphate derivatives are also carried out by
a host cell enzyme, thymidylate kinase. AZT-MP is,
however, a relatively poor substrate for this enzyme, in
part because the larger size of the 3′-azido group of
AZT-MP compared with the 3′-0H group of thymi-dine monophosphate hinders the interaction of AZT-MP with the active site of thymidylate kinase. HIV RT
has three distinct enzymic activities:
1. An RNA-dependent DNA polymerase,
2. A ribonuclease (H),
3. A DNA-dependent DNA polymerase.
Respectively, these activities:
1. Copy the plus-strand RNA of the virus to pro-duce a minus-strand DNA,
2. Remove the RNA template,
3. Synthesize the plus-strand of DNA using the
minus-strand DNA as a template.
Reverse transcription is a dimeric enzyme with
subunits of 65 kDa (labeled p66) and 51 kDa (p51).
Crystallographic analysis of the enzyme, which was
achieved only after overcoming considerable difficul-ties in crystallizing the protein, reveals that both sub-units contain domains referred to as palm, fingers,
thumbs and connection. An additional C-terminal do-main in the p66 subunit provides the RNAase activity
whereas the polymerase function is located in the palm
domain of p66. The p66 palm domain, together with
the p66 fingers and thumbs, form a cleft that binds the
template-primer complex in proximity to the poly-merase-active site. The polymerase is believed to carry
out both RNA- and DNA-dependent processes. The
triphosphate of AZT effectively competes with the en-dogenous thymidine triphosphate at the polymerase-active site, resulting in the incorporation of AZT-monophosphate into the viral DNA. Further extension
of the DNA chain is then blocked because the absence
of a hydroxyl group in the 3′ position of the ribose
moiety of AZT precludes the formation of the next 3′-5′-phosphodiester bond in DNA .
Because there is no reverse transcriptase in unin-fected host cells, it might appear that AZT would be
specifically active against the viras infection. Unfortu-nately, the clinical use of AZT is beset with problems.
First, virus replication is only reduced to about 10% of
the normal rate, largely because of the relatively inef-ficient conversion of AZT-MP to the antiviral triphos-phate. The incomplete inhibition of viral replication
facilitates the rapid emergence of AZT-resistant mu-tants. There is also a major problem of bone marrow
toxicity, which is probably due to the interference of
AZT phosphates with the pyrimidine metabolism of
host cells and with host cell DNA synthesis.
The other anti-HlV drugs shown in Figure 4.7,
with the exception of foscarnet, are all considered to
act in a manner similar to that of AZT. They are pro-gressively phosphorylated to the triphosphates and
then compete with the con-esponding endogenous nu-cleotides at the polymerase-active site of RT for incor-poration into viral DNA. The absence of the essential
hydroxyl group at the ribose 3′-position in all the com-pounds again results in premature temiination of the
chain. Tenofovir disoproxil fumarate (TDF) is an
unusual nucleotide prodrag; the fumarate moiety facil-itates diffusion of the drug across cell membranes.
Cellular esterases then release the tenofovir mono-phosphate entity, which is converted to the active
74
4,4 Antibacterial inhibitors of topoisomerases
triphosphate. Several of the RT inhibitors damage the
host mitochondria, apparently owing to an affinity of
the triphosphate derivatives of the drugs for DNA
polymerase y of mitochondria. Tenofovir triphosphate
exhibits very low affinity for this enzyme, at least in
vitro, indicating that the drug may be less toxic in vivo
than other RT inhibitors. The inhibition of RT by the
chemically very simple drug foscamet results from a
stractural analogy with pyrophosphate which enables
the drug to compete with the endogenous nucleoside
triphosphates at the pyrophosphate binding site on the
RT, thus blocking the growth of nucleic acid chains.
Foscarnet is a broad-spectmm antiviral drag with use-ful activity against cyclomegalovirus infection of the
retina and acyclovir-resistant herpes, in addition to its
anti~HIV activity.
4.3.2 Non-nucleoside inhibitors of HIV
reverse transcriptase (NNRTIs)
The discovery of this class of compounds heralded a
new phase in the exploitation of RT inhibition in anti-HIV therapy. Not only are NNRTIs novel chemicals
(Figure 4.8) quite different from the nucleoside and
nucleotide inhibitors (NRTIs), they also provide an es-cape from the specific problem of resistance caused by
mutations at, or near to, the active center of the poly-merase. Unlike the NRTIs, the NNRTIs are noncom-petitive inhibitors that interact with the enzyme at a
site distant from the active center. The first NNRTI to
be developed was nevirapine (Figure 4.8). X~Ray crys-tallography shows that this compound binds in a
pocket located between two p-sheets of the palm of the
p66 subunit some 10 A away from and just under floor
of the polymerase active center. The internal face of
the pocket is lined with hydrophobic amino acids, in-cluding leucine, valine, tryptophan and tyrosine. The
NNRTIs are chemically diverse and yet appear to share
a generally common mode of binding that is probably
largely facilitated by the hydrophobic nature of the
binding pocket and flexibility in the protein chains that
form it. The interaction process between enzyme and
inhibitors is probably accompanied by confomiational
rearrangements in the drug molecules themselves as
they move from the solution phase into the binding
pocket. Inhibition of RT activity results from adverse
changes in the shape of the active center which derive
from the conformational rearrangements involved in
the binding of the NNRTI in the binding pocket. This
explains the noncompetitive mode of inhibition ex-erted by NNRTIs.
Valuable as the introduction of NNRTIs has been
in the treatment of HIV infection, the drugs have
brought their own particular problems. Resistance re-sulting from new mutations affecting RT away from
the active center soon emerged (Chapter 7), and the
of adverse side effects. Neverthe-less, the NNRTIs are well established as an essentia!
component of the current HAART regimes.
4 0, liotirl^ffori:^ i isilniriifOffi’^S; fi f 9 9S Sr9,s 9 %B 9U C« %^ %^^ B 9 C9 B 9 9 9 S S 9 9U S %%^ B ^ %M S
%J’ SJB %flV 5 «3^^# 5 i i% ^ a %M ^%^^ ^
The replication of double-stranded (duplex) DNA in
prokaryotes and eukaryotes is a remarkably complex
process involving an elaborate suite of proteins and en-zymes. The whole process, from bacteria to mammals,
shares many common features which indicate a highly
conserved nature throughout a long evolutionary his-toty. Even so, it is increasingly clear from genomic
studies that there are significant differences among the
corresponding enzymes of divergent species. It is
therefore rather surprising that relatively few leads
have emerged either from natural sources or from syn-thetic chemistry which target the enzymes of DNA
replication that provide specific antimicrobial drags.
Although the increasing research attention being paid
to species differences among the replicative enzymes
may eventually improve this situation, the only suc-cessful drugs so far are the species-specific inhibitors
of DNA topoisomerases.
DNA cannot exist in bacterial cells as an ex-tended double-helical molecule. The length of bacter-ial DNA is about 1.300 pm and typically the cell into
which it fits is about 1 |.im in diameter. Clearly there
must be a high degree of ordered quaternary stractm^e
in the DNA to accommodate it within cells. This is
achieved by negatively supercoiling the DNA; i.e. the
supercoiling is left-handed, in contrast to the right-handed winding of the double helix. Special enzymes
75
Inhibitors of nucleic acid biosynthesis
and proteins induce torsional stresses in the molecule.
In this way, the enzymes alter the three-dimensional
shape of DNA while maintaining its primary structure
and the genetic information encoded in it. These en-zymes are also essential for DNA replication and tran-scription. Initiation of DNA replication can only start
if the DNA is negatively supercoiled. This is because
negatively supercoiled molecules of the nucleic acid
are easier to ‘melt’ locally than relaxed DNA in order
to generate a single-stranded template region. This en-ables the protein referred to as DnaA to promote the
interaction of DNA with the site of origin of replica-tion. Second, when a circular supercoiled DNA mole-cule is replicated, the two daughter molecules become
interlocked, or ‘catenated’, and without a means of re-moving the supercoils, the separation of the progeny
(decatenation) is impossible.
The enzymes that facilitate these topological
changes are known as topoisomerases, four types of
which have been identified in bacteria. The type II
topoisomerase, or DNA gyrase, is the only one of the
four that can introduce negative supercoils. It consists
of two 97 kDa GyrA chains and two 90 kDa GyrB
chains and is a major target for several classes of anti-bacterial drugs. The coiTesponding enzyme in mam-malian cells is not significantly targeted by the anti-bacterial inhibitors of DNA gyrase. The decatenation
of interlocked daughter DNA molecules in bacteria is
catalyzed by another enzyme, topoisomerase IV.
The supercoiling reaction begins with a segment
of DNA, approximately 120 base pairs (bp) in length,
wrapping itself around the tetrameric complex of the
gyrase. The GyrB subunit allows passage of the DNA
segment into the interior of the enzyme using en-zyme-bound ATP as an energy source. Both strands of
DNA are then cleaved by GyrA. The 5’~phosphate ter-minal of each cleaved strand is covalently bonded to
the hydroxyl group of tyrosine-122 in each of the
GyrA subunits. This link is essential to prevent the
free rotation of the cut DNA strands. The enzyme next
permits a segment of double-stranded DNA to pass
through the gap of the broken sequence. The ends of
the cleaved strands are then brought together and re-sealed by the ligase activity of the enzyme. Finally,
the GyrB subunit catalyzes the hydrolysis of the
bound ATP, permitting the release of the processed
DNA segment.
4.4.1 Quinolones
These compounds compose one of the most important
groups of wholly synthetic antibacterial drugs in cur-rent medical use. Nalidixic acid and oxolinic acid (Fig-ure 4.9) are the so-called first-generation quinolones,
whose spectrum of antibacterial action is confined to
Gram-negative bacteria. The introduction of a fluorine
atom at position C-6 in the second-generation com-pound ciprofloxacin (Figure 4.9) resulted in a marked
increase in potency and extended the antibacterial
spectrum to important Gram-positive pathogens. Sub-stitution with basic substituents to counteract the acid-ity of the carboxyl group, as in gemifloxacin (Figure
4.9), further increased the potency against streptococci
and staphylococci. Cuixent interest in the quinolones is
largely directed at developing compounds with activ-ity against bacteria resistant to the earlier quinolones.
The antibacterial activity of the quinolones is pri-marily due to inhibition of DNA gyrase. When the iso-lated enzyme is incubated with DNA and a quinolone,
the supercoiling reaction is arrested at the point where
the cut ends of the DNA strands are covalently linked
to the hydroxyl groups of the tyrosine-122 residues of
GyrA. The re-ligation of the broken strands is blocked
and the supercoiling reaction can be said to have been
frozen midway. This results in the accumulation of
double-stranded nicks in the bacterial genome and
may also prevent the essential movements of DNA and
CjHj
GOGH
Oxolinic acid Nalidixic acid
GOGH
Ciprofloxacin  Gemifloxacin
FIGURE 4.9 Examples of quinolone antibacterial drugs
that inhibit bacterial topoisomerases.
7i
4,4 Antibacterial inhibitors of topoisomerases
RNA polymerases along the DNA template. The bac-tericidal action of the quiriolones probably arises from
a combination of these effects.
Much attention has been devoted to unravelling
the complexities of the enzymic mechanisms of DNA
gyrase—the binding sites and modes of inhibition of
various antibacterial inhibitors of the enzyme. The
study of mutations affecting the activity of DNA gy-rase and its resistance to inhibitors, combined with
data from X-ray analysis of crystallizable domains of
the two subunits, has provided at least partial descrip-tions of the catalytic process and how it is blocked by
antibacterial drags. The following discussion concen-trates largely on the topic of drug action; the reader
should consult references listed under ‘Further read-ing’ for detailed descriptions of gyrase stnicture and
function.
The primary site of quinolone action is assigned
to the G3’rA subunit because the most common muta-tions that confer resistance to these drugs are found in
a region of GyrA refeiTed to as the quinolone resist-ance-determining region or QRDR. For example, re-placement of serine-83, which lies close to the active
site where DNA is bound by tryptophan, gives about a
20-fold increase in resistance to many different quino-lones. Quinolones bind strongly to gyrase complexed
with DNA but only weakly to either the enzyme or
DNA alone, suggesting that the drugs interact simulta-neously with both the protein and nucleic acid. In fact,
quinolones are believed to bind to a pocket consisting
of the QRDR and a segment of DNA which is also
bound to it. A recent refinement of this model for the
interaction of ciprofloxacin proposes that the planar
character of the bicyclic moiety of the drug enables
two molecules of the compound to stack, or intercalate
(see later discussion) between the bases of the DNA
bound at the active site. Alternatively, the drag might
actually displace a cytosine residue from the double
helix opposite the bond cleaved by the enzyme (Figure
4.10). The drug is now positioned to interact with sev-eral amino acid residues of GyrA, including serine~83
and aspartate-87. Some mutations in GyrB which can
also confer quinolone resistance suggest that a limited
region of this subunit may contribute to the structure of
the binding pocket. Crystal structures of a 59~kDa
fragment of the N-teniiinal domain of GyrA and a 43-kDa fragment of the N-terminal domain of GyrB are
A(i!|
FIGURE 4.10 A possible model of the quinolone binding
pocket, A(i): The drug (gray rectangle) intercalated into the
DNA double helix. A(il): In this alternative niodel of interca-lation, the drug has displaced a cytosine residue which is
‘flipped out’ of the helix. (B): The binding pocket. The GyrB
subunit is positioned close to the quinolone resistance-deter-mining region of the GyrA subunit, allowing asp;irtate-426
and lysine~447 to interact with the bound quinolone mole-cule. The DNA axis is perpendiculfir to the plane of the dia-gram, find the approximate position of the sugar-phosphate
backbone is shown by the encircled Ps.The drag interacts
with the distorted region of DNA at the active site, and the
bottom edge of the quinolone can also interact with serine-83 and aspartate~87 of GyrA. [Taken with permission from J,
Heddle and A. Maxwell Antimicrobial Agents and Chemo-therapy 46, 1805-1815 (2002).]
available. However, a full crystallographic analysis of
a DNA-gyTase-DNA-quinolone complex is still awaited.
Only then will the details of the ciuinolone binding site
be finally revealed.
Although the type 11 topoisomerase is believed to
be the major target for quinolones, the type IV enzyme
77
Inhibitors of nucleic acid biosynthesis
is also inhibited by some fluoroquinolones, although
to a lesser extent than the type II enzyme. Indeed in
Staphylococcus aureus the principal target for quino-lones is believed to be topoisomerase IV. In Strepto-coccus pneumoniae the inhibition of both DNA gyrase
and topoisomerase IV contribute to the antibacterial
action of quinolones. The amount of the contributions
varies with the individual quinolone.
Type IV topoisomerase does not induce negative
supercoiling of DNA, but plays an important role in
the partitioning of DNA during cell division. Its prin-cipal catalytic function is to ensure the decatenation of
DNA that is essential for the correct separation of
DNA into the daughter cells. Despite the distinct func-tions of the two enzymes, there is extensive amino acid
sequence homology between them, which may under-lie their susceptibility to inhibition by quinolones. The
type IV enzyme is a heterotetrameric protein com-posed of two subunits, ParC and ParE. ParC and ParE
are homologous to GyrA and GyrB with 36 and 40%
sequence identity, respectively (in Escherichia coli).
Currently there are no details of the interaction be-tween quinolones and type IV topoisomerase, but be-cause of its homology with DNA gyrase, the drugs
may bind to both enzymes in a generally similar way.
Quinolones stimulate DNA cleavage by the type IV
enzyme while at the same time inhibiting the re-ligation action.
Novobiocin
Cyclothialidine
FIGURE 4.11 Naturally occurring inhibitors of the func-tion of the P subunit of bacterial DNA gyrase.
4.4.2 Inhibitors of the GyrB subunit of DNA
gyrase: coumarins and cyclothialidine
The disturbing frequency of mutations to quinolone
resistance that arise in the GyrA subunit drives con-siderable interest in the GyrB subunit as an attractive
alternative target for drug action. The coumarins, ex-emplified by novobiocin (Figure 4.11) and cyclothiali-dine (Figure 4.11), are members of structurally distinct
families of naturally occurring inhibitors of DNA neg-ative supercoiling; their target is the ATP hydrolyzing
activity of GyrB. Studies on the kinetics of inhibition
of DNA gyrase by both types of compound indicate
potent competition with ATP, with inhibitor constant
(K^) values in the range of 10 ^ to 10 “^ M. However,
examination of the ATPase activity of isolated GyrB
subunits indicated that it did not follow Michaelis-Menten kinetics, thus raising doubts about the concept
of competitive inhibition. These doubts were sup-ported by the absence of any significant stractural sim-ilarity between the inhibitors and ATP. Furthermore,
point mutations in GyrB that cause resistance to the
coumarins were found to lie at the periphery of the
ATP binding site. Fortunately, the uncertainty was
eventually resolved by X-ray crystallographic data on
the complexes of the GyrB subunit with novobiocin,
cyclothialidine and adenyIyi-p-y~imidodiphosphate
(an analogue of ATP). Although the three ligands are
stmcturally distinct and bind to the enzyme in very dif-ferent ways, the X-ray analysis shows that there is
some overlap of their binding sites. The concept of
competitive inhibition of ATP binding to the B subunit
by both the coumarins and cyclothialidine may there-fore be correct. As would be expected, the interactions
of novobiocin and cyclothialidine with the enzyme are
78
4,5 Inhibitors of DNA-dependeiit RNA polymerase
complex and the reader is referred to the research
paper Usted under ‘Further reading’ for detailed infor-mation. To summarize, in the case of novobiocin, the
noviose sugar unit contributes to extensive hydrogen
bonding with key amino acids of the enzyme and water
molecules associated with the protein. Flydrophobic
interactions involving both the sugar and coumarin el-ements add to the stability of the complex. The novo-biocin molecule does not lie flat in the complex but is
bent around on itself in a manner that has been likened
to a staple. This is due to the confomiation of the pep-tide bond between the coumarin ring and the hydroxy-benzoate group being in the cis conformation rather
than in the expected trans conformation. The binding
contribution of the hydroxybenzoate group is appar-ently the least significant because a chemically modi-fied derivative of novobiocin lacking this group still in-hibits both DNA supercoiling and the ATPase activity.
The antibacterial activity of this compound is, how-ever, much reduced and it is possible that the 3′-isopentenyl~4’~hydroxybenzoate group facilitates the
entry of novobiocin into bacterial cells.
The binding of cyclothialidine to the GyrB sub-unit is stabilized largely by the formation of water-me-diated hydrogen bonds between the hydroxyl groups
of the substituted resorcinol group of the inhibitor and
key amino acids that line the binding site which over-laps with that for ATP. The clinical history of novo-biocin and other coumarins has been disappointing,
owing to several factors. The coumarins are poorly ab-sorbed in oral doses and their antibacterial activity is
limited mainly to Gram-positive organisms, probably
because of limited penetration into Gram-negative
cells. Drug-resistant mutants emerge readily from ini-tially drug-sensitive populations of Gram-positive bac-teria. Finally, although there is no functional ecjuiva-lent of DNA gyrase in mammalian cells, mammalian
topoisomerase II, which shares some sequence homol-ogy with gyrase, is susceptible to inhibition by novo-biocin. In contrast, cyclothialidine is much more spe-cific for the bacterial enzyme, offering hope that the
design of specific inhibitors of GyrB function may
lead to novel and effective antibacterial drugs. How-ever, cyclothialidine penetrates bacterial membranes
very poorly and is only weakly antibacterial in conse-quence. Recent chemical modification of cylcothiali-dine, however, has produced several compounds with
much improved antibacterial activity in vitro and
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The transcription of RNA from DNA is, of course,
common to both prokaryotic and eukaryotic organisms
and involves enzymes known as DNA-dependent RNA
polymerases. It was therefore somewhat suiprising to
discover that an important group of antibacterial an-tibiotics, the rifamycins isolated from Streptomyces,
show remarkable specificity for the inhibition of bac-terial DNA-dependent RNA polymerase. One of these
compounds, rifampicin, or rifampin (Figure 4.12), is a
potent antibiotic and a mainstay of treatment for tuber-culosis in combination with other drugs. Rifampicin is
active against many Gram-positive bacteria, but is less
N N-CH ,
Rifampicin
Streptovaricin D
FIGURE 4.12 Two antibiotics that selectively inhibit
DNA-dependent RNA synthesis in bacteria. Rifampicin is a
semisynthetic member of the rifamycin group. Strepto-varicin D is related in structure to tlie rifamycins; jointly the
ril’amycins and streptovaricins are known as ausamycins.
79
Inhibitors of nucleic acid biosynthesis
effective against Gram-negatives because of limited
access to the target enzyme in these organisms. Chem-ically the rifamycins are closely related to the strepto-varicins (Figure 4.12). The two groups of antibiotics
appear to have a similar mode of action, but unlike the
rifamycins, the streptovaricins are not in medical use.
Both groups strongly inhibit RNA synthesis in sensi-tive bacteria and in cell-free extracts by binding to and
inhibiting DNA-dependent RNA polymerase. The
dmgs neither bind to nor inhibit the corresponding
mammalian enzyme.
The RNA polymerase isolated from Escherichia
coli is a large (450 kDa), complex enzyme consisting
of four kinds of subunits: a, p, p’ and CT. The complete
or holoenzyme has the composition (ttjpp’cr) together
with two tightly bound zinc atoms. The function of the
<J subunit is to locate a promoter site where transcrip-tion is initiated. The <J subunit then dissociates from
the rest of the enzyme, leaving the core enzyme
(a-,pp’) bound to the DNA template via the p’ subunit.
The p subunit canies the catalytic site for the internu-cleotide bond foimation and is the target for antibiotic
inhibition. This was demonstrated by studies with
RNA polymerase isolated from rifampicin-resistant
bacteria. Recent X-ray analysis of the holoenzyme
from Thermophilus aquaticus revealed a DNA-bind-ing channel in the active site cleft of the p subunit and
an RNA exit channel.
Structural analysis of the RNA polymerase from
Escherichia coli indicates that there are two distinct
substrate binding sites on the p subunit. The ‘i’ or ini-tiation site, is template-independent and recognizes
only purine nucleoside triphosphates. The second site,
called the i + 1 site, has no nucleotide preference. The
initiation of transcription is marked by the formation
of an intemucleotide bond between the nucleotides
bound to the i and i + 1 sites. Rifampicin and strac-turally related antibiotics have long been known to
block initiation. The binding of the antibiotic is a two-stage process:
all dissociation constant for the interaction is very low,
3 nm, indicating tight but not covalent binding. The
crystal structure of the core of RNA polymerase (from
Thermophilus aquaticus) complexed with rifampicin
shows that the antibiotic binds in a pocket of the p sub-unit deep within the channel which accommodates the
DNA-RNA hybrid but is more than 12A from the ac-tive site. The napthol ring of the antibiotic forms van
der Waals interactions with hydrophobic side chains of
adjacent amino acids in the pocket. The analysis also
shows the potential for hydrogen bonds between the
five polar groups of rifampicin and neighbouring
amino acids. Rifampicin inhibits the polymerase by
sterically obstracting the path of the growing RNA
chain after two, or at the most three, nucleotides have
been added. The formation of the first intemucleotide
bond is not inhibited by rifampicin, and if chain
growth progresses beyond the second or third phos-phate diester bond before the addition of the drug, fur-ther chain elongation is insensitive to the action of
rifampicin.
Another complex antibiotic, streptolydigin, is
also a specific inhibitor of bacterial RNA polymerase.
However, it inhibits chain elongation as well as the ini-tiation process and increases the stability of purified
RNA polymerase-DNA template complexes. The p
subunit of the polymerase core enzyme bears the strep-tolydigin binding site, and the increased stability of the
enzyme-template-antibiotic complex delays the pro-gress of the enzyme along the template without affect-ing the accuracy of the transcriptional process.
Despite the evidence obtained in vitro for the
mode of action of streptolydigin, studies of its effects
on intact Escherichia coli cells indicate that in vivo
streptolydigin may accelerate the temiination of RNA
chains. The rate of elongation of RNA chains is unaf-fected, but streptolydigin may destabilize the tran-scription complex in vivo, thus permitting premature
attachment of termination factors. Streptolydigin has
not found a clinical application.
R + E O RE  RE*
The first stage is a fast bimolecular reaction, followed
by a second, slower unimolecular process involving a
confomiation change in the enzyme which is neces-sary for the inhibitory action of rifampicin. The over-4.6 Inhibition of nucleic acid syntliesis by
interaction witli Di^A
As we have seen, the synthesis of nucleic acids can be
interrupted by blocking the supply of essential nu-80
4.6 Inhibition of nucleic acid synthesis iiy interaction with DNA
cleotides, by the termination of chain extension fol-lowing the incorporation of some nucleotide analogues
into the nucleic acid structure, and by the direct inhibi-tion of certain polymerases. The fomiation of a com-plex between the inhibitor and DNA, either covalent or
noncovalent, may disrupt template function in replica-tion and transcription. Some of the best-known exam-ples of this group have useful anticancer activity and
include actinomycin D, bleomycin and mitomycin C.
These compounds are powerfully cytotoxic to mam-malian and microbial cells alike, and therefore have no
application in antimicrobial therapy and will not be
further discussed. A possible role for intercalation in
the inhibition of bacterial topoisomerases by
quinolones has already been mentioned. Several other
antimicrobial agents are known to intercalate into the
double helix of DNA, although this may not always be
the primary basis for their antimicrobial activity. The
intercalation phenomenon also has some useful practi-cal applications in the laboratory.
4.6.1 Acridines, phenanthridines and
choroquine
The medical history of the acridine dyes extends over
some 90 years since proflavine (Figure 4.13) was used
as a topical disinfectant on wounds during the First
World War. Proflavine is too toxic to be used as a sys-temic antibacterial agent, but the related acridine,
mepacrine (Figure 1.2), found wide application as an
-CH-(CHj)3N * — H
CH3 ^C ,
C2H5
•H
C2H5
Chloroquine
Ethidium
FIGURE 4.13 Three compounds thai intercalate with DNA.
antimalarial drug before its replacement by more ‘pa-tient friendly’ drugs. Chloroquine (Figure 4.13) is still
an important antimalaiial agent although, as we shall
see in Chapter 6, its interaction with DNA is no longer
considered to be the primary basis of its action against
the malarial parasite. The phenanthridine ethidium
(Figure 4.13) has some application as a ttypanocide in
veterinary medicine. All these compounds are charac-terized by flat, planar fused ring systems which are the
key to their intercalating property.
The compounds bind to the nucleic acids of living
cells and the phenomenon forms the basis of the tech-nique known as vital staining, since the nucleic acid-dye complexes exhibit characteristic colours when ex-amined by fluorescence microscopy. The dyes also
bind readily to nucleic acids in vitro, and the visible
absorption spectra of the ligand molecules undergo a
metachromatic shift to longer wavelengths.
Two types of binding to DNA are recognized: a
strong primary binding which occurs in a random man-ner in the molecule, and a weak secondary binding.
The strong primary binding occurs only with DNA, al-though many other polymers bind the dyes by the sec-ondary process. The primary binding to DNA, which is
mainly responsible for the ability of the drugs to inhibit
nucleic acid synthesis, depends upon the intercalation
of the rigidly planar molecules between the adjacent
stacked base pairs of the double helix of DNA.
The evidence for this unusual type of interaction
is provided by various measurable physical changes in
the DNA:
1. Solutions of DNA show increased viscosity.
2. There is a decrease in the sedimentation co-efficient of DNA detemiined by ultracen-trifugatioQ, which indicates a reduction in its
buoyant density.
3. The thermal stability of DNA, i.e. the tem-perature at which the double helix begins to
unwind or ‘melt’, is increased.
The extent of these changes is proportional to the
amount of drug intercalated into the double helix. In
the case of the anticancer drug actinomycin D, there is
direct X-ray crystallographic evidence for intercala-tion into an oligo-deoxyribonucleotide. The DNA-compound complex is both straighter and stiffer than
the uncomplexed nucleic acid and these changes raise
81
Inhibitors of nucleic acid biosynthesis
the viscosity of solutions of DNA treated with interca-lating agents. The reductions in sedimentation coeffi-cient and buoyant density of DNA following intercala-tion result from a reduction in the mass per unit length
of the nucleic acid. For example, a proflavine molecule
increases the length of the DNA by about the same
amount as an extra base pair, but because proflavine
has less than half the mass of the base pair, the mass
per unit length of the complexed DNA is decreased.
The increased thermal stability of intercalated DNA is
probably due in part to the extra energy needed to re-move the bound compound from the double helix in
addition to that required to separate the strands.
To peimit the insertion of an intercalating mole-cule into DNA, it is believed that a local partial un-winding of the double helix associated with the normal
molecular motions within the macromolecule pro-duces spaces between the stacked base pairs into
which the planar polycyclic molecule can move. A
model advanced many years ago and still generally ac-cepted shows schematically how polycyclic stmctures
may intercalate between the stacked base pairs (Figure
4.14). The hydrogen bonding between the base pairs
remains undisturbed, although there is some distortion
of the smooth coil of the sugar phosphate backbone be-cause the intercalated molecules maintain the double
helix in a partially unwound configuration. It is be-FIGURE 4.14 Diagram to represent the secondarj’ struc-ture of normal DNA (left) and DNA containing intercalated
molecules (right). The stacked bases of the nucleic acid are
separated at intervals by the intercalators (black), resulting in
some distortion of the sugar-phosphate backbone of the
DNA. [Reproduced with permission irom L. Leniian (1964)
J. CeUComp. Physiol. 64, Suppl. 1 (1964). Copyright owned
by Wiley-Liss, Inc., a subsidiaiy of John Wiley & Sons.]
lieved that the distortion of the double helix, together
with the hindrance to strand separation, are major fac-tors in blocking DNA replication and transcription.
The details of specific drag-DNA interactions
depend largely on the structures of the individual
drags. Intercalation does not involve the formation of
covalent bonds between the compound and DNA. In
general terms, the complex is probably stabilized by
electronic interactions between the planar ring systems
of the compounds and the heterocyclic bases of the
DNA above and below the drug. The complexes
formed by proflavine and ethidium may also be stabi-lized by hydrogen bonding between their amino
groups and the charged oxygen atoms of the phosphate
groups in the sugar-phosphate backbone. In the case of
chloroquine, the projecting cationic side chain may
form a salt link with a phosphate residue.
In ceitain tumour vinises and bacteriophages, in
the kinetoplasts of trypanosomes and in bacterial plas-mids (Chapter 8), double-stranded DNA exists as cova-lently closed circles. Circular DNA, covalently closed
via the 3′-5′-phosphodiester bond, is characteristically
supercoiled because the circular molecule is in a state of
strain. The strain is relieved and the supercoils often dis-appear when single-stranded breaks or ‘nicks’ are pro-duced by endonuclease action. Closed circular DNA
has an unusual affinity for intercalating molecules
which, because they partially unwind the double helix,
also reduce the supercoiling of the DNA. If the unwind-ing proceeds beyond a certain point, as more and more
drug is added the DNA begins to adopt the supercoiled
form again, but in a direction opposite from that of the
uncomplexed DNA. At this point the affinity of the
closed circular DNA for the intercalated molecules de-clines until it is less than that of nicked DNA. The di-minished affinity of closed circular DNA for ethidium at
high concentrations of the drag permits a convenient
separation of closed circular DNA from nicked DNA
because the sedimentation coefficient and buoyant den-sity of DNA with a lower content of intercalated com-pound are significantly higher. This effect is useful in
the isolation of closed circular DNA on a preparative
scale.
It is also possible that the initially higher affinity
of supercoiled DNA for intercalating molecules may
in part account for their specificity of action against or-ganelles and organisms that contain circular DNA.
82
4.6 Inhibition of nucleic acid synthesis iiy interaction with DNA
Thus the treatment of bacteria with acridines can in-duce the loss of plasmids from cells. The mitochondria
of certain strains of yeast are severely and irreversibly
damaged by growth in the presence of ethidium, ap-parently owing to drug-induced mutations which af-fect the mitochondrial DNA. The kinetoplast of try-panosomes is also seriously affected by intercalating
agents, DNA synthesis in this organelle being selec-tively inhibited. Eventually the kinetoplast disappears
altogether. Since this adversely affects the life cycle of
trypanosomes, it is possible that the selective attack on
the kinetoplast may underlie the trypanocidal activity
of intercalating drugs such as ethidium.
Further reading
Angehrn, P. et al. (2004). New antibacterial agents derived
from the DNA gyrase inhibitor cyclothialidine. / .
Med.Chem. 47. 1487.
Baca, A. M. (2000). Ciystal structure of Mycobacterium tu-berculosis 7,8, dihydropteroate synthase in complex
with pterin monophosphate: new insight into the enzy-matic inechfmisrn and sulfa-dnig action../. Mol. Biol.
302,1193.
Campbell, E. A. et al. (2001). Structural mechanism for ri-fampicin inhibition of bacterial RNA polymerase. Cell
104, 901.
Champoux, J. J. (2001). DNA lopoisomerases: structure,
function and mechanism. Aimu. Rev. Biochem. 70, 369.
Heddle, J. and Maxwell, A. (2002). Quinolone-bludlug
pocket: role of GyrB. Aiitimicrob. Agents CItemotlier.
46, 1805.
Hitchings, G. H. (1983). Inhibition of folate metabolism in
chemotherapy. Handb. Exp. Plmr/nacol. 64, 11.
Lewis, R. J. et al. (1996). The nature of inhibition of DNA
gyrase by the coumaiins and the cyclothialidines re-vealed by X-ray crystallography. EMBO J. 15, 1412.
Maxwell, A. and Lawson, D. M. (2003). The ATP-binding
site of Type II topoisomerase as a target for antibacter-ial drugs. Curr. Topics Med. Chem. 3, 283.
Meneudez-Arias, L. (2002). Targeting HIV: autiretroviral
therapy and development of drug resisttmce. Trends
Pharmacol. Sci. 23, 381.
Rastelli G. et al. (2000). Interaction of pyrimethamine, cy-cloguanil, WR99210 and their analogues with Plas-modium falciparum dihydrofolate reductase: stnictural
basis of antifolate resistance. Bioorg. Med. Chem. 8,
1117,
Ren, J. et al. (1995) High-resolution structures of HIV-1 Re-verse Transcriptase from four RT-inhibitor complexes.
Nat. Struct. Biol. 2, 293.
Rongbao, Li etal. (2000). Three-dimensional structure of M,
tuberculosis dihydrofolate reductase reveals opportuni-ties for the design of novel tuberculosis drugs,,/. Mol.
Biol. 295, 307.
Tarn, R. C. etal. (2002), Mechanisms of action of ribavirin in
antiviral therapies. A«r/vir, Chem. Chemother 12, 261.
Vassylyev, D. G. et al. (2002). Crj’stal structure of a bacter-ial RN,A, polymerase holoenzyme at 2.6×4 resolution.
A’fltMrc417, 712.
Wilson, W. D. and .Tones, R. L. (1981). Intercalating drugs:
DNA binding and molecular- pharmacology. Adv. Phar-macol. Chemother. 18, 177.
Yuvaniyama, T et al. (2003). Insights into antifolate resist-ance from malarial DHFR-TS structures. Nat. Stuct.
Biol. 10, 357.
83
CliBptcr fi¥6
^ 88^ ^
The process of protein biosynthesis, io which the in-formation encoded by the four-letter alphabet of nu-cleic acid bases is translated into defined sequences of
amino acids linked by peptide bonds, is an exquisitely
complex process involving more than 100 macromole-cules. Amino acid-specific transfer RNA molecules,
messenger RNy\s and many soluble proteins are re-quired, in addition to the numerous proteins and three
types of RNA that make up the ribosomes. Although
many general features of the protein synthetic machin-ery are similar in prokaryotic and eukaryotic organ-isms, several naturally occurring compounds and cur-rently one series of wholly synthetic compounds
specifically inhibit bacterial protein synthesis and pro-vide us with drugs of considerable therapeutic value. It
is intriguing, therefore, tiiat ‘nature’ has been much
more successful than synthetic organic chemistry in
producing compounds tiiat discriminate between bac-terial and mammalian protein synthesis. Inhibitors
specific for protein synthesis in fungi are as yet un-known, probably because the similarities between the
mechanisms in fungal and mammalian cells are too
close to permit this degree of discrimination. We pro-vide a brief outline of ribosomal structure and the se-quence of events in protein biosynthesis. The subse-cjuent discussion is mainly concerned with the modes
of action of inhibitors of bacterial protein synthesis in
clinical use.
5 1 Riho*50iiip*5
These remarkable organelles are the machines upon
which polypeptides are elaborated. There are three
main classes of ribosomes, identified by their sedi-mentation coefficients in the ultracentrifuge. The 80S
ribosomes are confined to eukaryotic cells, while 7()S
ribosomes are characteristic of prokaryotic cells. A
species of 5()-55S ribosome in mammalian mitochon-dria resembles bacterial ribosomes in functional or-ganization and antibiotic sensitivity; analogous small
ribosomes also occur in the chloroplasts of green
plants. The SOS paiUcle dissociates reversibly into 6()S
and 40S subunits, and the 70S ribosome into SOS and
30S subunits, when the Mg^”^ concentration of a sus-pending solution is reduced. Both SOS and 70S ribo-somes are composed exclusively of protein and RNA
in mass ratios of approximately 50:50 and 35:65, re-spectively. There are three distinct species of RNA in
most ribosomes, with sedimentation coefficients of
29S, 18S and 5S in SOS particles from animal cells;
25S, 18S and 5S in SOS particles from plant cells; and
23S, 16S and 5S in 70S particles. The 55S ribosomes
contain two RNA species that sediment at about 16S
and 12S, but probably no 5S RNA. The protein com-position of ribosomes is impressively complex. The
30S subunit oi Escherichia coli ribosomes contains 21
proteins (‘S’ proteins) and the 50S (Targe’) subunit
85
Inhibitors of protein biosynthesis
contains 34 proteins (‘L’ proteins). The amino acid se-quences of all of these proteins are now known.
Great progress has been made in understanding
how the ribosome is constructed, and a consensus
model of the gross structures of the subunits of the 70S
ribosome, based upon electron microscopic images, is
represented in simplified form in Figure 5.1. Further-more, success in obtaining high-quality crystals of ri~
bosomes from certain bacteria, including Thermus
therinophilus, Deinococcus radiodurans and Haloar-cula rnarismortui (an archaeobacterium) has facili-tated high-resolution X-ray analyses of the two riboso-mal subunits (Figure 5.2) and their interactions with
several antibiotics of major medical importance. Al-though none of these bacteria are pathogenic, the gen-erality of the 70S ribosomal structure makes it reason-able to conclude that the data from their ribosomes are
applicable to those of pathogenic bacteria, especially
those from Deinococcus radiodurans, which are more
sensitive to antibiotics than the archaeobacterium ribo-somes. It has proved more difficult to crystallize ribo-somes from bacteria such as Escherichia coli or
Staphylococcus aureus because these ribosomes tend
to deteriorate rapidly under the conditions optimal for
crystal formation. However, a lower resolution (be-tween 9 and lOA) structure of the 70S Escherichia coli
ribosome is now available. The detailed and complex
crystallographic data are beyond the scope of this
book, and the interactions between ribosomes and
their antibiotic inhibitors will therefore be discussed in
outline only. Detailed descriptions are available in ref-erences listed under ‘Further reading.’
There are two main functional regions in ribo-somes, known as the translational and exit domains.
Both ribosomal subunits contribute to the translational
domain; mRNA binding and its interaction with
aminoacyl-tRNAs, i.e. decoding, occur on the 30S
subunit in the region called the platform, whereas pep-tide bond formation is catalyzed by the peptidyl trans-ferase activity located on the central protuberance of
the SOS subunit. The lengthening peptide chain leaves
the 508 subunit via the exit domain which is found on
the side of the subunit opposite to the peptidyl trans-ferase site. The topographies of the various elongation
and initiation factor binding sites are also well under-stood and represent the culmination of many years of
effort by a number of groups using highly sophisti-cated techniques. Similar features have been recog-nized in eukaryotic ribosomes, which bind to the rough
endoplasmic reticulum at the exit site.
For many years it was believed that ribosomal
proteins played the leading role in decoding mRNA
and in peptide bond synthesis. However, it is now clear
that ribosomal RNA is critically involved in decoding
(16S rRNA) and in peptide bond synthesis (23S
rRNA), with ribosomal proteins being required to
maintain the functional three-dimensional stmctures
of the RNA molecules. As we shall see, these discov-eries have major implications for the sites of action of
several antibiotics that inhibit protein biosynthesis.
Cenfrai proln.iuersrsce
Cleft
\ \
‘•ans:3tioi3
Platform
L,<i! do^iair.
30S subunit  SOS subunit  70S ribosome
FIGURE 5.1 Simplified representation of the subunits of a prokaryotic ribosome and their cooperative interaction to form
the functional 70S particle based upon images obtained by electron microscopy. (This diagram was kindly provided by Paul
J. Franklin.)
86
5,2 Stages in protein biosyntliesis
^ ^ V
I ‘,”/. :/<‘J ^
«s»<# \* # <m^
FIGURE 5.2 High-resolution structures resolved by X-ray crystallographic analysis of the two ribosomal subunits. The 30S
subuiiit was ciystallized from Thermus thermophilus and the SOS subuiiit from Deinococcits radiourans. The RNA chains
are represented as blue-gray (30S) or gray (SOS) ribbons and the protein main chains are shown in different colors. CP, cen-tral protuberance; P, platfonn. A, P and E (in red) indicate the sites where tRNA molecules bind to the large and small sub-units. [Reproduced with permission from T. Aiierbach et al. Current Drug Targets. Infectious Disorders 2,169-176 (2002).]
ff ISHC %B ^ %Vr9 % ^ Qfl? v ^ a n n §«# 5 %J ‘ %<%F in n S«P S ^sff^ ^ W n n 3» 3 a QB ? v ^ n v ^
5.2.1 Formation of aminoacyl-transfer RNA
Each amino acid is converted by a specific aminoacyl-tRNA synthetase to an aminoacyladenylate which is
stabilized by association with the enzyme:
Aminoacyl-tRNA synthetase
ATP + amino acid (aa) <-> aa-AMP-Enz + PP;.
Each amino acid-adenylate-enzyme complex then in-teracts with an amino acid-specific tRNA to form an
aminoacyl-tRNA in which the aminoacyl group is
linked to the 3′-OH ribosyi moiety of the 3 ‘ temiinal
adeoosyl group of the tRNA by a highly reactive ester
bond:
aa-AMP-Enz + tRNA ^ aminoacyl-tRNA-n AMP-n ENZ
The subsequent stages in prokaryotic protein biosyn-thesis are outlined in Figure 5.3.
5.2.2 Initiation
Three protein factors, IFl , 1F2 and 1F3, loosely associ-ated with 70S ribosomes are concerned with initiation.
IF l enhances the rate of ternary complex fomiation be-tween rnRNA, initiator tRNA and SOS ribosomal sub-units. IFl also has a role in promoting the dissociation
of 70S ribosomes released from previous rounds of
polypeptide synthesis into 30S and SOS subunits. Fac-tor Il;’3, which then binds to the 30S subunit, is also
needed for the binding of mRNA. The complex con-taining the SOS subunit, 1F3 and mRNA is joined by
IF2, GTP and the specific initiator tRNA, ,’V~formyi-inethionyl-tRNAp (fMet-tRNAp), the role of rF2 being
to direct the binding of fMet-tRNAp to a specific ini-tiator codon, usually AUG but occasionally GUG. IFl
and IF2 are now ejected from the complex, a process
dependent on the hydrolysis of one molecule of GTP
to GDP and inorganic phosphate. The next stage in-volves the detachment of IF3 in the presence of a SOS
subunit to permit the formation of the 70S ribosome.
87
Inhibitors of protein biosynthesis
GDP + Pi
FIGURE 5.3 Diagrammatic summary of the main stages in tlie biosynthesis of proteins on 70S ribosomes. The scheme
should be read clockwise starting at the top. L I + 1, 1 + 2 represent the initiator and successive codons; T is the tenninator
codon on mRNA; fMet, aaj, aaj, are i¥-formyIniethione and two other amino acids: tRNAp, tRNA j , tRNAj, are specific trans-fer RNAs. The involvement of the various protein cofactors referred to in the text is also indicated.
88
5,2 Stages in protein biosyntliesis
The association of the 508 and 30S subunits is be-lieved to involve interactions between their protein and
RNA chains. Initiation on 80S ribsomes resembles that
on 70S ribsomes except that eukaryotic initiation in
vivo uses unformylated Met-tRNA^’^’. in addition,
there are at least nine eukaryotic initiation factors
whose interplay is much more complex than that in
prokaryotic organisms.
5.2.3 Peptide bond synthesis and chain
elongation
The consensus view of synthetic sequence rests
largely on the concept of three distinct sites on tiie ri-bosonie, called the acceptor (A) site, the donor or pep-tidyl (P) site and the exit (E) site. The A site is the pri-mary decoding site where the codon of the mRNA first
interacts with the anticodon region of the specific
aminoacyl-tRNA. The fMet-tRNAp, however, binds
directly to the P site. The binding of the next aminoa-cyl-tRNA to the A site requires protein factors EF-T,
and EF-Tj^. EF-T^^ binds GTP and then forms a ternary
complex with aminoacyl-tRNA. This complex binds
to the acceptor site, with accompanying hydrolysis of
one molecule of GTP. GTP hydrolysis is not essential
for the binding of aminoacyl-tRNA, but in its absence
the bound aminoacyl-tRNA is not available for pep-tide bond formation. The role of the stable factor, EF-T^, is to regenerate E^F-T^-GTF from EF-T^-GDP by
stimulating the exchange of bound GDP for a mole-cule of free GTP. Appai’ently EF-T^ forms a high-affinity intemiediate complex with EF-T^, and GDP is
lost from this intermediate. An important and charac-teristic feature of the ribosome is the fidelity with
which it translates the genetic code of the mRNA into
the correct amino acid sequence. The interaction en-ergy of base pairing leads readily to mismatching of
codon and anticodon. Ribosomes, however, reject
mismatches with an error frequency as low as lO””””’.
The binding of the ternary complex of aminoacyl-tRNA-EF-Tu-GTP to a cognate mRNA codon at the A
site results in a conformational change in the ribosome
from an ‘open’ state to the ‘closed’ state, which then
drives the irreversible chemical changes leading to
peptide bond formation. In the event of a mismatch
between the codon of mRNA and the tRNA anticodon,
the conformational change in the ribosome does not
occur and it remains in the open state. The mis-matched aminoacyl tRNA then dissociates from the A
site. As we shall see, these events are critical to an un-derstanding of the miscoding activity of aminoglyco-side antibiotics.
After correct matching of aminoacyl tRNA and
the mRNA codon at the A site, the scene is set for the
formation of the first peptide bond. The carboxyl
group of the A’-foixnylmethionine attached to the P site
through its tRNA is ‘donated’ to the amino group of
the adjacent amino acid at the A site to form a peptide
bond. The formation of the peptide bond is catalyzed
by peptidyl transferase, which is a complex component
of the 508 subunit located at the central protuberance
of the 508 subunit. Although several ribosomal pro-teins, including L2, L15, L16 and L27, are located in
the vicinity of the peptidyl transferase site, the cuiTent
view is that 238 rRNA is responsible for catalyzing
peptide bond formation. The reader is referred to the
relevant papers listed under ‘Further reading’ for de-tails of the complex structures of both 168 and 238
rRNA. 8uffice it to say that the peptidyl transferase
function is probably located in the region of the 238
rRNA known as domain V, which also harbors the
binding site for protein L27. The dipeptide which is
formed at domain V remains attached through its C-teixninal to the second tRNA at the A site. The dipep-tidyl tRNA is then translocated from the A to the P site,
still linked to the mRNA (through the codon-anti-codon interaction). The third consecutive codon of the
mRNA is now exposed at the A site by the relative
movement of the ribosome towards the 3′ end of the
mRNA. The translocation step requires factor EF-G
and the hydrolysis of another molecule of GTP. EF-G
binds to the L7/L12 region of the large subunit. This
area is implicated in GTP hydrolysis mediated by EF-1\^, EIF-G and IF2. After peptide bond formation, the
deacylated tRNA transfers to the E site, which pro-motes its ejection from the ribosome. It is not clear
whether there is base pairing at the E site between the
mRNA codon and the anticodon of the tRNA. The
growing polypeptide chain passes into and through an
exit tunnel which stretches approximately lOOA from
the peptidyl transferase site through the ribosome to
the point of exit.
89
Inhibitors of protein biosynthesis
5.2.4 Chain termination and release  5.3 Puromycin
The signal for temiination of the polypeptide chain is
given by the appearance of one of three temiinator
codons—UA A, UAG or UGA—at the A site. The com-plete polypeptide is detached from the tRNA at the C~
terminal amino acid, a step that requires peptidyl trans-ferase activity and the release factors RFl, RF2 and
RF3. Factors RFT and RF2 are concerned with the
recognition of specific terminator codons, RFl recog-nizing UAA and UAG and RF2, UAA and UGA. Both
the binding of RFl and RF2 to the ribosomes and their
release require RF3, which also promotes the cleavage
of peptidyi-tRNA to release the completed protein
chain. Hydrolysis of GTP is also involved in the release
reaction. Release from eukaiyotic ribosomes involves
only one codon-recognizing release factor and requires
the cleavage of GTR The formyl groups of the fMet
ends of prokaryotic polypeptides ai’e removed by a spe-cific enzyme and in many proteins the methionine
residue is also removed. After release of the completed
polypeptide, the ribosome is liberated fi-om the mRNA
and deacylated tRNA by the combined action of GTP,
EF-G and ribosome release factor (RRF), which permits
dissociation into 30S and 508 subunits. 1F3 then binds
to the 30S subunit. This prevents reassociation until a
full initiation complex has once more been completed.
The antibiotic puromycin is a unique inhibitor of pro-tein biosynthesis, since the dmg itself reacts to fomi a
peptide with the C-tenninal of the growing peptide
chain on the ribosome, thus prematurely terminating
the chain. This remarkable property gave puromycin
an important role in the elucidation of the mechanism
of peptide bond fomiation and, as we shall see, in
defining the point of action of several other inhibitors
of protein biosynthesis.
The structural similarity of puromycin to the ter-minal aminoacyladenosine moiety of tRNA was noted
many years ago (Figure 5.4) and this proved to be the
key to understanding its actions. Aminoacyladenosine
is the temiinal residue of tRNA in both prokaryotic and
eukaryotic organisms. Puromycin therefore terminates
protein synthesis equally effectively on 70S and 808
ribosomes and the antibiotic has no therapeutic value.
The structural analogy of puromycin with aminoacy-ladenosine led to the demonstration that the amino
group of the antibiotic forms a peptide bond with the
acyl group of the temiinal aminoacyladenosine moiety
of peptidyl-tRNA attached to the ribosome. No further
peptide bond formation is possible because of the
chemical stability of the C—N bond which links thep-methoxyphenylalanine moiety of puromycin to the nu-HsC^i^^CHj
HO-CH^Q
Base Cy Cy
Terminus of aminoacyl- tRNA
FIGURFi 5.4 Structural tmalogy between
puromycin and the aminoacyl terminal of
transfer RNA. Cy represents cytosine and R
represents the rest of ttie amino acid molecule.
90
5.4 Inhibitors of aminoacyl-tRNA formation
cleoside residue. Peptidyl-purom3’cin then dissociates
from the ribosorne.
Provided that the peptidyl-tRNA is in the P site on
the ribosome, its reaction with puromycin has no other
requirement than a nonxially functioning peptidyi
transferase activity. Puromycin does not, however,
react with peptidyl-tRNA at the A site; in this situation
factor EF~G and GTF must be added in order to effect
translocation of the peptidyl-tRNA to the P site. Only
then is peptidyl-puromycin fomied and released from
the ribosome. The puromycin reaction occurs fairly
readily at 0° C, whereas nomial chain elongation is
negligible at this temperature, suggesting that puro-mycin has a considerable competitive advantage over
aminoacyl-tRNA in reacting with the peptidyl-tRNA.
The puromycin reaction will in fact proceed under
greatly simplified conditions, requiring only SOS ribo-somal subunits, the oligonucleotide CAACCA~(fMet)
to replace the peptidyi tRNA normally found at the P
site, and Mg-‘*’ and K”^ ions. This simple system,
known as the fragment reaction, allows the separation
of peptide bond fomiation from the more complex
process of translation. It has been extremely useful in
the investigation of those antibiotics suspected of in-hibiting peptide bond synthesis.
Derivatives of puromycin indicate that a single
benzene ring in the side chain is necessary for activity;
replacement of the/J-methoxyphenylalanine with pro-line, tryptophan, benzylhistidine or any aliphatic
amino acid results in a marked loss of activity. The L-phenylalanine analogue is about half as active as
puromycin, while the D-phenylalanine analogue is
completely inactive. Replacing the p-methoxypheny-lalanine residue with the 5′-benzyl-L-cysteine analogue
results in only a minor toss of activity, which may be
due to the increased distance between the benzene ring
and the free NH2 group caused by the additional S and
C atoms. Since puromycin substitutes for all aminoa-cyl-tRNAs equally well, the sufficiency of a single
benzene ring in the amino acid moiety of puromycin
and its analogues is puzzling. The aromatic ring may
be involved in a hydrophobic interaction with the ter-minal adenosine of peptidyl-tRNA at the donor site,
thus contributing to the formation of an intimate com-plex between puromycin and peptidyl-tRNA prior to
the formation of a peptide bond. In view of the struc-ture of the aminoacyladenosine of the tRNA terminal.
the requirement for linkage of the amino acid moiety
to the ribose 3′ position of puromycin is, however, not
unexpected. Puromycin substituted in the 5′ position
of the ribose with cytidylic acid is an effective peptide
chain terminator, and there is an absolute requirement
for cytidine in this derivative. Presumably this substi-tution extends the stractural analogy with tRNA.
5.4 liihibitoi’s of aminoacyl-tRNA formation
Several naturally occurring and synthetic analogues of
amino acids inhibit the fomiation of the aminoacyl-tRNA complex. Close analogues may become attached
to the appropriate tRNA and subsequently become in-coi”porated into abnomial proteins. Among these are
ethionine, norleucine, A’-ethylglycine and 3,4-dehy-droproline. Several naturally occurring antibiotics,
such as borrelidin, furanomycin and indolmycin, com-petitively antagonize the incorporation of the coixe-sponding amino acids, i.e. threonine, isoleucine and
tryptophan, respectively, into aminoacyl-tRNA. Most
of these inhibitors of aminoacyl-tRNA fomiation lack
specificity against micro-organisms and hence have no
useful medical application, although indolmycin is
said to be specific for prokaryotic tryptophanyl-tRNA
synthetase. However, by far the most important in-hibitor of aminoacyl-tRNA synthesis is the antibiotic
mupirocin or pseudomonic acid A (Figure .5..5), which
is produced by Psciidomonas fhioresccns. Mupirocin
has excellent activity against several species of Staph-ylococcus, and is especially useful against the danger-ous methicillin-resistant Staphylococcus aureus
(MRSA). The antibiotic has limited activity against
Gram-negative bacteria but includes in its spectrum
FIGURE .5.-5 Mupirocin (pseudomonic acid A).This antibi-otic, which has useful topical activity against methicillin-re-sistant Staphylococcus aureus and Streptococcus pyogenes,
is a specific inhibitor of bacterial isoleucyl-tRNA synthetase.
91
Inhibitors of protein biosynthesis
Haemophilus influenzae, Neisseria gonorrhoeae and
Neisseria meningitidis. Unfortunately, although mu~
pirocin is well absorbed after oral doses, it is rapidly
metabolized in the body to the inactive monic acid and
its cHnical use is therefore confined to topical applica-tions. It is effective in the treatment of the skin infec-tion impetigo caused by Staphylococcis aureus and
Streptococcus pyogenes. Mupirocin is also useful in
eliminating MRSA from the nasal passages of hospital
staff and vulnerable patients.
The antibacterial activity of mupirocin depends
upon its specific inhibition of isoleucyl-tRNA syn-thetase. Biochemical data indicate that the compound
is a competitive inhibitor of the isoleucyl adenylate al-though the chemical structures are significantly differ-ent. However, X-ray crystallography of isoleucyl-tRNA synthetase from Thermus therrnophilus shows
that the antibiotic binds to the catalytic cleft of the en-zyme, confirming that it does indeed compete with
isoleucyl adenylate. The antibacterial specificity of
mupirocin, which rests on the fact that the compound
has little or no inhibitory activity against eukaryotic
isoleucyl-tRNA synthetase, is apparently explained by
differences in just two amino acids of the bacterial and
eukaryotic enzymes. First, histidine-581 in the bacter-ial enzymes is replaced by an asparagine or serine in
eukaryotes. Second, leucine-583 in Thermus therrno-philus, or phenylalanine-583 in many other bacterial
species, is replaced by isoleucine in the eukaryotic en-zymes. The crystallographic data suggest that the pres-ence of isoleucine in eukaryotic isoleucyl-tRNA syn-thetase markedly weakens the binding of mupirocin
and renders the antibiotic ineffective as an inhibitor of
the human form of the enzyme.
5.5 Inhibitors of initiation and translation
5.5.1 Streptomycin
This naturally occuning antibiotic is a member of the
aminoglycoside group and has the complex chemical
stracture illustrated in Figure 5.6. Streptomycin was
discovered in the early 1940s and was the first drug re-ally effective against tuberculosis, although it is less
commonly used in the treatment of this disease today.
It is a broad-spectrum antibiotic, active against a range
OH C=
HO—C^^- ^ V
H2 /^-J^^^O H
NH,
I “
:=N H
I
•3 NH
O. .Oo-^^^’^ ^ N—C—NH
HO
HO—C
%
N- _
H OH H II
NH
FIGURE 5.6 The aminoglycoside streptomycin, the first
effective antitiibercuiar drug.
of Gram-positive and Gram-negative bacteria, but its
use is limited by several problems. First, the drug is ef-fective only when given by injection because its ab-soiption from the gastrointestinal tract is very poor.
Second, along with other aminoglycosides, strepto-mycin may cause permanent deafness, owing to UTC-versible injury to the eighth cranial nerve, and may
also cause kidney damage, although fortunately the
latter is usually reversible. Third, bacterial resistance
to this antibiotic develops readily.
Unusually for an inliibitor of protein biosynthe-sis, streptomycin is bactericidal rather than bacterio-static. Cell death is preceded by marked effects on pro-tein biosynthesis which are specific for the 7()S
ribosomes of bacteria:
1. Stfeptomycin strongly inhibits the initiation of
peptide chains. The drug also slows the elon-gation of partly completed chains, although
even at high concentrations of streptomycin,
chain elongation is not completely suppressed.
Peptidyl transferase activity is unaffected.
These effects on initiation and elongation afe
attributed to a distufbance of the functions of
both A and P sites by streptomycin.
2. Studies canied out on the effects of strep-tomycin in cell-free systems from bacteria
using synthetic polynucleotides as messen-gers clearly show that streptomycin induces
codon misreading. Thus the antibiotic in-hibits the incoiporation into peptide linkages
of phenylalanine directed by poly(U) his-tidine and threonine directed by poly(AC),
and arginine and glutamic acid directed by
poly(AG).
92
S,5 Inhibitors of initiation and translation
In contrast, streptomycin may, under some condi-tions, stimulate the iiicoiporatioii of amino acids in the
presence of S3’nthetic messengers which do not nor-mally code for these amino acids. For example, while
streptomycin inhibits the incoiporation of phenylala-nine in the presence of poly(U), the incoiporation of
isoleucine and serine is stimulated. Streptomycin also
induces poly(C) to promote the incoiporation of thre-onine and serine instead of proline. All these observa-tions indicate that streptomycin distorts the proofread-ing selection of the con-ect aminoacyl-tRNA by the
ribosome. However, the misreading is not random and
the following rules are more or less observed:
1. In any mRNy\ codon, only one base is mis-read, usually a pyrimidine located at the 5′
end or middle position of the codon.
2. There is no misreading of the base at the 3′
end.
3. Misreading of purines is rare and the occur-rence of these in a codon decreases the chance
of misreading the codon.
The induction of misreading of the genetic message by
streptomycin probably underlies the well-known
ability of this antibiotic to suppress certain bacterial
mutations.
Site of action of streptomycin
Streptomycin binds tightly but not irreversibly to 70S
ribosomes, with a K^ of 10″””” M. There is also low-affinity binding {K^_j >10″””‘*M), which is probably irrel-evant to the action of streptomycin. For many years it
was thought that the ribosomal binding site for strepto-mycin had been identified by experiments with bacte-rial mutants highly resistant to the antibiotic. Ribo-somes prepared from streptomycin-sensitive and
streptomycin-resistant Escherkiiia coH were dissoci-ated into 30S and 508 subunits by lowering the Mg^^
concentration in the medium. A ribosomal subunit
‘cross-over’ experiment showed that reassociated 70S
particles composed of 30S subunits from resistant cells
and 508 subunits from sensitive cells were resistant to
streptomycin. In the opposite cross, i.e. 30S subunits
from sensitive cells and SOS subunits from resistant
cells, the resulting 70S ribosomes were streptomycin
sensitive. This indicated that the target site of strepto-mycin was on the 308 subunit, a view strengthened by
the finding that radiolabelled streptomycin bound
specifically to the 308 subunit but not to the 508 sub-unit of sensitive ribosomes. Streptomycin did not bind
to the 30S subunit from resistant cells and did not in-duce misreadings of mRNA translated with resistant
ribosomes. It was then found that in the resistant ribo-somes, protein SI2 was mutated. Several resistant
variants were isolated with various single amino acid
replacements. Thus lysine-42 was replaced by as-paragine, threonine or arginine, while in another mu-tant lysine-87 was replaced by arginine alone. Flow-ever, studies with 308 subunits treated with protein
extractants to remove the S12 protein showed that it is
not essential for protein synthesis nor is it an absolute
requirement for streptomycin binding.
The accumulating evidence for a central role for
ribosomal RNA (rRNA) in protein biosynthesis has
been accompanied by the discovery of the importance
of interactions between rRNA and several antibiotics
and their ability to interfere with the biosynthetic
process. Until recently, the crucial technique used to
reveal the significance of rRNA was ‘footprinting’.
This detects the ability of antibiotics bound to ribo-somes to protect the bases of specific nucleotides of
16S and 23S rRNA against chemical modification,
usually alkylation, by reagents such as dimethyl sul-fate. Such protection is considered to be evidence of
specific interactions or binding between the antibiotics
and functionally significant domains in rRNA which
would otherwise be attacked by the chemically reac-tive reagent. When streptomycin binds to 70S ribo-somes in the presence of dimethyl sulfate, elec-trophoretic analysis of 16S rRNA following its
hydrolysis to individual nucleotides shows that the an-tibiotic affords protection for the bases of nucleotides
911 to 915, although protection is incomplete even
when the streptomycin-ribosome binding is fully sat-urated. Useful as the footprinting method has been in
the study of ribosome-antibiotic interactions, it has
now largely been superseded by the remarkable in-sights achieved by X-ray crystallography. In the case
of the aminoglycosides, the crystal stmcture of the 30S
subunit from Thermus thermophilus complexed with
streptomycin, paromomycin and spectinomycin has
been solved to 3A . The analysis shows that strepto-mycin is tightly bound to the phosphate backbone of
q’2
Inhibitors of protein biosynthesis
helix-44 of the 16S rRNA, a major component of the A
site, through salt bridges and hydrogen bonds. The
four nucleotides implicated in streptomycin binding
by footprinting were confirmed by the X-ray data.
There are additional interactions with heiix~27 and
with lysine~45 of the S12 protein. It is now considered
that streptomycin binds tightly to the A site on the 30S
subunit, where it stabilizes the closed conformation of
that site. X~Ray crystallography shows that strepto-mycin increases the number of interactions between
the ribosomal shoulder and the central part of the 30S
subunit. By promoting stabilization of the closed state
of the ribosome, which would normally only be
achieved by correct matching of codon and anticodon,
the antibiotic permits mismatching to occur, with re-sultant misreading of the genetic code. The wild-type
S12 protein contributes to the stability of the three-di-mensional structure of the A site on 16S rRNA. Muta-tions in S12 that confer resistance to streptomycin no
longer permit the antibiotic to exert its miscoding ac-tion at the A site.
Bactericidal action of streptomycin
Streptomycin and stracturally related aminoglycosides
are unusual among inhibitors of protein biosynthesis in
causing the death of bacteria. Most other inhibitors
merely arrest bacterial growth, which resumes when
the antibiotic is removed from the microbial environ-ment. It seems likely that the ability of the aminogly-cosides to induce ribosomal misreading of mRNA is
an important factor in their bactericidal action. The re-sulting aberrant proteins probably cause a variety of
disordered activities within the cell, including dis-ruption of the normal functions of the cytoplasmic
membrane and the outer membrane of Gram-negative
bacteria, leading to irreversible changes in cellular
metabolism.
5.5.2 Other aminoglycoside antibiotics
In addition to streptomycin, there are several other
aminoglycosides that are useful in antibacterial
chemotherapy. These include neomycin, kanamycin,
gentamicin (Figure 5.7), tobramycin, amikacin, and
netilmicin, the last two of which are semisynthetic
modifications of naturally occurring antibiotics de-signed to minimize enzymic inactivation by resistant
bacteria (Chapter 9). Like streptomycin, these antibi-otics are usually given by injection and are valuable in
the treatment of serious Gram-negative infections.
Paromomycin is unusual in finding its primary use
against amebic infections of the intestinal tract.
Several of these antibiotics have effects on pro-tein biosynthesis that differ fi-om those of strepto-mycin. For example, gentamicin, kanamycin and
neomycin exhibit three separate concentration-de-pendent effects on isolated ribosomes:
1. At concentrations below 2 |ig ml”^ there is
strong inhibition of total protein synthesis as-sociated with inhibition of the initiation step,
but little induction of mRNA misreading.
2. Between 5 and 50 |i,g ml^^ there is mis-reading, especially by reading through the
termination signals. Protein synthesis may
therefore actually increase through the accu-mulation of abnonnally long polypeptides as
the ribosomes continue past the end of one
message and on to the next.
3. Higher antibiotic concentrations re-establish
inhibition of protein synthesis.
F>ach ribosomal subunit has one strong binding
site for kanamycin as well as a number of weak bind-ing sites. Gentamicin and neomycin compete with
kanamycin, suggesting similar binding domains
which must be distinct from the streptomycin-binding
region since there is no comparable inhibition with
streptomycin. This conclusion is supported by foot-printing studies with neomycin, kanamycin and gen-tamicin which indicate a pattern of protection different
from that of streptomycin. Neomycin, kanamycin and
gentamicin protect adenine (A)-1408, guanine (G)-1491 and G-1494 of 16S rRNA from chemical modi-fication by dimethyl sulfate. These bases are located
close to the decoding region of the 3′ end of 16S
rRNA, and two of them (A-1408 and G-1494) are also
protected from chemical probing by tRNA bound to
the ribosomal A site. The data are consistent, there-fore, with the miscoding eifects of neomycin, kana-mycin and gentamicin. The basis of the different con-centration-dependent effects of the antibiotics is
uncertain but may be associated with progressive sat-94
S,5 Inhibitors of initiation and translation
HjN O^^^J^ /
HOH^C o-^^^jT^
HO-MO-NHjl O—I OH
HO
H,NH,C H
.O ^
CH2NH2
HO-HO-HO
HO
Neomycin B
CHjOH
O
Kanamycin A
MH_ I n—I r
r ^ NH,
H2N 0^;~^- A /
HOH2C o-^^^p^
HO
NHji 0—1 OH
CH2NH2
Paromomycin
H3C
Gentamicin C,g
OH
CH3NH i H , _.CH3
HNCH
Spectinomycin
FIGURE 5.7 ExEUTiples of other EUTiiiioglycoside antibiotics. Spectinomycin is more appropriately described as an aminocy-clitol antibiotic because it contains an inositol ring witli two of its OH groups substituted by niethylamino groups.
uration of their ribosomal binding sites with
ing concentration.
Crystallographic analysis of the complex of paro-momycin and the 30S subunit from Thermus ther-mophiUus reveals that the drug binds in the major
groove of heiix~44 of 16S rRNA. A nuclear magnetic
resonance study of the interaction of gentamicin with
a fragment of helix-44 indicated that it binds in much
the same way as paromomycin. This probably applies
to the other aminoglycosides as well. The binding to
helix~44 accords with the miscoding activity of these
aminoglycosides. X-Ray crystallography has revealed
that subtle distinctions between their effects on mis-coding and those of streptomycin are due to differ-ences of detail in their various binding interactions
with 16S rRNA.
9S
Inhibitors of protein biosynthesis
Spectinomycin is usually included in the amino-glycoside group, even though it lacks an amino sugar
residue (Figure 5.7). Unlike the previously mentioned
aminoglycoside antibiotics, its action is bacteriostatic
rather than bactericidal. The effects of spectinomycin
on protein synthesis are also markedly diiferent from
those of the other aminoglycosides. While it inhibits
protein synthesis in bacterial cells and in cell-free sys-tems containing 70S ribosomes, spectinomycin does
not induce misreading of mRNA. Spectinomycin has
no effect on codon recognition, peptide bond forma-tion, or chain termination and release. The antibiotic
inhibits the translocation of peptidyl-tRNA from the A
site to the P site. The rigidity of the spectinomycin
molecule, conferred by the fused ring system, is
thought to be relevant to the inhibitory action. The X-ray analysis of the spectinomycin~30S subunit com-plex referred to earlier shows that the drug binds in the
minor groove of helix~34 of 168 rRNA, making a sin-gle contact with a 2’~hydroxyl group and hydrogen
bond links with several other bases, especially with G-1064 and cytosine(C)-1192. Its binding mode is thus
considerably different from the other aminoglyco-sides. The rigid spectinomycin molecule may steri-cally hinder a movement of the head region of the 30S
subunit believed to be involved in the normal translo-cation event. With the emergence of p-lactamase~pro-ducing Neisseria gonorrhoeae, spectinomycin has be-come useful in the treatment of gonococcal infections.
Postscript: do aminoglycosides inhibit
trans-translation in bacteria?
The recently discovered phenomenon of fran^-transla-tion rescues stalled ribosomes and contributes to the
degradation of incompletely synthesized polypeptides.
7)-an5-translation is unique to prokaryotes and is es-sential for the viability of some bacteria, for example,
the important pathogen Neisseria gonorroeae. In this
process a species of messenger RNA, termed transfer
messenger RNA (tniRNA, or SsrA RNA or lOSa
RNA), acts first as tRNA being aminoacylated at the 3′
end with alanine and catalyzed by alanyl-tRNA syn-thetase. An alanine residue is thereby added to the end
of the stalled protein chain. The tmRNA then reverts to
its messenger function and translation resumes, not on
the original messenger RNA, but at an internal position
in the tmRNA. The arrested protein synthesis is re-leased, ribosome recycling resumes and the previously
stalled protein chain is now tagged at the C-terminal
with a sequence which signals its subsequent degrada-tion. Experiments in vitro show that several aminogly-cosides, neomycin B being the most potent, block
fi-on^-translation by binding to the tRNA domain of
tmRNA. This interaction disrupts the normal confor-mation of tmRNA and renders it incapable of efficient
aminoacylation with alanine by alanyl-tRNA syn-thetase. If aminoglycosides inhibit franj-translation in
intact bacterial cells, it would interfere with the dis-posal of miscoded and truncated proteins and would
therefore contribute significantly to the antibacterial
action of these antibiotics. Evidence for or against
such an in vivo action is awaited with interest.
5.5.3 Tetracyclines
Several members of this group are illustrated in Figure
5.8. The tetracyclines are broad-spectrum antibiotics
with additional activity against rickettsial organisms,
mycoplasmas and certain protozoa. The bacteriostatic
activity of the tetracyclines results from the direct in-hibition of protein biosynthesis. Unlike most other
therapeutically useful inhibitors of protein biosynthe-sis, the tetracyclines inhibit both 70S and SOS ribo-somes, although 70S ribosomes are more sensitive.
However, the tetracyclines are much more effective
against protein synthesis in bacterial cells than against
mammalian cells, largely because of the ability of sen-sitive bacteria to concentrate tetracyclines within their
cytoplasm (Chapter 7).
Studies of the effects of the tetracyclines on the
tRNA-ribosome interaction show that they inhibit the
binding of aminoacyl-tRNA to the A site on the ribo-some but have little eftect on binding to the P site ex-cept at high drag concentrations. The binding of fMet-tRNAp to the ribosome is about one-tenth as sensitive
to tetracycline as the binding of other aminoacyl-tRNAs, since fMet-tRNAp binds to the P site rather
than to the A site. The tetracyclines do not directly in-hibit formation of the peptide bond or the translocation
step except at high concentrations. They have no effect
on the hydrolysis of GTP to GDP required for the func-tional binding of aminoacyl-tRNA to the A site. Possi-96
S,5 Inhibitors of initiation and translation
H3C , ^O H
CONH ,
N (CH3);  N (CHs)^
CONH,
H3C,. /O H
OH N (CHjjj
OH
OH O OH O
Oxytetracycline
CONH,
CH, OH  N(CH3)2
OH
Doxycycline
CONH2
FIGURE 5.8 Four major tetracy-cline broad-spectrum antibiotics.
Minocycline is effective against
some bacteria that are resistant to tlie
other drugs.
bh’ the tetracyclines uncouple GTP hydrolysis from
the binding reaction. Tetracyclines also inhibit peptide
chain temiination and release by blocking the binding
of the release factors at stop codons in the A site. How-ever, it is unlikely that the effects on termination and
release contribute significantly to the antibacter 10.1 tiC~
tion of tetracyclines.
There is a single, moderately high-affinity bind-ing site for tetracycline on the 30S subunit, with a K^^
of approximately 1 |.lM. There are also many low-affinity sites which are not considered essential to the
inhibitory action of the drug. Photoaffinity labelling
studies with a photoreactive derivative of tetracycline
revealed extensive labelling of protein S7, which is lo-cated near the region of contact between the two ribo-sonial subunits. Footprinting studies apparently impli-cated several residues of 16S rRNA in the binding of
tetracyclines. However, doubts regarding the rele-vance of the footprinting data were raised by the ob-servation that one of the residues, A-892, protected by
tetracycline from alkylation by dimethyl sulfate, was
not similarly protected by minocycline and doxycy-cline, which also inhibit ribosomal function in the
same way as tetracycline. Furthermore, the multiple
binding interactions of tetracyclines with the 30S
subunit cause conformational changes in 16S rRNA
which may affect the susceptibility of nucleic acid
bases to alklylation and the S37 protein to photoaffin-ity labelling.
Fortunately, X-ray crystallography of tetracycline
bound to the 30S subunit has clarified the nature of the
binding interaction. Although six tetracycline binding
sites were identified, the most important site lies in a
pocket formed by the head of the 30S subunit at the A
site. The tetracycline molecule interacts with the sugar
backbone of helix-34 through a magnesium ion. At
this site the antibiotic sterically interferes with the lo-cation of the anticodon loop of aminoacyl-tRNA and
prevents its binding to the niRNA codon at the A site.
The crystallographic data therefore agree well with the
classic biochemical results on the action of tetracy-cline on ribosomal function. The relevance of the other
five tetracycline binding sites identified by crystallog-raphy to the action of tetracycline is not clear, but they
may contribute synergistically to the overall suppres-sion of bacterial protein synthesis.
The features of the tetracycline molecule recpired
for antibacterial activity have been worked out in some
detail. Because the permeation mechanism for the
entry of tetracyclines into bacterial cells (Chapter 7)
may have its own structural requirements, it should not
be assumed that the structural features of the tetracy-cline molecule required for antibacterial activity are
the same as those for the inhibition of ribosomal func-tion. It is possible that some tetracycline derivatives
which inhibit ribosomes may lack antibacterial activ-ity because of a failure to achieve an inhibitory con-centration within the bacterial cell.
97
Inhibitors of protein biosynthesis
The more limited investigations of the stractural
requirements for the inhibition of protein synthesis on
isolated ribosomes reveal several modifications in
structure (Figure 5.9) that significantly affect inhibi-tory activity:
1. Chlorination of the 7 position significantly
increases inhibitory activity.
2. Epimerization of the 4-dimethylamino group
significantly decreases activity.
3. Both 4a,12a-anhydro- and 5a,6-anhydro-tetracyclines are much less active than
tetracycline.
4. Ring opening of the teti^acycline nucleus to
give the iso derivatives and the a and p isomers
of apo-oxytetracycline destroys activity.
5. Replacement of the amidic function at Cj
with a nitrile group results in a marked loss of
potency.
It has long been suspected that the ability of the
tetracyclines to chelate polyvalent cations like Mg-+ is
relevant to the inhibition of protein biosynthesis. The
crystallographic evidence described here now gives
strong support for this view. Free Mg-“*” ions in the bac-terial cytoplasm may also complex with tetracycline
and limit its ability to interact with ribosomal Mg-“*”.
The 11,12p-diketone system, the enol (positions 1 and
3) and the cai-boxamide at position 12 have all been
implicated as possible chelation sites. The glycyltetra-cycline tigicycline (Figure 5.10) is also an effective
chelating agent. Another suggestion, based on circulai”
dictooism studies on the 7-chlortetracycline com-plexes with Ca-“*” and Mg^”^, is that chelation requires
the bending of ring A back towards rings B and C so
that the oxygen atoms at positions 11 and 12 together
with those at positions 2 (amidic oxygen) and 3 form a
coordination site into which the metal atom fits.
5.6 Inhibitors of peptide bond formation and
translocation
5.6.1 Chloramphenicol
Chloramphenicol (Figure 5.11) is a naturally occuning
antibiotic that is now entkely produced by chemical
HjC,^ ^OH
N (CH3)^  N(CH3),
OH
CONH,
N (CH3)2
OH
CONH,
CONH,
Isotetracycline
FIGURE 5.9 Tetracycline derivatives
with greatly reduced antibiotic activity.
98
S,6 Inhibitors of peptide bond formation and translocation
N(CH3);
O
H,C I H
‘ CH3
N (CH3)2
OH
CONH,
OH O
FIGURE 5.10 Tigicycliiie is a novel tetracycline intro-duced to combat bacteria resistant to earlier tetracyclines.
synthesis, it was one of the first liroad-spectratn antilii-otics to be discovered and has excellent bacteriostatic
activity against both Gram-positive and Gram-nega-tive cocci and bacilli, as well as rickettsias, mycoplas-mas and Chlainydia. Unfortunately, its ever-widening
use in some parts of the world without effective med-ical controls revealed serious side effects associated
with the bone marrow in some patients. A concentra-tion of 25-3 0 Llg of chloramphenicol for mf “”-^ of blood
maintained for 1-2 weeks leads to an accumulation of
nucleated erythrocytes in the maiTow, indicating an in-terference with the normal red cell maturation process.
Nomial erythropoiesis usually resumes after with-drawal of the drug, but very occasionally, i.e. not more
than I in 20,000 cases, a more serious defect develops
in the marrow which leads iiTeversibly to the loss of
both white and red cell precursors. The biochemical
basis for chloramphenicol-induced fatal aplastic ane-mia has not been established. However, an action of
the drug on mitochondrial ribosomes, which more
closely resemble 70S than 80S ribosomes, with possi-ble effects on the mitochondrial function of key stem
cells in the marrow, cannot be ruled out. The very low
incidence of the irreversible form of chloramphenicol
toxicity indicates a special sensitivity in those few in-dividuals who succumb to it. Chloramphenicol therapy
is therefore now restricted to serious infections for
which there is no effective alternative. These include
FIGURE 5.11 Chloramphenicol. Ttie active form is the 13-threo stereoisomer.
typhoid and meningitis caused by bacteria resistant to
p-lactam antibiotics, in cases where the patient is aller-gic to these drugs or where use is restricted to topical
application, for example in superficial infections of
the eye.
The bacteriostatic action of chloramphenicol is
due to a specific inhibition of protein biosynthesis on
70S ribosomes; it is completely inactive against SOS ri-bosomes. Studies with radioactively labelled chloram-phenicol show that it binds exclusively to the SOS sub-unit to a maximum extent of one molecule per subunit.
The binding is completely reversible. Structurally un-related antibiotics such as erythromycin and lin-comycin, that also interfere with the function of the
SOS subunit, compete with chloramphenicol for the
binding region, whereas inhibitors of the 30S subunit
function, such as the aminoglycosides and tetracy-clines, do not. Biochemical evidence strongly indi-cates that chloramphenicol blocks peptide bond for-mation by inhibiting the peptidyl transferase activity
of the SOS subunit. Thus chloramphenicol inhibits the
puromycin-dependent release of nascent peptides from
70S ribosomes and also inhibits the 508 subunit-cat-alyzed puromycin fragment reaction.
Earlier studies concentrated on the identification
of proteins thought to be involved in the binding of
chloramphenicol to the 508 subunit. The technique of
affinity immune electron microscopy suggested that
the peptidyl transferase region of the ribosome, con-taining the proteins LIS , LI S and L27, contributed to
the binding of chloramphenicol. Because the vast ma-jority of known enzymes are proteins, it is understand-able that the early work on the action of chlorampheni-col sought to identify a protein or proteins associated
with peptidyl transferase activity that would provide
the target site for the antibiotic. However, the discov-ery that domain V of 23S rRNA catalyzes peptide
bond formation led to a reassessment of the nature of
the target for chloramphenicol. Although the results of
footprinting studies suggested that several nucleotides
of 23S rRNA were involved in the interaction of chlo-ramphenicol with its binding site, once again it re-mained for X-ray crystallography to reveal more pre-cise details of the binding site. Deinococcus
radiodurans SOS subunits complexed with chloram-phenicol confirmed that the drug is bound to a hy-drophobic crevice in the peptidyl transferase region of
99
Inhibitors of protein biosynthesis
the A site. The X-ray data provide evidence that the
two oxygen atoms of the nitro group of chlorampheni-col, the 1-hydroxyl, 3-hydroxyl and 4′-carboxyi
groups all have the potential to hydrogen bond with
several specific nucleotide bases in a loop of domain V
of 23S rRNA. A Mg^”*” ion may also be involved in the
bonding of the 3-hydroxyl with adjacent nucleotide
bases. The antibiotic is thus exquisitely placed to block
the coiTect positioning of the incoming aminoacyi
tRNA and so inhibit the formation of the next peptide
bond. An analogous study with 508 subunits from the
archaeobacterium Haloarcula rnarismortui placed the
binding site away from the peptidyl transferase center
in another hydrophobic crevice at the entrance to the
polypeptide exit tunnel. Because pathogenic bacteria
and Deinococcus radiodurans belong to the eubacte-ria, it seems likely that the binding data from Deino-coccus may be more relevant to the therapeutic action
of chloamphenicol than the Haloarcula data, although
it can be argued that binding to both sites may impor-tant in the antibacterial action of the drug.
aminoacyi tRNAs at the active center, clindamycin dis-rupts substrate binding and hinders the path of the
growing peptide chain. The binding site of clin-damycin partially overlaps with that of chlorampheni-col at the A site, but the drug also interacts with the P
site. Crystallographic data show that clindamycin
binds to a specific loop of domain V of 23S rRNA.
There is no evidence of any interactions with riboso-mal proteins. The three hydroxyl groups of the sugar
moiety of clindamycin are all positioned in the crystal
structure to form hydrogen bonds with specific nu-cleotide bases of the 23S rRNA. The ability of clin-damycin to interact with the A site is probably due to
the proline moiety of the drug, which is seen to over-lap with the phenyl ring of chloramphenicol at the A
site. The carbon atom of the methyl group at the teimi-nal of the sugar side chain residue is positioned to in-teract with a nucleotide base at the P site, possibly in-volving van der Waals and hydrophobic forces. The
sulfur atom of clindamycin may also interact with the
P site.
5.6.2 Clindamycin  5.6.3 IVIacrolides and l<etolides
This is a semisynthetic member of the naturally occur-ring lincosamide antibiotics. Clindamycin (Figure
5.12) is clinically useful against staphylococcal and
streptoccoccal infections and against anaerobic patho-gens such as Bacteroides spp. It is poorly active
against Gram-negative bacteria. Like chlorampheni-col, clindamycin inhibits the peptidyl transferase ac-tivity of the 50S subunit. However, whereas chloram-phenicol interferes with the correct positioning of
FIGURE 5.12 Clindamycin is a narrow-spectrum antibac-terial drug effective against Gram-positive bacteria .
The original macrolide antibiotic, erythromycin (Fig-ure 5.13), is a complex, naturally occurring compound
effective against many Gram-positive bacteria, my-coplasmas and chlamydia, but less active against
Gram-negative pathogens. Semisynthetic derivatives,
e.g. azithromycin, brought additional benefits, includ-ing useful activity against Haemophilus influenzae, an
important Gram-negative pathogen of the upper respi-ratory tract. The most important recent introductions
have been the ketolides like telithromycin (Figure
5.13) in which the cladinose sugar moiety at position
C-3 is replaced with a keto group. A major advantage
of the ketolides is that they retain activity against some
bacteria that have become resistant to the older
macrolides.
Despite the various chemical differences among
macrolides and ketolides, the drags share a common
mode of action; they inhibit the function of the 50S
subunit by blocking the access of the growing peptide
chain to the entrance of the polypeptide exit tunnel.
Thus, although they do not directly inhibit peptidyl
transferase activity, macrolides and ketolides effec-100
S,6 Inhibitors of peptide bond formation and translocation
Erythromycin
3 ^ N ^
Telithromycin
FIGURE 5.13 En’thromycin, a ‘mediiim-speclrum’ anti-bacterial dnig of the niacrolide family and telithromycin, a
semisynthetically modifed macrolide antibiotic, are repre-sentative of a new series of ketolides.
lively suppress protein biosynthesis in bacteria. They
are without effect on the 80S ribosomes of mammalian
cells. Details of the actual binding site for erythromy-cin on the .5()S subunit emerging from X-ra}’ crystal-lography data pinpoint the potential for multiple hy-drogen bond interactions between the desosamine ring
at the 5 position of the macrolactone moiety of the an-tibiotic with nucleotides of domain V of 23S rRNA.
This domain is a major component of the exit tunnel.
Van der Waals forces may also contribute to the stabil-ity of the binding interaction. A commonly encoun-tered form of bacterial resistance to erythromycin de-pends on A’-dimethylation by the inducible enzyme
jV-methyl transferase at the N-6 position of a specific
adenine residue of 23S rRNA: A-2058 in Escherichia
coli, A~2()86 in Bacillus stearothennophilus and A-2058 in Bacillus subtilis (see Chapter 9 for further de-tails of the A’-methyl transferase system). The crystal
structure of the SOS subunit shows clearly that methyl
groups in the indicated position prevent hydrogen
bonding between A-2()58 (of Deinococcus radiodu-rails) and the 2′ hydroxyl group of the desosamine ring
of erythromycin and other macrolides. The methyl
groups would very likely also sterically hinder access
of the antibiotic to its binding site. This illustrates a
pleasing concordance between X-ray studies and clas-sic biochemical work on antibiotic resistance. It is in-teresting that humans and all other mammals have gua-nine instead of adenine at position 2058 in 2.3S rRNA,
which effectively prevents macrolides and Itetolides
from binding to SOS ribosomes.
The crystal structure of telithromycin, bound to
the Deinococcus radiodurans SOS subunit, shows that
the antibiotic also blocks the ribosomal exit tunnel.
The interactions involve domains 11 and V of 23S
rRNA and provide tighter binding than macrolide an-tibiotics—an observation borne out by direct binding
studies. The tighter binding of telithromycin to ribo-somes may account for its improved activity against
macrolide-resistant bacteria harbouring the A’-di-methylase enzyme.
5.6.4 Streptogramins
The streptogramin antibiotics were discovered more
than four decades ago but until recently were of more
academic than practical interest. The growing menace
of bacterial resistance to antibiotics, however, raised
the possibility that some members of this large group
of complex compounds could be clinically useful.
Streptogramins are of two structurally distinct types
which are produced as mixtures by certain Strepto-myces. Type A streptogramins are polyunsaturated
cyclic lactones that resemble the macrolides. Type B
streptogramins are cyclic hexadepsipeptides. More re-cently, semisynthetic modifications of the naturally
occuning streptogramins have yielded significant im-provements in clinically important properties such as
water solubility. Dalfopristin and quinupristin (Figure
5.14) are examples of semisynthetic derivatives of
type A and type B compounds, respectively. A combi-nation of dalfopristin and quinupristin is proving valu-able in the treatment of serious and potentially life-threatening infections caused by methicillin-resistant
101
Inhibitors of protein biosynthesis
O I H
Dalfopristin
N(CH3)2
lized with the streptogramin A antibiotic, virgini-amycin M, revealed that the conformational change in
the subunit induced by this antibiotic results from its
binding to portions of both the A and P sites. It will be
of interest to see whether this mode of binding of type
A streptogramins is replicated in ribosomes belonging
to bacterial species other than archaeobacteria like
Haloarcula marismortui. iV-Dimethylation of A-2058
in domain V of 23S rRNA or its replacement by gua-nine or uridine leads to resistance to the type B strep-togramins, indicating a role for this adenine residue in
the binding interaction. It is interesting that modifica-tion or replacement of A-2058 causes resistance to
stracturally diverse compounds, including macrolides,
lincosamides and type B streptogramins.
^^^X
O
Quinupristin
FIGURE 5.14 Dalfopristin and quinupristin are strep-togramin antibiotics tliat, when combined, cause irreversible
inliibition of bacterial protein biosynthesis.
Staphylococcus aureus and vancomycin-resistant en-terococci (VRE).
Remarkably, the two types of antibiotic act syner-gistically to give iiTeversible inhibition of protein
biosynthesis and consequently a bactericidal eftect.
The synergism arises from the distinct actions of the
different streptogramins on ribosomal function. Type
A compounds block the binding of aminoacyl-tRNA
and peptidyl-tRNA to the A and P sites, respectively.
Type B compounds hinder the interaction of peptidyl-tRNA only with the P site. Some form of conforma-tional change in ribosomal stracture is induced by type
A compounds and enhances the affinity of the 508 sub-unit for type B streptogramins. X~Ray analysis of the
SOS subunit from Haloarcula marismortui cocrystal-5.6.5 Oxazolidinones: novel synthetic
inhibitors of peptidyl transferase
The antibacterial activity of oxazolidinones was dis-covered more than 20 years ago, but toxicity problems
with the early compounds prevented their further de-velopment. Fortunately, a re-examination of the series
in the early 1990s led to the synthesis of much less
toxic compounds and the discovery of linezolid (Fig-ure 5.15). Linezolid has excellent activity against dan-gerous Gram-positive infections caused by MRSA and
VRE. Because linezolid is representative of the first
chemically novel series of antibacterial drags to
emerge in several decades, there is now intense inter-est in this area of chemistry. Linezolid and most other
oxazolidinones developed so far lack useful activity
against Gram-negative pathogens, probably because
the drug efflux pumps in these bacteria are very effec-tive in clearing the compounds fi’om bacterial cyto-FIGURE 5.15 Linezolid was the first clinically active oxa-zoiidinone antibiotic. It is effective against Gram-positive
bacteria.
102
S,6 Inhibitors of peptide bond formation and translocation
plasm (see Chapter 7). Much attention is therefore cur-rently focused on synthesizing novel oxazolidinones
with good activity against Gram-negative pathogens
such as Haemophilus influenzae. The Enterobacteri-acae may provide an even greater challenge.
Following the revival of interest in oxazolidi-nones, it was soon discovered that the basis of their tm-tibacterial action is the inhibition of protein biosynthe-sis. Locating the precise site of action has proven more
difficult. It became clear that oxazolidinones bind ex-clusively to the bacterial SOS subunit and prevent com-plexation with its 30S partner, mRNA, initiation fac-tors and fMet-tRNAj.. This appeared to place the
inhibitory mechanism in a separate category from the
existing inhibitors of protein biosynthesis which act at
points beyond the fomiation of the preinitiation com-plex. However, later experiments suggested that oxa-zolidinones inhibit the binding of fMet-tRNAp to the
peptidyl transferase at the P site of the SOS subunit. A
study of the effects of mutations affecting nucleotides
of 23S rRNA which confer resistance to oxazolidi-nones found that the nucleotides are restricted to a con-fined space between the A and P sites in the three-di-mensional structure of the SOS subunit. This space
partially overlaps the P site. Another recent investiga-tion used a photoactivated, radioiodine-labelled oxa-zohdinone derivative to identify drug-binding sites in
ribosomes actively engaged in protein synthesis (in
contrast to isolated purified ribosomes). The com-pound cross-linked with A-2602, which is known to be
a critical base in the peptidyl transferase active center.
Protein L27, believed to be closely associated with, al-though not functionally involved with peptidyl trans-ferase, was also labelled. Even allowing for possible
artefacts arising in photoaffinity labelling experi-ments, i.e. spurious labelling of irrelevant ‘bystander’
molecules, the weight of evidence from both the af-finity labelling and mutational studies points to the
peptidyl transferase center as the primary site of oxa-zolidinone action. Interference with the correct align-ment of fMet-tRNAp at the P site could explain the dis-ruption of the formation of the preinitiation complex.
The surprisingly low binding affinity of oxazo-lidinones for isolated, purified SOS subunits has so far
precluded an X-ray crystallographic analysis of the
drug-ribosome interaction. However, the considerable
potential for the clinical application of oxazolidinone
antibiotics will no doubt continue to focus attention on
the precise details of their mechanism of action.
5.6.6 Fusidic acid
Fusidic acid belongs to a group of steroidal antibiotics
(Figure S.16) which inhibit the growth of Gram-posi-tive but not Gram-negative bacteria. The inactivity of
fusidic acid against Gram-negative bacteria may be
due to inadequate penetration into the bacterial cyto-plasm, since the drug inhibits protein synthesis on iso-lated ribosomes from Gram-negative bacteria. Alter-natively, fusidic acid may be rapidly extruded from
Gram-negative cells by one or more of the drug-efflux
pumps (Chapter 7). Fusidic acid is used topically in
some countries to treat Gram-positive infections of the
skin that cause impetigo. Occasionally the drug may
be given systemically to combat potentially life-threat-ening infections caused by MRSA and VRE. However,
fusidic acid is not available in the United States.
The addition of fusidic acid to 70S ribosomes in
vitro prevents the translocation of peptidyi-tRNA from
the A site to the P site and eventually suppresses the
EF-G-dependent hydrolysis of GTP. The inhibition is
overcome by the addition of excess EF-G. The resist-ance of some strains of bacteria to fusidic acid appears
to be associated with a change in EF-CJ, since the fac-tor prepared from resistant cells normally catalyzes
translocation in the presence of the antibiotic. All this
points to factor FIF-G as the target protein for fusidic
acid. Fusidic acid-hypersensitive mutant forms of EF-G have been used in an effort to locate the binding site
CH,
FIGURE 5.16 Fusidic acid is a steroidal antibiotic with a
narrow spectnim of action against Gram-positive bacteria.
103
Inhibitors of protein biosynthesis
for the drug. However, it seems unlikely that the amino
acids involved in these mutations could all make con-tacts with the drug. Instead it is believed that the muta-tions probably modify the confomiation of EF-G and
enhance its affinity for fusidic acid. A current model
for the action of fusidic acid proposes that the initial
EF-G-ribosome interaction triggers a burst of GTP hy-drolysis which either uncovers or even generates the
binding site for fusidic acid on EF-G. A stable com-plex is fomied of F>F-G-GDP-fusidic acid and the ribo-some which is unable to release EF-G for a further
round of translocation and GTP hydrolysis. Fusidic
acid also inhibits protein synthesis on SOS ribosomes
in a similar manner by stabilizing the EF-2-GDP-ribo-some complex (ElF-2 corresponds to the prokaryotic
EF-G). The lack of toxicity of fusidic acid against
mammalian cells is probably because the drug does
not achieve a high enough intracellular concentration
in mammalian cells to form the stabilized complex.
5.6.7 Cycloheximide
Occasionally referred to as actidione, cycloheximide
(Figure 5.17) is toxic to a wide range of eukaryotic
cells, including protozoa, yeasts, fungi and mam-malian cells, and its lack of selectivity precludes any
clinical use. It is included here because of its unusual
specificity of action against SOS ribosomes, with no ef-fect on 70S ribosomes. Cycloheximide is therefore
mostly used as an experimental tool to inhibit protein
synthesis in eukaryotic cells, and occasionally to ex-clude fungi from bacterial cultures.
There are considerable variations in the sensitiv-ity of SOS ribosomes from different species of yeasts to
FIGURE 5.17 Cyclolieximide is an unusually specific in-hibitor of SOS ribosomes.
cycloheximide, which have been exploited to explore
its site of action. For example, ribosomes from Sac-charornyces cerevisiae are strongly inhibited by cyclo-heximide, while those from Sacchawmyces fragilis
and Kluyveromyces I act is are resistant. Cross-over ex-periments with the 60S and 40S subunits from Sac-chawmyces cerevisiae and Sacchawmyces fragilis
showed that sensitivity to cycloheximide resides in the
60S subunit. Analysis of the proteins of the 60S sub-unit from Kluyveromyces lactis revealed that protein
L41 differs from the corresponding protein in Saccha-romyces cerevisiae, suggesting that L41 may be in-volved in the interaction of the antibiotic with sus-ceptible ribosomes. A recent study found that L41
contributes to the efficiency of protein synthesis in
Saccharomyces cerevisiae but is not essential. Mutants
in which L41 was deleted had an increased rate of
elongation, indicating that L41 in some way normally
regulates the rate of elongation. The L41-deficient mu-tants were more resistant to cycloheximide, suggesting
that the compound inhibits the translocation of pep-tidyl-tRNA from the A to the P site. Other results show
that two other ribosomal proteins, L42 and L28, also
contribute in some way to the interaction of cyclohex-imide with SOS ribosomes. Footprinting studies reveal
that cycloheximide protects two guanine residues
lying within a loop region of 28S rRNA of the larger
ribosomal subunit which is associated with the hydrol-ysis of GTP and the ribosomal interaction with elonga-tion factor EF-2. This is consistent with the inhibition
by cycloheximide of the translocation step, and it
seems likely that the antibiotic hinders the function of
EF-2. The involvement of proteins L41, L42 and L28
in the action of cycloheximide may be linked to the
role of these proteins in maintaining the confomiation
of 28S rRNA so as to afford a nucleic acid binding site
for the antibiotic.
Further reading
Auerbacli, T. et al. (2002). Antibiotics tai’geting ribosomes:
crj’staliographic studies. Cum Drug Targets Infect.
Disord. 2. 169.
Ban, N. et al. (2000). The complete atomic stracture of the
large ribosomal subunit at 2.4A resolution. Science
289, 905.
104
S,6 Inhibitors of peptide bond formation and translocation
Bashan, A. et al., (2003). Structural basis of the ribosomal
niacliiner}’ for peptide bond formation, ti’anslocation
and nascent chain progression. Molec. Cell. 11, 91.
Berisio, R. el al. (2003). Structural insight into the antibiotic
action of telithromycin against resistant nnitants. / .
Bact. 185, 4276.
Bobkova, E. V. et al. (2003). Catalytic properties of mutant
23S ribosomes resistant to oxazolidinones. / . Biol.
Chem. 278, 9802.
Bonfiglio, G. and Furneri, P. M. (2001). Novel streptogramin
antibiotics. Exp. Opin. Investlg. Drugs. 10, 185.
Carter, A. P. eJal. (2000). Functional insights from tlie struc-ture of the 30S ribosomal subunit and its interactions
with antibiotics. Nature 4©7, 340.
Chopra, I. and Roberts, M. (2001). Tettacycline antibiotics:
mode of action, applications, molecular biology and
epidemiology of bacterial resistance. Microbiol.
Molec. Biol. Reviews 65, 232.
Colcat, J. R. et al. (2003). Cross-linking in the living cell lo-cates the site of action of oxazolidinone antibiotics../.
Biol. Chem. 278, 21972.
Corvaisier, S., Bordeau, V. and Felderi, B. (2003). luhibidon
of transfer RNA aminoacylation and fra/is-tKmslation by
aminoglycoside antibiotics. / . Biol. Chem. 278, 14788.
Dresios, L et al. (2003). A dispensable yeast ribosomal pro-tein optimizes peptidyl transferase activity and affects
translocation../. Biol. Chem. 278, 3314.
Hansen, J. L. ctai (2003). .Structure of five antibiotics boimd
at the peptidyl transferase center of the large ribosomal
subunit./. Mot.Biot. 330, 1061.
Livermore, D. (2003). Linezolid in vitro : mechanisms and
antibacterial spectrum../. Antimicrob. Chemother. 51,
Suppl. S2, ii9.
Martemyanov, K. A. et al. (2001). Mutations in the G–do–main of elongation factor G from Thermus ther-mophilus affect both its interaction witli GTP and fu-sidic acid../. Biol. Chem. 276, 28774.
Nakama, T., Nurecki, O. and Yokoyama, S. (2001). Struc-tural basis for the recognition of isoleucyl-adenylate
and an antibiotic mupirocin by isoleucyl-tRNA syn-thetase. ./. Biol. Chem. 276, 47387.
Ogle, J. M. et al. (2003) Insights into the decoding mecha-nism from recent ribosome stmctures. Trends Biochem.
Sci. 28, 259.
Poehlsgaard, .1. and Douthwaite, S. (2002). The macrolide
binding site on the bacterial ribosome. Curr. Drug Tar-gets Infect. Disord. 2, 67.
Schluenzen, F. et al. (2001). Structural basis for the interac-tion of antibiotics with the peptidyl transferase center
in eubacteria. Nature 413, 814.
Vannuffel, P. and Cocito, C. (1996). Mechanisms of action
of streptogramins and macrolides. Drug-s 51, Suppl.
1. 20.
Zhanel, G. C. et al. (2002). The kelolides. Drugs 62, 1771.
105
Chsiiifpi’ ^’^iM
The antimicrobial agents described so far have beeo
arranged according to their primary effects on cellular
metabolism and biosynthesis. This approach accounts
for many of the most important dnigs in current an-timicrobial therapy. There are, however, several valu-able drugs whose various modes of action fall outside
those most commonly encountered. In this chapter we
describe important examples of compounds with un-usual actions. The drugs are arranged according to
their therapeutic targets antibacterial, antifungal, an-tiviral and antiprotozoal—although some of the anti-bacterial drugs also have useftil activity against certain
protozoal pathogens.
The discovery of the naturally occurring nitroarornatic
antibiotic chloramphenicol (Figure. 5.11) raised the
possibility that other organic nitro compounds would
have antimicrobial activity. The five compounds
shown in Figure 6.1 are perhaps the most useftil and
commonly used antimicrobial drugs among the many
nitro compounds that have been screened. Some other
nitro compounds have important applications in the
eradication of parasitic nematode worms, but these fall
outside the scope of this book.
Metronidazole is valuable in the treatment of in-fections caused by strictly anaerobic bacterial patho-gens such as Bacieroides fragilis and Clostridium dif-ficile, and against Helicobacter pylori, a bacterium
causally liniced to peptic ulcer disease and gastritis
which inhabits the stomach in an acid environment of
low oxygen tension. The spectrum of metronidazole
extends to several protozoal parasites, including Giar-dia lamblia, Entamoeba histolytica and Trichomonas
vaginalis. Nitrofurazone is a broad-spectrum antibac-terial agent, useful as a topical treatment for infected
bums and sidn grafts. Nitrofurantoin is sometimes de-scribed as a urinary antiseptic, owing to its value in
clearing infections of the urinary tract caused by
(jram-negative pathogens such as Pseudomonas aeru-ginosa, Serratia marscescens and Proteus rnirabilis.
Furazolidone also has antibacterial activity, but its
principal use is against certain protozoal infections, in-cluding Giardia lamblia. Although benznidazole is in-cluded in Figure 6.1, its only application is in the treat-ment of South American trypanosomiasis, or Chagas’
disease (see later discussion).
The nitroheterocyclic drugs are all subject to re-duction to bioactive molecules or radicals by ceUuiar
enzymes of the target pathogens. Their spectrum of ac-tion is mainly a ftmction of the redox potentials of the
consituent nitro groups. Nitrofurazone, nitrofurantoin
and furazolidone have relatively high redox potentials
of between -250 and -270 mV, whereas metronidazole
has a much lower potential of 480 mV. The first three
drugs can be reductively activated by a wide range of
enzymes such as the NAE)P(H)-dependent nitro reduc-tases. Owing to the much lower redox potential of
metronidazole, the drug can only be activated by the
pyruvate-ferredoxin oxido reductases and hydroge-107
Drugs with other modes of action
CHjCHjOH
Metronidazole
~N NH
“^ 0
Nitrofurantoin
0,N
*—’ o
Benznidazole  Nitrofurazone
Furazolidone
FIGURE 6.1 Nitrolieterocyclic drugs with activity against
anaerobic bacteria and some protozoan parasites.
liases of anaerobic bacteria and protozoa such as Tri-chomonas vaginalis. However, in all cases it is consid-ered that the nitro groups are reduced to a short-lived
nitro radical anion:
R”  -NOj <~> R- -NO ,
or other short-lived products, including chemically re-active hydroxylamine derivatives. The bioactive prod-ucts of the reductive process of metronidazole attack
DNA, causing single- and double-stranded breaks and
base mutations. Although the precise nature of the
damage to DNA is not clear, there is a preferential at-tack of the reactive metabolite on thymidine residues
and other pyiimidines. Bacteria with mutations that
adversely affect excision repair and DNA recombina-tion are more sensitive to metronidazole. Damage to
DNA is believed to be the cause of cell death and lysis
in bacteria and protozoal parasites. The situation ap-pears to be rather different with the three other nitro-heterocylic drugs. Their bioactive products do not
cause fragmentation of DNA, although they are
weakly mutagenic. It is possible that the reductive ac-tivation of these drugs generates short-lived intermedi-ates that target other essential biochemical processes,
although further research is needed to investigate this
possibility.
The model presented here accounts for the activ-ity of metronidazole against anaerobic bacteria. The
situation is less clear in the case of organisms which
survive under conditions of low, rather than zero, oxy-gen tension, such as Helicobacter pylori. One sug-gestion is that in the presence of oxygen, the nitro
radical anion is converted back to metronidazole and
superoxide:
R—NO •– ^ R” -NOj 4- O2′
The superoxide generated in this process is then con-verted by the enzyme superoxide dismutase to hydro-gen peroxide and molecular oxygen. In the presence of
a transition metal, either iron or copper, a series of re-actions, known collectively as the Haber-Weiss reac-tion, give rise to the highly reactive hydroxyl radical,
OH*, which also damages DNA, again resulting in cell
death. The ability of nitroheterocyclic drugs to damage
DNA increases the risk of harmful mutagenic effects in
the infected patient.
6.2 A unique antifungal antibiotic—
griseofulvin
Fungal infections of the skin and nails (‘ringworm’)
are commonly caused by various species of Trichophy-ton. Such infections usually respond well to topically
applied drugs of the azole class (Chapter 3). However,
where the infection is widespread, oral treatment with
azoles or terbinafme may be necessary. In cases where
the fungus is both widespread and resistant to these
drags, the physician may turn to griseofulvin (Figure
6.2). This is an antibiotic produced by Penicillium
griseofulvum which causes the tips of fungal hyphae to
become curled and suppresses further growth. At the
molecular level, it binds to the intracellular protein
tubulin and possibly to the accessory proteins involved
in the polymerization of tubulin to form microtubules
(the microtubule-associated proteins, MAPs). Micro-tubules participate in the movements of subcellular
organelles and in the separation of daughter chromo-somes during mitosis in eukaryotic cells. A character-istic property of microtubules is their rapid assembly
and disassembly which is due to the reversible poly-merization of the constituent tubulin. This dynamic in-108
6.3 Antiwiral agents
H3C—0
Griseofulvin
FIGURE 6.2 Griseofulvin, a unique antifungal antibiotic
that disiTipts the function of microtubules.
stability, as it is called, is crucial to the mitotic process.
The binding of griseofulvin to tubulin and the MAPs in
some way hinders the assembly and disassembly of the
microtubules and thereby disrupts cell proliferation.
The selectivity of griseofulvin for dermatophytes
is not fully understood since the antibiotic also binds to
mammalian tubulin. Nevertheless, the many differ-ences in the amino acid sequences of, for example, the
tubulin of the yeast Saccharomyces pombe and that of
mammalian brain tubulin may permit differential bind-ing of griseofulvin to the tubulin of fungal cells under
the conditions of antifungal therapy.
Griseofulvin has recently been shown to dismpt
the function of tubulin in mitosis in the protozoan par-asite Trichomonas vaginalis. In view of concern about
the potential mutagenicity of metronidazole, griseoful-vin might provide an alternative treatment for infec-tions caused by this organism.
6 “ft ffi^ i ^ B’i%i^i i^«Bi i ss ^% i s 8^ ffl*^ B’ijf Sr\vi a Qii w i n %J& i C9 %r« QB? 5 5 %^&
6.3.1 Inhibitors of tlie protease of the human
immunodeficiency virus
In Chapter 4 we reviewed inhibitors of HIV reverse
transcriptase. While these drugs have provided a major
advance in the treatment of AIDS, their long-term ef-ficacy in monotherapy is always threatened by the re-markable ability of HIV to generate drug-resistant mu-tants. To combat this tendency, inhibitors of reverse
transcriptase are usually given in combination with
other inhibitors of HIV replication. Outstanding
among the latter agents is an expanding group of com-pounds that inhibit the HIV-specific protease.
The viral translation products fonxied during the
replicative phase of the virus are long polypeptide
precursors which are then specifically cleaved to re-lease several mature proteins. In this way the virus gen-erates a number of distinct proteins from a single
niRNA molecule. The enzyme responsible for the
cleavage process, HIV protease, is itself first formed as
a zymogen precursor protein. HIV protease, encoded by
the viral po! gene, belongs to the aspartyl class of pro-teases. Enzymes of this mechanistic type are maximally
active in the pH range 4.5-6.5 but nevertheless retain
significant activity at the cytoplasmic, neutral pH.
As the nature of HIV and its replicative cycle
were revealed during the 1980s, the importance of HIV
protease as a potential target for antiviral activity be-came apparent. Considerable attention had already
been given to the design of inhibitors of another as-partyl protease, renin, and the peptidic inhibitors of
this enzyme provided a good starting point for the de-velopment of anti-HIV protease compounds. However,
like many of the renin inhibitors, the early HIV pro-tease inhibitors were flawed by extremely low solubil-ity, which resulted in poor uptake and tissue distribu-tion (‘bioavailability’) after administration. Then in
1989 X-ray crystallography revealed the three-dimen-sional structure of HIV protease, which greatly facili-tated the design of nonpeptidic inhibitors. Like the
peptides, the nonpeptidic compounds are also compet-itive inhibitors of the natural substrate. Unlike the ear-lier peptidic inhibitors, the best of the nonpeptidic
compounds have acceptable solubility and bioavail-ability. As a result, a range of HIV protease inhibitors
is now in clinical use and new compounds continue to
be developed. The four examples given in Figure 6.3
are potent selective inhibitors that produce rapid de-creases in the number of viruses in the blood and, sig-nificantly, an increase in the circulating CD4 h’mpho-cytes that are so critical to effective immune defence
against microbial infections.
HIV protease inhibitors are now an established
component of the HAART regime (see Chapter 4) in
combination with at least two different inhibitors of
HIV reverse transcriptase. This therapy can reduce the
circulating viral RNA in 90% of patients to levels that
are not detectable by the polymerase chain reaction for
109
Drugs with other modes of action
Ritonavir
CONH+but
Indinavir
C(CH3)3
NH
O
Saquinavir Lopinavir
FIGURE 6.3 Inhibitors of HIV protease used in ttie tlierapy of AIDS in combination witii otlier antiretrovirai drugs.
up to one year. Ominously, however, replication of the
viras may persist in lymph nodes despite sustained
drug therapy.
None of the cuiTently available anti-HIV drags
are free of side effects, and these can make it difficult
for patients to maintain the strict regime of drug taking
that is necessary to sustain the effectiveness of
HA ART. An unexpected side effect of the protease in-hibitors is the suppression of insulin secretion from the
pancreatic islet cells in response to a rise in circulating
glucose levels. This leads to insulin resistance, hyper-lipidemia, and in an unfortunate minority of patients,
type n diabetes. The mechanism of this serious prob-lem is being vigorously pursued with a view to design-ing new inhibitors of HIV protease that are free from
effects on the insulin response.
6.3.2 Inhibition of HIV entry into host cells
Because of the remarkable ability of HIV to acquire re-sistance to virtually any drug it is confronted with, the
search for inhibitors of other key stages in the life
cycle of the viras continues. HIV gains entry to cells in
a three-step process: (a) The viral envelope protein
Env binds to the CD4 glycoprotein receptor on the sur-face of CD4+ T cells, macrophages, dendritic cells and
monocytes, (b) The binding of the homotrimeric Env
to CD4 induces a confomiational change in the Env
subunit gpl20 that allows concomitant binding to ei-ther of the cell surface chemokine receptors, CCR5
and CCR4. (c) Coreceptor binding triggers exposure of
a hydrophobic fusion peptide at the N-terminal of the
Env subunit gp41, which then inserts into the mem-brane of the host cell, causing fusion of the viral mem-brane with the host membrane. This membrane fusion
process ensures the release of the viral core protein and
the RNA strands into the cytoplasm. Both the binding
steps and the membrane fusion process are currently
being targeted in dmg design programs and several
compounds are under clinical evalution in HIV-in-fected patients, with promising results. The fusion in-hibitor enfuvirtide (T20, Fuzeon), which is a synthetic
peptide with the sequence Ac-YTSLIHSLIEESQN
110
6.3 Antiwiral agents
QQEKNEQELLELDKWASL-NH2, binds to a region
of gp41 which is exposed transiently after Eriv binds to
CD4 and thereby disrupts the ensuing steps in the fu-sion-viral entry process.
6.3.3 Antiinfluenza drugs
Influenza is potentially a very dangerous infection; the
great influenza pandemic of 1918-1919 is said to have
Icilied some 21 million people worldwide. While the
subsequent pandemics have generally not approached
the severity of the 1918—1919 outbreak, there is con-tinuing concern that even relatively mild, localized
epidemics can cause considerable social and economic
dislocation and significant mortality. The main de-fense against influenza is annual immunization with
inactivated, killed virus vaccines, constantly modified
to take account of the propensity of the influenza vims
for mutation and recombination. Despite the reason-able success of the vaccination programs, there is a
general recognition that effective drugs are also
needed, especially in view of the continuing threat of
the sudden emergence of a major pandemic caused by
a highly virulent mutant.
Amantadine and rimantadine
These closely related compounds (Figure 6.4) have
both prophylactic and therapeutic activity against in-fluenza AT infection, although they are ineffective
against the less common type B virus. They prevent
influenza Aj infection as long as dosing is continued,
Amantadine
FIGURE 6,4 Two drugs active against influenza type A
viruses with a unique action against the ion channel function
of the viral protein M,.
and the duration of the disease appears to be shortened
even if the drugs are started after the infection has
begun. In vitro, the compounds inhibit replication of
the influenza virus after the virus has entered the host
cell but before the process of viral uncoating begins.
The mature influenza virus particle is enveloped in a
lipid membrane that contains three integral proteins:
hemagglutinin, neuraminidase and a protein desig-nated as M,. This latter protein, which is a homo-oligomer, has been identified as the specific molecular
target for amantadine. During a viral attack on suscep-tible cells, the virus particles, or virions, enter the cells
by endocytosis and are then incoiporated into the en-dosomal compartment. At this stage a tetramer of the
Mj protein functions as a highly selective proton
channel across the virion membrane. A specific histi-dine residue in the M^ protein (His-37) is essential for
proton selectivity. A proton flux from the endosome
through the channel into the virion interior ensures the
reduction in internal pH that is recjuired for the un-coating of the virion. The ion channel activity of M-,
has been confirmed with recombinant M-, protein in-serted into the membranes of Xenopus oocytes. The
antiviral action of amantadine and rimantadine results
from a specific interaction between the drugs and the
M-, protein which abrogates its ion channel function.
Amantadine and rimantadine can be thought of as hy-drophobically stabilized surrogates for H”” ions that
compete for proton binding at the lone electron pairs
oflTis-37.
The usefulness of amantadine and rimantadine as
antiinfluenza drugs is limited by their side effects, es-pecially those affecting the central nervous system. In-deed, the primary medical use of amantadine is as an
adjunct in the treatment of Paiiinson’s disease.
Inliibitors of viral neuraminidase in the
treatment of influenza
Amantadine and rimantadine have never achieved
widespread use for the treatment and prevention of in-fluenza. An alternative approach has developed in-hibitors of viral neuraminidase which have the advan-tage of being effective against both influenza type A
and type B vimses. The two neuraminidase inhibitors
cuiTently available, zanamivir and oseltamivir (Figure
6.5), are most effective when taken as soon as possible
111
Drugs with other modes of action
Zanamivir  Oseltamlvir
O.^ ^OH
u p NH OH
o
DANA: 2,3,-dehydro-2-deoxy-A/-acetylneuraminic acid
FIGURE 6.5 Inhibitors of viral neu-raminidase witli activity in early-stage
infections caused by influenza type A
and type B viruses. DANA is a transition
state intermediate in the catalytic action
of the enzyme used in the design of
inhibitors.
after the symptoms of ‘flu first appear. The subsequent
duration of the infection is significantly shortened and
the risk of serious complications, such as bronchitis, is
reduced.
The attachment of the influenza virases to the
surface of target cells and the subsequent release of
progeny viruses is mediated successively by two gly-coprotein spikes on the viral coat: hemagglutinin and
neuraminidase. Hemagglutinin binds to the sialic acid
moieties of surface receptors on the target cells and ini-tiates viral adsorption and penetration. After replica-tion inside the cells, progeny virions budding from the
cell membrane bind via their hemagglutinin spikes to
the cell surface receptors and to other viras particles.
The viral neuraminidase now cleaves the terminal
sialic residues from the receptors and releases the
newly formed virases from the cell membranes and
from each other. Neuraminidase may also facilitate the
migration of viruses through the mucin layer which
covers the respiratory tract.
The elucidation of the three-dimensional struc-ture of neuraminidase and its interaction with the cell
surface receptors during the 1990s paved the way for
the design of inhibitors of the enzyme. The structure of
the first inhibitor, zanamivir, is analogous to that of a
transition state analogue believed to be involved in the
catalytic process, 2,3-dehydro-2-deoxy-iV-acetylneu-raminic acid (DANA, Figure 6.5). Zanamivir binds to
amino acids (glutamic acid-119 and arginine-292) in
the active center of the enzyme which are conserved in
both type A and type B viruses. The cleavage of the
terminal sialic acid residues of the cell receptors is in-hibited, suppressing the release of progeny virions and
their spread to other cells.
Zanamivir must be administered by inhalation
into the respiratory tract. The stmcturally similar os-eltamlvir has the advantage of being effective if admin-istered orally as an ethyl ester prodrug which is con-verted to the active inhibitor after absorption by liver
esterases.
6.3.4 Interferon
Originally discovered in 1957 as a naturally occur-ring antiviral agent, interferon is now used as the
generic name for a family of proteins involved in host
defences against certain viral and parasitic protozoal
infections, hiterferons (IFNs) also affect the immune
system, cell proliferation and differentiation and thus
have a useful, although limited, antitumour activity.
The therapeutic opportunities for the interferons
112
6,4 Antiprotozoal agents
have expanded considerably with the advent of tech-nology to provide substantial quantities of recombi-nant proteins.
The nomenclature for IFNs, based on amino acid
sequence data, defines four groups: IFN~a and IFN-O)
(previously IFN-a-1 and lFN-a~2), IFN-p and IFN-y.
Only IFN-{X is used therapeutically as an antiviral drug
and it is valuable in the treatment of hepatitis B and C.
IFN-{X is also useful against hairy cell leukemia and
AlDS-related Kaposi’s sarcoma, actions that may de-pend upon its antiproliferative rather than its antiviral
property. IFN-a itself constitutes a family of related
proteins encoded by at least 14 functional genes.
The antiviral activity of IFN-a is mediated
through a complex cascade of events, beginning with
the binding of the protein to its specific receptor em-bedded in the cytoplasmic membrane of the cell. The
IFN-a receptor consists of at least two subunits, IFN-AR1 and IFN-AR2. A current model of IFN-a recep-tor function proposes that the Rl and R2 subunits are
associated, respectively, with two distinct protein tyro-sine kinases, Tyk2 and JAKl (a ‘Janus’ kinase). The
binding of IFN-a to the receptor results in the activa-tion of these kinases which then phosphorylate the
gene transcription factors STATl and STAT2 on spe-cific tyrosine residues. The activated STAT molecules
form heterodimers that translocate to the nucleus in as-sociation with an additional factor, IFN regulatory fac-tor. The resulting complex, termed IFN-stimulated
gene factor-3, then interacts with the IFN-stimulated
response element to modulate the transcription of
more than 300 genes. To add to the complexity, there
is also evidence for the involvement of additional tran-scriptional factors, STAT3 and STATS.
Although many proteins may contribute to the an-tiviral action of IFN-a, which includes suppression of
penetration, uncoating, transcription, translation and
virus assembly, attention has largely focused on the
roles of three key proteins induced by the signal trans-duction process:
1. 2′,5′-oligoadenylate synthetase, otherwise
known as (2′-5′)(Aj^) synthetase, which is ac-tive only in the presence of double-stranded
RNA (dsRNA, an intermediate or byproduct
of viral replication),
2. RNAase L, and
3. dsRNA-dependent protein serine kinase.
The (2′-5′)(A^|) synthetase exists in several isoenzymic
forms and catalyzes the conversion of ATP to a series
of AMP oligomers linked by 2′-5′ rather than the usual
3′-5′ phosphodiester bonds. The 2′-5′ A oligomers (up
to 15 units in length) then activate the latent fomi of
RNAase L. The active form of this endonuclease
hydrolyzes both niRNAs and rRNAs at sequences con-taining UU and UA. The destruction of RNA mole-cules contributes to both the antivkal and antiprolifer-ative actions of IFN-a.
As mentioned earlier, IFN-a is useful in the treat-ment of hepatitis C, usually in combination with rib-avirin (Chapter 4). Recent evidence indicates that the
hepatitis C virus interferes with the lAK-STAT signal-ing sequence induced by IFN-a, which may depress
the effectiveness of the drug through reduced tran-scription of the IFN-a-stimulated genes.
6 £j, JS i ^ w i i ^ S*tf^ H fi^’!P* d l rf^ i Sft C? Oi i ^ w ^
6.4.1 Antimalarial drugs
Quinolines
The statistics for malaria are appalling: some 2.5 bil-lion people are at risk from the disease with 300-500
million new cases every year, of whom as many as 2
million will die. The first effective antimalarial drug
was quinine (Figure 6.6), which is present in the bark
of the cinchona tree of South America. Later chemi-cally related but wholly synthetic drugs include
chloroquine (Figure 4.14), mepacrine and mefloquine
(Figure 6.6). Drugs that intefere with the folic acid
metabolism of malarial parasites are described in
Chapter 4.
Of the ciuinoline drugs, chloroquine has been a
mainstay of both the prophylaxis and treatment of
malaria for more than 60 years. Unfortunately, resist-ance to this drag is now widespread (Chapter 9). Early
studies suggested that the antimalarial action of
chloroquine might depend upon its ability to bind to
DNA by intercalation (Chapter 4), leading to inhibi-tion of DNA replication and transcription. However,
although it is still possible that the intercalative prop-erty of chloroquine contributes to its antimalarial
113
Drugs with other modes of action
H3C-.HCI
Mepacrlne
N”””””–””’^CH,
Halofantrine
FIGURE 6.6 Four well-established antimalarial drags.
action, the principal effect of the drug is to disrupt the
ability of the parasite to cope with heme released dur-ing the metabolism of hemoglobin.
At a certain stage in the complex life cycle of
Plasmodium parasites, the protozoans invade the red
cells of the host. The trophozoites, as the parasitic cells
are called at this stage, digest more than 80% of the he-moglobin of the infected red cells in lysosomal vac-uoles to obtain amino acids for their own development.
The digestive process releases the cytolytic porphyrin
heme within the parasitic cell. The trophozoites pro-tect themselves against the toxic effect of heme in two
ways. One way is by facilitating polymerization of
heme into an inert, insoluble crystalline substance
called hemozoin, or p-hematin. In this molecule the
iron atom of one molecule of heme is coordinated to
the propionate carboxyl residue of the next heme. The
resulting dimers form chains linked by hydrogen
bonds. There is still debate as to whether this polymer-ization and crystallization process is mediated by an
enzyme or protein of the trophozoites or whether it is
a spontaneous reaction dependent solely on the chem-ical nature of heme. In the second method, a signifi-cant proportion of the heme released by the digestion
of hemoglobin escapes incorporation into hemozoin.
The residual heme is oxidatively degraded by hydro-gen peroxide foimed as a by-product of the oxidation
of Fe^”*” to Fe^^ immediately following its release from
hemoglobin. Another fraction of heme is degraded by
interaction with glutathione catalyzed by glutathione
reductase.
The positive charge on the chloroquine molecule
is probably responsible for its accumulation within the
acidic environment of the food vacuoles of the parasite
to concentrations as high as 100 pM. There are several
histidine-rich proteins (HRPs) in the digestive vac-uoles of Plasmodium falciparum that promote the
polymerization of heme. One of these proteins, HRP-II, has 51 His-Hls-Ala repeats that may provide the
binding sites for the 17 molecules of heme that bind to
1 molecule of HRP-II. Chloroquine inhibits the HRP-Il~mediated formation of hemozoin. However, against
114
6,4 Antiprotozoal agents
this must be set the finding by other investigators that
chloroquine also inhibits hemozoin formation in the
absence of any added proteins. Whether hemozoin
synthesis is protein mediated or is a spontaneous phe-nomenon within the trophozoites, it seems hkeiy that
the inhibitory effect of chloroquine on the process de-pends on an interaction between the drug and the heme
molecule or minimal heme oligomers.
Chloroquine also competitively inhibits the de-gradative processes that eliminate heme not captured
by hemozoin formation. The drug may fomi a complex
with heme that blocks peroxidation by hydrogen per-oxide and its reaction with glutathione. Toxic chloro-quine-heme complexes accumulate and contribute to
the death of the parasitic cells.
Despite the chemical similarity of the quinoline
drags shown in Figure 6.6, there are differences in
their modes of antimalarial action. While chloroquine,
mepacrine and mefloquine all inhibit hemozoin forma-tion, the inhibitory actions of mefloquine and quinine
on the ability of the protozoan to accumulate hemoglo-bin in its digestive vacuoles may also contribute to the
antimalarial action of these drugs. After many years of
effort, the final details of the antimalarial actions of the
quinoline drugs still remain to be defined. As we have
seen, the drugs inhibit several aspects of trophozoite
function which would contribute to a synergistic attack
on cell survival.
Although halofantrine (Figure 6.6) is not a mem-ber of the quinoline group, its prinicipal mode of anti-malaiial action is probably broadly similar to that of
the quinolines, i.e. it blocks the elimination of the cy-tolytic heme. There is in vitro evidence that halo-fantrine inhibits the glutathione-mediated degradation
of heme. Halofantrine has potentially dangerous side
effects on cardiac function which may be associated
with drug-mediated inactivation of the essential potas-sium channels in the cytoplasmic membrane of my-ocardial cells. It is conceivable that interference with
ion channel function, such as the proton pump of the
protozoan food vacuoles, could also contribute to the
antimalarial action of halofantrine.
Artemisinin
An infusion of the leaves of the plant Artemisia annua
is an ancient Chinese herbal remedy for fevers, includ-ing malaria. The active principle, aitemisinin (Figure
6.7), is highly active against the malarial parasite.
Artemisinin and its more convenient water-soluble de-rivative, artesunate (Figure 6.7), are proving to be use-ful new weapons in the fight against malaria, particu-larly against the chloroquine-resistant and cerebral
forms of the disease. These drugs rapidly kill all the
asexual stages of the most dangerous malarial parasite,
Plasmodium falciparum. Artemisinins are usually
given in combination with inhibitors of folic acid me-tabolism or with mefloquine.
Artemisinin and its close structural analogues dif-fuse readily into red cells infected by the paiitsite. In-fected red cells are found to contain up to 100 times
more artemisinin than uninfected cells. The endoper-oxide bridge of the drug molecules then reacts with the
ferrous iron atom of heme released from digested he-moglobin. The chemistry of the interaction of artem-isinin with heme and the susbequent generation of re-active carbon-centered free radicals is complex, and
further details can be found in a reference in ‘Further
reading.’ Flowever, the essential role of the endoperox-ide bridge is evidenced by the fact that derivatives
lacking this feature are inactive. Molecular modeling
studies suggest that the endoperoxide bridge can
achieve close proximity to the heme iron. The interac-tion with the Fe-^”^ atom catalyzes the breakdown of
artemisinin into a complex cascade of unstable inter-mediates, the details of which are still debated. The
balance of evidence suggests that a carbon-centered
H,C
FIGURE 6.7 Antimalarial agents derived from the tradi-tional Chinese herbal medicine Artemisia annua, usually
given in combination with other antimalarial drugs.
115
Drugs with other modes of action
radical is generated which alklylates essential proteins
and leads to ttie death of the parasite. However, it is
also possible that active oxygen species are generated
during the breakdown of artemisinin, which would
cause peroxidation of lipids in the vacuolar membrane
of the parasite and subsequent dissolution of the
vacuole.
Recently a more specific explanation has been
advanced for the antimalarial action of the artemisin-ins. The carbon-centered free radical generated by the
interaction of artemisinin with heme is revealed as a
potent inhibitor of the Ca^”^-dependent ATPase
(SERCA) of the sarcoplasmic and endoplasmic reticu-lum of the protozoan cells. This enzyme, referred to as
PfATP6, is not inhibited by quinine or chloroquine. A
direct link between the enzyme inhibition of Pf ATP6
and cell death has not been established, although there
is a correlation between the inhibitory potency of a
range of artemisinin derivatives and their ability to kill
the parasite. The alkylation and enzyme inhibition of
Pf ATP6 may well synergize with the effects of the free
radical on other, as yet undefined, proteins to achieve
the lethal action of artemisinins. The malarial parasite
develops resistance to artemisinin-type drags with
considerable difficulty, which further suggests that the
antimalarial action of these drags is multifactorial.
Atovaquone
Although hydroxyanthraquinones have been known
for 60 years or more to inhibit the respiration of malar-ial parasites, the introduction of the hydroxy-napthaquinone derivative atovaquone (Figure 6.8) for
the treatment of malaria is relatively recent, hi combi-nation with an inhibitor of dihydrofolate reductase,
such as proguanil (Figure 4.3), atovaquone is highly
effective in the prevention of malaria caused by Plas-modium falciparum. The combination is largely free of
the side effects that can make compliance with other
antimalarial drag regimes difficult. Atovaquone is also
used to treat another protozoal infection caused by
Toxoplasma gondii and the dangerous respiratory fun-gal pathogen Pneumocystis carinii.
The primary inhibitory action of atovaquone is
against the respiratory chain in mitochondria, leading
to an interraption in the supply of ATP. The target site
is the ubiquinone-cytochrome b and c reductase re-gion of the respiratory chain, i.e. the cytochrome fee,
complex. The complex catalyzes electron transfer
from ubiquinone to cytochrome c and at the same time
translocates protons across the mitochondrial mem-brane. The complex contains prosthetic groups, cy-tochrome b, cytochrome c, and a nonheme iron-sulfur
protein. A partial structural similarity between ato-vaquone and ubiquinone suggests that there is compe-tition between the two molecules at the cytochrome
dc, complex. Because of the technical problems asso-ciated with culturing malarial parasites in vitro, it is
difficult to isolate mitochondria from the parasites in
sufficient amounts to investigate the interaction be-tween atovaquone and its target site. Fortunately,
there is a high degree of amino acid sequence similar-ity between the cytochrome b of Plasmodium falci-parum and that of the yeast Saccharomyces cere-visiae. The cytochrome bc^ complex is readily
purified from the yeast and the details of its crystal
structure are known.
Structural modeling with the yeast preparation re-vealed that atovaquone potentially competes for the
occupation of the ubiquinol oxidation pocket, of the
bc^ complex. The soluble domain of the iron-sulfur
protein is proximal to the oxidation pocket, and recent
molecular modeling studies based on the crystal struc-ture of the ftc, complex, together with biochemical
spectroscopic experiments, point to hydrogen bonding
between the hydroxyl group of the drug and histidine-181 of the iron-sulfur protein. The quinone carbonyl
group of the drug is seen to hydrogen bond with gluta-mate-272 of the cytochrome b component when a
bound water molecule is introduced into the model to
form a bridge between the quinone carbonyl group and
glutamate-272.
The model of the interaction of the atovaquone-cytochrome fee, complex provides some insight into
the potential for the selective inhibition of mitochon-drial function in the microbial parasites. The ef loop of
cytochrome b contains amino acid residues in close
contact with the atovaquone-binding pocket. Position
275 in the loop is occupied by leucine in Saccha-romyces cerevisiae and in Pneumocycstis carinii, but
is replaced by phenylalanine in the human protein.
Modeling of this replacement indicates that pheny-lalanine causes significant steric hindrance to the
binding of atovaquone, which could account for the
116
6,4 Antiprotozoal agents
H3C O
(CHjCHCCHj)^
CH,
I ‘
CCH,)_H
FIGURE 6.8 Alovaquone, an inhibitor of protozoan niilochondrial function recently
introduced into tlie propliyiaxis and therapy of malaria in combination witli inhibitors
of folic acid metabolism. Ubiquinone is shown here to illustrate its partial structural re-semblance to atovaquone.
markedly lower potency of the dmg against mam-malian mitochondria.
6.4.2 Antitrypanosomal drugs
There are two major forms of trypanosomiasis:
African sleeping sickness, caused by two species of
Trypanosoma brucei, and Chagas’ disease or South
American trypanosomiasis, caused by Trypanosoma
cruzi. African sleeping sickness has become resurgent
in sub-Saliaran Africa, causing an estimated 100,000
deaths each year. Chagas’ disease affects some 18 niil-Uon people in South America, with about 25% of the
population at risk of acquiring the disease. Unfortu-nately there are few drugs for the treatment of either
form of trypanosomiasis and most of them were devel-oped decades ago. Research into the chemotherapy of
trypanosomiasis remains relatively neglected. The ac-tion of another antitr>’panosomal drug.” ethidium, used
in veterinaiy medicine, is discussed in Chapter 4.
Suramin
First made available over 70 years ago, suramin (Fig-ure 1.2) is a large, polysulfonated molecule with six
negative charges at physiological pH. It is often the
drug of choice for the treatment of the early stages of
African sleeping sickness. In view of its molecular size
and highly charged nature, it is surprising that suramin
is able to gain access to the cytoplasm of the parasitic
cells. In fact, the compound binds avidly to serum pro-teins and it is believed that both free proteins and those
complexed with suramin enter trypanosomes by endo-cytosis. The selective toxicity of suramin for try-panosomes may in part be due to a more avid uptake of
serum proteins by trypanosomes compared with the
cells of the infected patient. The concentration of
suramin in trypanosomes can reach 100 jiM.
Smimiin binds to and inhibits a broad range of en-zymes derived from Trypanosoma brucei, including
dihydrofolate reductase, thymidine kinase and all the
enzymes of the glycolytic pathway. Although the IC5,,
values (the concentration of inhibitor needed to inhibit
an enzyme by 50%) are in the high range of KVlOO
\iM, they are much lower than those for the con-espon-ding enzymes from mammalian sources. The gly-colytic enzymes of the trypanosome, which generate
all of its ATP, are confined to membrane-bounded or-ganelles called glycosomes. Although the physical
characteristics of suramin make it unlikely to diffuse
into glycosomes, the drug probably binds to the newly
synthesized proteins during their cytoplasmic phase
prior to entry into the glycosomes. The normal
turnover of uncomplexed enzymes in the glycosomes
would be expected to lead to their gradual replacement
by suramin-bound enzymes entering from the cyto-plasm. This model is consistent with the observed pro-gressive slowing down of energy metabolism in cells
treated with suramin. The antitrypanosomal action of
suramin is unlikely to depend on the inhibition of a sin-gle enzyme, a conclusion that is supported by the fact
that resistance to suramin has not been a serious prob-lem despite many decades of use. A multifaceted mode
of action probably hinders the ability of trypanosomes
to develop resistance to the drug.
117
Drugs with other modes of action
Pentamidine
This diamidine compound (Figure 6.9), like suramin,
is useful in treating the early stages of African try-panosomiasis. By exploiting a protozoal aminopuiine
transport system, pentamidine is concentrated within
the trypanosomes. Despite being relatively nonspecific
in its interaction with macromolecules, pentamidine
probably exerts its primary antitrypanosomal action by
attacking and selectively cleaving the DNA of the
kinetoplast.
IVIelarsoprol
Otherwise known as melarsen oxide, melarsoprol
(Figure 6.9) is a trivalent organic arsenical drug that
has been used to treat sleeping sickness since 1949.
Unlike suramin and pentamidine, it is useful against
late-stage disease, although treatment is fraught with
the risk of serious side effects, especially a potentially
lethal encephalopathy. Melarsoprol also enters the try-panosome via an aminopurine transporter.
Although African trypanosomes incubated with
melarsoprol die within minutes, the mechanism of ac-tion is not clear and may have more than one aspect to
it. Several glycolytic enzymes are inhibited by melar-soprol, including phosphofructokinase (K- < I pM),
fractose-2,6-diphosphatase (i.e. Kj = 2 (iM) and, to a
lesser extent, pyruvate kinase (K- > 100 tiM). Melarso-prol also disrupts the function of a unique trypanoso-mal biochemical, tiypanothione [N\N^-bis{gla-tathionyl)-spemiidine], with which the drag forms a
stable adduct. Trj’panothione is a major cofactor in the
control of the redox balance between thiols and disul-fides in trypanosomes. The adduct with melarsoprol
inhibits trypanothione reductase (K- = 17.2 |iM),
which is a key enzyme regulating the redox state of
trypanothione itself. However, the relatively high Kj
casts some doubt on the relevance of the inhibition of
trypanothione reductase to the antitrypanosomal ac-tion of melarsoprol. Furtheimore, incubation of Try-panosoma brucei cells with the drug leads to only a
minor conversion of reduced trypanothione to its
adduct with melarsoprol.
Melarsoprol is typical of organic arsenical agents
in its ability to fonn adducts with thiols and may there-fore inhibit many enzymes with essential thiol groups
or that require thiol-containing cofactors. Thus al-though the major effect of melai’soprol is probably to
suppress glycolysis in African trypanosomes, the inhi-bition of other enzymes may well contribute to the
overall antitrypanosomal action. The toxicity of melar-soprol to the patient is also almost certainly due to the
avidity of the drag for thiols.
HjN^^ N  /
^ /N . ^N—( \ /) As
-CHjOH
CHF,
I ‘
H,—C-C H2NCH2CH2CH2—C-COOH
Melarsoprol  Eflornlthine
Pentamidine
FIGURE 6.9 Drugs used to treat African trypanosomiasis.
118
6,4 Antiprotozoal agents
Eflornithine
DL-a-difluoromethylornithine (DFMO) or eflornithine
(Figure 6.9) was origiiiariy designed as a ‘suicide’ in-hibitor of ornithine decarboxylase (ODC) for use as an
antitumour drug. ODC is involved in the biosynthesis
of the poiyamines putrescine, spermidine and sper-mine, vi/hich are essential for cell division in eukary-otic cells. Depletion of cellulai- poiyamines caused by
the inhibition of ODC results in the suppression of mi-tosis and cell proliferation. In addition to its potential
as an anticancer agent, eflornithine has also proved to
be an effective dmg against African tr3’panosoniiasis.
Flowever, adverse side effects, which are similar to
those encountered with other cytotoxic drugs, are com-mon during antitrypanosomal therapy with eflor-nithine. Fortunately these effects are reversible after
treatment ceases. When given in high doses for 14
days, eflornithine is effective against both early- and
late-stage disease.
The basis of the trypanosome-selective action of
eflornithine hinges on marked differences in the
turnover rates of ODC in mammals and the try-panosome. Eflornithine is an irreversible inhibitor of
mouse ODC, forming a covalent adduct with cysteine-360. Since human ODC shares a 99% sequence iden-tity with the mouse enzyme, it can be reasonably as-sumed that the human enzyme is also inhibited by
eflornithine in the same way. The K- against mouse
ODC is 39 |.lM, compared with 220 |i,M against the
coiTesponding enzyme from Trypanosoma briicei.
This might lead one to expect that the drug would be
more effective against mouse and mammalian cells in
general than against trypanosomes. Flowever, while
the trypanosomal enzyme is highly stable with mini-mal intraceriular turnover, mammalian ODC has a
half-life of only 20 min, placing it among the most rap-idly metabolized of eulcaryotic proteins. This, com-bined with the rapid elimination of eflornithine from
the body, results in a single dose of the drug exerting
only transient inhibition of the constantly renewed
ODC of the host. By contrast, there is sustained inhibi-tion of the trypanosomal ODC, resulting in depletion
of putrescine and spermidine as well as of trypanothi-one (recall that the latter is a conjugate of glutathione
with spermidine). The drug-treated trypanosomes
cease dividing and become incapable of changing their
variant surface glycoprotein (VSG). Normally, contin-ual changes in VSG provide the basis of the remark-able ability of trypanosomes to evade immunological
detection and destruction by the host. When changes in
the VSG are prevented, the paiitsite becomes vulnera-ble to immunological attack, which assists in the reso-lution of the infection.
The antitrypanosomal dnigs described here are
effective only against the African forms of trypanoso-miasis. There are even fewer drugs effective against
Chagas’ disease. The preferred approach is prevention
of the disease by controlling the insect vector for Try-panosoma cruzi, the reduviid bug, which infests
poorly constructed housing. Nevertheless, several
drugs ai^e being tested against Chagas’ disease, the best
of which is the nitroheterocyclic compound benznida-zole (Figure 6.1). The action of this drug is similar to
that of the compounds discussed in Section 6.1, i.e. it
is reductively activated in the protozoan cell to gener-ate a free radical which attacks DNA and other macro-molecules in the cells. However, full details of the ac-tion of benznidazole in Trypanosoma cruzi have not
been defined. Treatment of Chagas’ disease with ben-znidazole and other nitroheterocyclic drugs is far from
satisfactory, owing to the risk of DNA damage to the
patient during the sustained doses needed to eliminate
the parasite.
Fiil”tllPI ° f^^flillf l %aB9>BB%^B B «^9 3 %a B a a %1
Chaudhuri, A. R. and Ludefia, R. F. (1996). Griseofulvin: a
novel interaction with hrain tubulin. Biochem. Pharma-col 51, 903.
Cohen, J. L. (1996). Protease inhibitors: a tale of two com-panies. Science 272, 1882.
Dachs, G. v., Abatt, V. R. and Woods, D. R. (1995). Mode of
action of metronidazole and a Bacteroides fragilis
inetA resistance gene in Escherichia coli. J. Antimi-crob. Chemother. 35, 483.
Duong, F. H. (2004). Hepatitis C virus inhibits interferon sig-naling through up-regulation of protein phosphatase
2A. Gastroenterology 126, 263.
Egan, T. J., Ross, D. C. and Adams, R A. (1994). Quinoliue
antimahirial drugs inhibit spontaneous formation of P-hematin (malaria pigment). FEBS Lett. 352, 54.
Ekstein-Ludwig, U. et al. (2003). Artemisinins target
SERCA ol Plasmodium falciparum. Natwe 424, 957.
119
Drugs with other modes of action
Jefford, C. W. (2001). Why artemisinin and certain synthetic McKimm-Breschkin, J. L. (2002). Neuraminidase inhibitors
peroxides are potent antinialariais: implications for the for the treatment and prevention of influenza. Expert.
mode of action. Curr. Med. Chem. 8, 1803. Opin. Pharmacother. 3, 103.
KerrJ.M.ela/. (2003). Of JAKs,STATs, blind watchmaker, Menendez-Arias, L. (2002). Targeting HIV: antiretroviral
jeeps and trains. F£iJS Leff. 546, 1. therapy and development of drug resistance. Trends
Kessl, J. L. et ai. (2003). Molecular basis for atovaquone Pharmacol. Sci. 23, 381.
binding to the cytochrome bc^ complex. /. Biol. Chem. Raether, W. and Hanel, H. (2003). Nitroheterocyclic drugs
278, 31312. with broad spectrum activity. Parasitol Res. 90, S19.
Lear, J. D. (2003). Proton conduction through the M2 protein Sanchez, C. P. and Lanzer, M. (2000). Changing ideas on
of the influenza A virus: a quantitative, mechanistic chloroquine in Plasmodium falciparum. Curr. Opin.
analysis of experimental data. FEES Lett. 552, 17. infect. Dis. 13, 653.
Matthews, T. et ai. (2004). Enfuvirtide: the first therapy to Urbina J. A. (2002). Chemotherapy of Chagas’ disease.
inhibit the entry of HIV-1 into host CD4 lymphocytes. Cun: Pharm. Des. 8, 287.
Nat. Rev. Drug Discov. 3. 215.
120
Chapter sewen
To inhibit microbial growth, drags must reach and
maintain inhibitory concentrations at the target sites
wliich usually reside in the cytoplasm or are embedded
in the cytoplasmic membrane. Antimicrobial agents
traverse the permeability barriers provided by cell
walls and membranes that separate the target sites
from the external environment by passive, or in some
cases, facihtated diffusion. In addition to structural
barriers hindering drug access to their targets, many
wild-type micro-organisms actively extrude inhibitory
compounds from the cytoplasm back into the external
environment via a battery of drug efflux pumps. The
structural permeability barriers and drug efflux sys-tems combine to modulate the level of intrinsic re-sistance to drugs in wild-type organisms. An isolated
target site prepared from dilTerent species of micro-organism may have sensitivities comparable to a spe-cific drug in vitro, whereas the intact cells may show
very different responses to the same drug. Higher lev-els of resistance acquired during sustained exposure to
drugs are caused by a range of genetically based mech-anisms discussed in later chapters, including reduced
drug access that is due to increased cell wall thiclcness,
loss of porins from the outer membranes of Gram-neg-ative bacteria and increased levels of expression of
drug efflux pumps. The aggregation of bacterial cells
in some situations to fonn bioiilrns may lead to re-duced susceptibility to drug action, and this has been
attributed to hindered drug access through the biofdm.
For example in patients with cystic fibrosis, Pseudo-rnorias aeruginosa forms bioiilms in lung which are
notoriously difficult to treat. However, the present con-sensus is that the altered dynamics of bacterial cell
growth in biofilms probably accounts for the lower
drug sensitivity and that biofilms do not in general
pose a significant diffusional barrier to drag HCCCSS.
a a \& %^ n i % ^ n C 9 a vJs %DV 5 5 5 5 Vb^C S 9J< 5 a 5 %iW 5>^5bj9 a n 5 % ^ a %3^ 5B %J^ % ^ n 5bS%«
dididiiUi i
7.1.1 The cytoplasmic membrane
The pemieability barrier provided by the cytoplasmic
membrane of micro-organisms depends on the charac-teristic lipid bilayer that is common to all biological
membranes. Drags cross this barrier either by passive
diffusion or by facilitated diffusion involving a biolog-ical carrier system.
121
Drug transport
Passive diffusion
The rates of passive diffusion of unchai-ged organic
molecules across lipid membranes are governed by
Pick’s law of diffusion and coiTelate reasonably well
v^ith their lipidMater partition coefficients. Pick’s law
is expressed by the equation:
where Vis the rate of diffusion (in nmolmg^’s”^),/! is
the surface area of the membrane (cm^mg^^), S^, and S^
are, respectively, the external and internal concentra-tions of free permeant, and P is the permeability coef-ficient (cm s”‘). The internal concentration of free drug
can be substantially affected by binding to Intracellu-lar macromolecular targets, metabolism to other chem-ical species or by changes in ionization that are due to
differences between internal and external pH values.
Lowering the Internal free concentration of a com-pound by any of these factors enhances the rate of in-ward passive diffusion by steepening the concentration
gradient of unbound drug. Diffusion across mem-branes is bi-directional, and the rates at which a com-pound diffuses into and out of a cell determine the time
at which a steady-state intracellular concentration of
the compound is achieved. In reality, however, a
steady-state intracellular drug concentration in an in-fecting micro-organism may be achieved only tran-siently, if at all, because of rapidly changing conditions
both inside the cell and in its external environment.
The greater the lipid solubility of a compound,
expressed as the partition coefficient, the more readily
it enters and diffuses across the lipid bilayer of the
membrane. However, when lipid solubility is so high
that a compound is essentially insoluble in water, it
may be unable to diffuse out of the lipid interior of the
memlirane into the aqueous environment of the cyto-plasm. This adversely affects the biological activity of
compounds with sites of action within the cytoplasmic
compartment but could enhance activity if the target
site is membrane-bound. The relationship between bi-ological activity and lipophilicity is expressed in the
‘Hansch equation’ (so named after the scientist who
formulated it):
log (I/ O = -/c(log Pf- + k’ log P + p<5 + k”,
where C is the molar concentration of the drug for a
standard biological response, in the case of antimicro-bial drugs usually the minimal inhibitory concentra-tion (MIC) or alternatively, the concentration needed
for 50% inhibition of growth or cell survival (IC5,,); P
is the partition coefficient; p and O are physicochemi-cal constants (Hammett constants) defining certain
electronic features of the molecule; and k, k’ and k” are
empirically determined constants. The equation indi-cates that within a chemically related series of biolog-ically active molecules having similar values of p and
a, an optimal partition coefficient is associated with
maximum biological activity. However, this relation-ship holds only for those agents that cross membranes
by passive diffusion and it may break down when bio-logically facilitated transport is involved or when per-meation occurs through water-filled pores, as in the
case of the porin channels in the outer membrane of
Gram-negative bacteria. Futhermore, the lipophilicity
of a drag may be the critical factor determining its
affinity for the target site because of the dominant con-tribution of hydrophobic forces to the dmg-target
interaction.
The Hansch equation has been applied to sets of
synthetic antibacterial compounds that penetrate the
bacterial envelope by passive diffusion. The results
show that the compounds most active against Gram-negative bacteria are generally less lipophilic (or alter-natively, more hydrophilic) than compounds with pri-mary activity against Gram-positive organisms. The
cytoplasmic membranes of the two classes of bacteria
are sufficiently similar to make it unlikely that they
could account for the differences in the partition coef-ficients of optimally active compounds. The explana-tion lies largely in the unique properties of the porin
channels in the Gram-negative outer membrane, which
facilitate the influx of hydrophilic compounds. We
shall return to this important topic later in the chapter.
The rates of passive diffusion of water-soluble
molecules across lipid membranes are usually very
low, although uncharged polar compounds with mo-lecular masses of less than 100 Da diffuse more read-ily. Nevertheless, as we shall see, certain hydrophilic
antimicrobial agents of much higher molecular mass
readily enter the cytoplasm. Ionized compounds dif-fuse across cytoplasmic membranes with difficulty,
unless the molecules contain compensatory lipophilic
122
7.1 Cellular permeability barriers to drug penetration
regions, because the strongly bound hydration shells of
ionized groups in aqueous solution hinder diffusion
across the lipid bilayer. The effect of ionization on the
activity of an antibacterial agent is well illustrated by
er3’thromycin. The pK^ of the basic dimethyiamino
group of this antibiotic is 8.8 and the concentration re-quired for antibacterial activity decreases markedly as
the pH of the bacterial medium is increased from neu-trality towaixls 8.8. Presumably only un-ionized eryth-romycin molecules, which represent an increasing pro-portion of the total erythromycin as the pK^ of the drug
is approached, diffuse into the bacteria.
Facilitated diffusion
A remarkable feature of cytoplasmic membranes is
their ability to transfer certain ions, nutrients, waste
products and toxins at much higher rates than are pos-sible by passive diffusion. This process is known as
facilitated transfer or facilitated diffusion. Characteris-tically, the rate of transfer of the permeant is propor-tional to its concentration over a limited range, beyond
which a limiting rate is approached. This is due to the
involvement of carrier proteins within the membrane
that transiently bind the permeants and ‘shuttle’ them
across the membrane. The rate of transfer increases
with increasing permeant concentration until all of the
carrier sites are saturated. This is in contrast to passive
diffusion, where the transfer rate is proportional to
permeant concentration over a much wider range. The
kinetics of facilitated diffusion are directly compara-ble with the Michaelis-Menten kinetics of enzymes
and their substrates. The following equation enables
the transfer rate across the cytoplasmic membrane, v,
to be calculated for a given concentration, C, of sub-stance S:
^ = v„ja + Kjcx
where C is the concentration of S either inside or out-side the cell, depending on the direction the solute is
being transferred; V^,^^ is the maximum rate of dif-fusion when all the carrier sites are occupied by S and
K^^ is the concentration of S at which half the maxi-mum number of carrier sites ai^e occupied. K^^ is
therefore correlated with the affinity of the carrier
molecule for S.
Facilitated transfer by itself results in the equili-bration of the permeant across the membrane. Flow-ever, when the transfer system is linked to an input of
‘energy’, usually the hydrolysis of ATP or the proton
motive force across the cytoplasmic membrane, the
pemieant is transferred across the membrane against
its concentration gradient. This is known as active
transport. Some facilitated transfer systems are highly
specific and only close structural analogues of the
natural permeant compete effectively for the transport
sites. In contrast, most drug efflux pumps exhibit
remarkably broad specificity in the range of com-pounds they remove from microbial cells (see later dis-cussion).
7.1.2 Tlie cell wails of bacteria and fungi
The function of the peptidoglycan cell wall of bacteria
and of the muhilayered glycoprotein-polysaccharide
fungal cell wall is to give shape and tensile strength to
microbial cells. These structures are mainly open net-works of macromolecules and generally do not offer
significant permeability baniers to compounds of mo-lecular mass less than 50 kDa. Even the thick peptido-glycan walls of Gram-positive bacteria are permeable
to antimicrobial peptides such as nisin and defensin,
which have a molecular mass of 3 kDa or more. How-ever, there are two important exceptions to the general
rule of high penneability of bacterial cell walls:
1. As discussed in Chapter 2, the cell wall of a
Gram-positive mycobacteria is characterized
by a high lipid content that is due to the pres-ence of long-chain mycolic acids on the outer
surface of the wall which are covalently
linked to the underlying arabinogalactan. The
extended hydrocarbon chains of the mycolic
acid molecules are tightly packed in a paral-lel array pei”pendicular to the cell surface.
The outemiost surface of the mycobacterial
cell also contains a range of other complex
lipids and waxes. This assembly has very low
fluidity and resembles the outer membrane of
Gram-negative bacteria. The mycobacterial
cell wall is therefore a formidable penneabil-ity barrier to antibacterial drugs and accounts
123
Drug transport
for the well-known resistance of Mycobac-terium tuberculosis to many antibiotics. The
important antituberculosis antibiotic rifampi-cin (Chapter 4) is relatively hydrophobic and
may gain access to its target in the cytoplasm
by diffusion though the lipid banier. Hy~
drophilic antituberculosis drugs such as iso-niazid and ethambutol that interfere with the
biosynthesis of the mycobacterial cell wall
could initially diffuse through porin channels
which are thought to penetrate the outer
membranelike stracture. Subsequent disrap-tion of cell wall biosynthesis may then fur-ther facilitate influx of a drug.
2. Clinically significant acquired resistance of
Staphylococcus aureus to vancomycin is as-sociated with a greatly thickened cell wall,
which it is suggested may trap the antibiotic
within the extended peptidoglycan mesh-work, hindering its access to the target sites
(see Chapter 9).
Because of their strongly polar, predominantly
negatively chai^ged nature, the teichoic acids of Gram-positive cell walls could, in principle, influence the
penetration of ionized molecules. The interaction of
water-soluble, positively chai^ged compounds, such as
the aminoglycosides, with teichoic acid might gener-ate locally high drug concentrations within the enve-lope, enabling the drugs to challenge the penneability
banier of the cytoplasmic membrane more etfectively.
In contrast, the entry of anionic molecules could be re-tai’ded by teichoic acid, although the exquisite sensitiv-ity of many wild-type Gram-positive bacteria to peni-cillins, which are organic anions, shows that the
repulsive effect of teichoic acid is not significant. It is
unlikely that the teichoic acids of Gram-positive bac-terial cell walls have any significant effect on the
steady-state intracellular- concentrations of antibiotics.
The Gram-negative outer membrane
In contrast to the thin peptidoglycan cell wall of Gram-negative bacteria, the outer membrane of these organ-isms is a significant contributor to the greater intrinsic
resistance of Gram-negative bacteria to many antibi-otics (Table 7.1). An indication that the outer mem-brane hinders dmg penetration came from studies with
TABLE 7.1
drugs
Differential sensitivity to typical antibacterial
Drugs active against
Gram-positives
and Gram-negatives
Drugs less active against
Gram-negatives
Tetracyclines
Streptomycin and
aminoglycosides
Sulfonamides
D-Cycloserine
Chloramphenicol
Fosfomycin
Many synthetic antiseptics
Nitroirirans
AmpiciUin and carbenicillin
Tliienamycin
Fluoroquinolones
Benzylpenicillin
Methicillin
Macrolides
Lincomycin
Rifamycins
Fusidic acid
Vancomycin
Novobiocin
Bacitracin
Gram-negative cells with defective envelopes. L-Phase
(or L-forms) of Proteus mirabiUs were found to be 100
to 1000 times more sensitive than intact cells to eryth-romycin and several other macrolides. There was a
smaller increase in sensitivity to other antibiotics, in-cluding streptomycin, chloramphenicol and the tetra-cyclines. Both the outer membrane and the peptidogly-can are also defective in T-forms, so that the relative
contributions of the various outer layers of intact bac-teria to the baiTier function were not certain in this
early work. However, later studies clearly defined the
outer membrane as a significant penneability baiTier to
some molecules.
Two major features of the Gram-negative outer
membrane distinguish it from the cytoplasmic
membrane:
1. Negatively charged lipopolysaccharide (LPS)
in the outer leaflet of the bilayer replaces the
glycerophospholipid of most other biological
membranes. The negative charge of the LPS
is partly neutralized by divalent cations,
mainly Mg^”*” and Ca'”*”, which are readily re-moved by chelating agents such as ethylene-diaminetetraacetic acid (EDTA).
2. Molecular diffusion across the complex
lipid-lipopolysaccharide bilayer of the outer
membrane is slow. In order to permit rapid
influx of essential nutrients and ions, the
124
7.1 Cellular permeability barriers to drug penetration
outer membrane is studded with water-filled
pores fomied by porin proteins. The porin
channels, which are unique to the outer mem-brane, are instrumental in pemiitting the ini-tial influx of certain hydrophilic antibacterial
compounds across the outer envelope. Porin
proteins consist of trimers of p-baiTels ar-ranged as antiparallel p-strands, commonly
16 in number, threading through the outer
membrane. At the inner face of the mem-brane, the strands are joined by short p-turns
and at the outer face by longer loops of amino
acids. Three types of porin channel have been
identified:
a. general channels with low penneant
selectivity,
b. permeant-selective channels with inter-nal specific binding sites, and
c. permeant-selective ‘gated’ channels that
only open upon the binding of the spe-cific permeant.
All three types of porin channel restrict
transit to compounds with molecular masses
of less than approximately 600 Da. The gen-eral OmpF porin of Escherichia coli is the
most thoroughly studied porin and was the
first membrane protein to be successfully
crystallized for X-ray analysis. It is closely
homologous to two other porins, PhoE,
which allows the influx of phosphate ions,
and OmpC. OmpF has pore dimensions (at its
narrowest) of 11 x 7A which allows the pas-sage of major nutrients and hydrophilic an-tibiotics with molecular masses of < 600 Da.
Although water-soluble antibiotics usually
pass through the general channels, the selec-tive porins are also used. The p-lactam antibi-otic imipenem, for example, diffuses through
the basic amino acid-specific channel OprD
in Pseudomonas aeruginosa. The movement
through the general porin channels of p-lac-tam antibiotics close to the molecular mass
limit is retarded by repulsive interactions be-tween the dmgs and the predominantly nega-tively charged amino acids lining the chan-nels. These interactions may hinder diffusion
by as much as 100-fold. The diffusion of
lipophilic molecules through the porin chan-nels is much more difficult because the
charged amino acid residues lining the nar-rowest regions of the channels orient their as-sociated water molecules in a direction that
hinders the passage of lipophilic permeants.
The importance of porin channels to the in-flux of hydrophilic antibacterial agents is
clearly demonstrated by the reduced suscep-tibility of porin-deficient mutants to antibi-otics, including some valuable semisynthetic
p-lactams.
In several species of Gram-negative
bacteria, most notably the potentially danger-ous opportunist pathogen Pseudomonas aer-uginosa, porin function is even more restric-tive. The high-flux channels of Escherichia
coli axe replaced in Pseudomonas aeruginosa
by a low-efficiency porin, OprF, that restricts
diffusion to about 1% of the rate through the
channels of other Gram-negative bacteria.
Only about 2% of OprF porins ai^e in an ac-tive, open state at any one time. The absence
of efficient porin channels and the low per-meability of the rigid LPS of its outer mem-brane are major contributors to the character-istic intrinsic resistance of Pseudomonas
aeruginosa to both hydrophilic and lipophilic
Although mycobacteria are fomially classified as
Gram-positive, the organization of the dense lipid ma-terial in the outer layers of the cell envelope resembles
the outer membrane of Gram-negative bacteria. As
previously discussed, the extreme impermeability of
the outer lipid coating of mycobacteria is the major de-terminant of the general resistance of these organisms
to antibacterial agents. How therefore do the effective
antituberculosis drugs (and indeed, nutrients) gain ac-cess to the bacterial cytoplasm? While slow diffusion
across the lipid bilayer is probable, recent discoveries
suggesting the existence of porins in the bilayer indi-cate the possibility of another route of access. A porin
protein, MspA, with high channel activity, has been
isolated from Mycobacterium smegmatis. Recently the
MspA protein was crystallized and subjected to X-ray
125
Drug transport
analysis. A homo-octameric structure was revealed
with a single central channel which would readily per-mit the passage of isoniazid, ethambutol and pyiiz-inamide and small, water-soluble nutrients (Figure
7.1). It remains to be seen whether comparable struc-tures can be defined in Mycobacterium tuberculosis.
Further research into this possibility would be relevant
to the development of new antituberculosis drugs.
It had been thought that the barrier function of the
outer membrane provided an adequate explanation for
the intrinsic resistance of Gram-negative bacteria to
many drugs. However, it is now recognized that even a
highly effective permeability barrier cannot com-pletely stem the influx of drugs. An interesting exam-ple is provided by hydrophilic p-lactams. These com-pounds cross the outer membrane of Escherichia coli
through the porin system and by slow diffusion across
the lipid bilayer. The half-equilibration time of p-lac-tams into the periplasmic space is less than 1 s. In
porin-deficient mutants, the only route of drug ingress
is by diffusion across the lipid bilayer. F>en in this sit-uation, the half-equilibration time is only a few min-utes. Thus although the minimal inhibitory concentra-tions of p-lactams against the porin-deficient cells are
significantly increased, there is still effective access to
the penicillin-binding proteins on the outer face of the
cytoplasmic membrane.
7.2 Multidrug efflux
Because the permeability banier of the lipid bilayer of
the outer membrane cannot fully account for the intrin-sic resistance of many Gram-negative bacteria to anti-bacterial agents, there must be some additional factor
at work. In fact, wild-type strains of many bacteria,
both Gram-negative and Gram-positive, extrade a
wide range of antibiotics, including p-lactams, tetracy-clines, chloramphenicol, macrolides and fluroquino-lones as well as antiseptic agents into the external
medium by drug efflux pumps. The drag efflux activi-ties of various strains of Pseudomonas correlate well
with their relative levels of antibiotic resistance. The
absolute levels of intrinsic resistance are determined
FIGURE 7.1 Stereo view from the external environment into the MspA porin chtmnel from Mycobacterium smegmatis. The
surfaces of polar amino acid residues are shown in green and nonpolar residues in yeUow. If the photograph is held approx-imately 50 cm from the eyes and attention is concentrated on the space between the two images, with practice a three-dimen-sional image can be seen between the two outer images. The images were generated from X-ray analysis of the crystalline
porin protein. [Taken with permission from M. Falier, M. Niederweis and G. E. Schulz Science 303, 1189 (2004).]
126
7,2 Multidrug efflux
by the synergism between the low permeability of the
outer membrane and the dnig efflux systems, which
together depress the prevailing intracellular drug con-centrations below effective inhibitory levels.
An extraordinary diversity of dmg efflux pumps
has been discovered in recent years, not only in bacte-ria, but also in fungi and protozoa and even in mam-malian cells. The ability of living cells to reverse the
influx of hai’niful chemicals has a long evolutionary
past and is a major contributor to the intrinsic resist-ance of cells. The upregulation and acquisition of drug
efflux systems by gene transfer in acquired drug resist-ance is discussed in Chapter 9. An extensive review of
the full range of drug efflux pumps is beyond the scope
of this book and several references are available in
Tnuther reading’ to take the reader deeper into this
complex field. However, it is evident that the many dif-ferent pumps which have been characterized can be
grouped into four major families:
1. the major facilitator family (MFS),
2. the resistance-nodulation-division (RND)
family,
3. the small multi-drug resistance (SMR) fam-ily.
4. the AlT-binding cassette (ABC) family.
The efflux pumps are able to drive a wide range
of toxic chemicals (Table 7.2) out of the cells against a
concentration gradient using canier molecules and are
therefore examples of active transport. The energy for
active transport is generated by the proton motive force
in the MFS, RND, and SMR pumps. As its name im-plies, the ABC family consumes ATP to drive drug ex-trusion. A fifth family, multidrug and toxic compound
extrusion (MATE), has recently been described in Es-cherichia coli.
MFS efflux pumps are found in both Gram-posi-tive and Gram-negative bacteria. The QacA system (a
14-transmembrane domain protein) in Staphylococcus
aureus extrudes antiseptic compounds such as
chlorhexidine and cetyltiimethylammonium bromide.
Nor.A, a 12-transmembrane domain protein expressed
in Staphylococcus aureus, extrudes fluoroquinolones,
chloramphenicol, antiseptics and a wide range of other
compounds.
Tiie presence of the outer membrane in Gram-negative bacteria requkes the cooperation of addi-tional proteins to ensure the export of chemicals across
this barrier. The membrane fusion protein (MFP) links
TABLE 7.2 Examples of antimicrobial efflux pumps. The
the range of substrates taken up by the pumps is often much
substrates list provides examples only and
wider
Pump type
ABC
MacAB-TolC
,A,bc23
Cdrl
Pfnidrl
SMR
EMRe
Mmr
MPS
QacA, QacB
MdfA
Mdri
RND
AcrAB/Tolc
MexAB/OprM
MexXY/OprE
Organism
Escherichia coli
Enteroccocusfaecalis
Candida albicans
Plasmodium falciparum
Escl’iericliia coli
Mycobacterium tuberculosis
Staphylococcus aureus
Escherichia coli
Candida albicans
Esciierichai coli
Pseudomonas aeruginosa
Pseudomonas aeruginosa
A ntimicrobiai substrates
Macrolides
Lincomycin
Azoles
Choroquine, artemisinin
Quaternary ammonium antiseptics.
Erythromycin
Chlorhexidine, quatemjiry ammonium
antiseptics
Norfloxacin, erythromycin, quatenijiry
ammonium antiseptics
Azoles, benomyl
P-Lactams. macrolides
p-Lactams, macrolides, Fluoroquinolones
Aminoglycosides
127
Drug transport
the efflux pump in the cytoplasmic membrane with the
outer membrane factor (OMF) which is embedded in
the outer membrane and extends through the
periplasm. The whole assembly provides a conduit for
the extrusion of toxic chemicals into the extracellular
space. Figure 7.2 illustrates how this system may be
arranged in the cell envelope; the mechanism of the
link with the energy-generating proton motive force of
the cytoplasmic membrane is not known.
Members of the RND family are mostly found in
Gram-negative bacteria, although evidence for them
has been detected in Gram-positive cells. The RND
pumps are nonspecific in their uptake of chemicals and
hence confer intrinsic resistance to a wide range of an-tibacterial agents (Table 7.2) There are 12 transcyto-plasmic membrane domains (TM) together with two
large extracytopiasmic domains between TM l and
TM2 and between TM7 and 8. The MFP and OMF pro-teins complete the assembly. The best-known RND
pump is the AcrB transporter in Escherichia coli. Here
the MFP protein is designated as Acr A and the OMF as
TolC. The AcrAB complex confers intrinsic resistance
to large lipophilic drags, such as erythromycin, fusidic
acid and detergents, that traverse the porin channels
with difficulty. Susceptibility to smaller antibiotics, in-cluding tetracyclines, chloramphenicol and fluoro-quinolones, which diffuse through the porin channels,
remains high. The rate of influx of these drags over-whelms the capacity of the AcrAB system to maintain
cytoplasmic concentrations below inhibitory levels.
Nevertheless, deleterious mutations affecting AcrAB
greatly enhance the sensitivity of Escherichia coli to a
antibacterials
outer membrane factor I porin channel
outer membrane
Membrane fusion protein
transporter protein 1 antibacterials
cytoplasmic membrane
transporter protein 2
cytoplasm
FIGURE 7.2 Suggested arrangement of the components of bacterial multidrug
efflux pumps in Gram-negative bacteria. RND and MPS pumps (transporter pro-tein I) use accessor}’ proteins in Gram-negative bacteria as chamiels or pores to
direct antibiotics out of the cells into the external environment. SMR and ABC
pumps (transporter protein 2) extrude antibiotics across the cytoplasmic mem-brane. In Gram-positive bacteria, the extruded antibiotics then diffuse directly
through the peptidoglycan cell wall to the cell exterior. The pumps also capture
tmtibiotics in the periplasm of Gram-negative bacteria. In Gram-negative bacte-ria, antibiotics delivered by the SMR tmd ABC pumps may diffuse outwards via
porin channels. RND, MPS and wSMR pumps are energized by the proton motive
force across the cytoplasmic membrane: ABC pumps are driven by the hydroly-sis of ATP. The mechanism of coupling energy consumption to pump function is
at present not known. The arrows indicate the direction of antibiotic fluxes.
128
7,3 Facilitated uptake of antimicrobial drugs
variety of drags. The AcrAB-TolC system of £s-cherichia coli is homologous with the MexAB-OprM
pump of Pseudomonas aeruginosa and Acr in Salmo-nella spp. and Haemophilus influenzae. Mutational in-activation of either MexA or OprM in wild-type Pseu-domonas aeruginosa results in a marlted increase in
cellular sensitivity to many antibacterial agents.
There is evidence for SMR efflux pumps in both
Gram-negative and Gram-positive bacteria and in My-cobacterium tuberculosis. The SMR pump, EmrE, in
Escherichia coli is capable of transporting a diverse
range of antibacterial agents (Table 7.2), but the details
of its organization are not clear.
The complete sequence of the genome of the
yeast Saccharomyces cerevisiae predicts that it con-tains 30 genes for ABC pumps and 28 genes for MPS
pumps, although to what extent these genes ai^e consti-tutively expressed and affect intrinsic drug resistance
is largely unknown. The role of ABC and MPS pumps
in acquired drug resistance in eukaryotic cells is re-viewed in Chapter 9. At least two ABC pumps have
been identified in Escherichia coli: arcAB and
MacAB-TolC, the latter contributing to the intrinsic re-sistance to niacrolide antibiotics. The bacterial
pathogen Enterococcus faecalis also expresses four of
these pumps that confer substantial intrinsic resistance
to the lincosamide antibiotics.
An extraordinary property common to all the ef-flux pumps is their broad substrate specificity, en-abling the extrusion of a diverse range of antimicrobial
compounds. Until recently, the structural basis for this
broad substrate specificity remained elusive. However,
the solution of the crystal structure of the AcrB pump
from Escherichia coli has now provided a remarkable
insight into this puzzle. The transmembrane protein
was crystallized both in isolation and complexed with
four structurally dissimilar compounds, including the
fluoroquinolone ciprofloxacin, the quaternary antisep-tic dequalinium, and rhodamine. AcrB is a ho-motrimer, which in addition to threading through the
cytoplasmic membrane, forms a large cavity facing the
cytoplasm. This cavity contains the ligand-binding do-main. The ligands bind to various positions in the cav-ity using different subsets of hydrophobic amino acids
lining the cavity. The binding interactions are therefore
believed to be mainly hydrophobic, perhaps also in-volving ai^omatic m-n interactions. The large size of the
central cavity and the predominance of hydrophobic
binding interactions with drug molecules largely ex-plains the broad range of antibacterial agents that the
AcrB pump extrudes from the bacterial cytoplasm. It
seems likely that similar ligand-binding motifs may
underly the broad specificity of many other drug efflux
pumps.
The upregulation of drug efflux systems in ac-quired drug resistance is considered in greater detail in
Chapter 8. It is important to realize, however, that the
contribution of constitutively expressed multidrug ef-flux pumps in wild-type bacteria to intrinsic drug re-sistance depends on the rate of the inward movement
of drugs across the cell envelope. If the barrier function
is breached by mutational changes or treatment with
EDTA, which chelates divalent cations and thereby
disrupts the LPS of the outer membrane of Gram-neg-ative bacteria, the efflux systems may be overwhelmed
by the inward rush of drug molecules, and intrinsic re-sistance will be lost. The influence of the various com-ponents of bacterial cell envelopes on the uptake of
drugs is summarized in Table 7.3.
a9k^ K C « % # a 5 a QfCS %i% ^ vj& %<1 &Jfi %Vr9 aftOs ? Vs^ S C 9 aaQoaann S % # a %flV & $ i %M 5 vji i ftcfiOdQ v ^
7.3.1 Antibacterial agents
As we have seen, the inward diffusion of antibacterial
agents across the outer membrane of Gram-negative
bacteria is essentially a passive process. Hydrophilic
compounds move through the water-filled poriii chan-nels, whereas lipophilic agents diffuse through the
lipid bilayer. Strongly charged cationic agents such as
quaternary ammonium antiseptics and chlorhexidine,
and polycationic antibiotics such as the polymyxins
and aminoglycosides, destabilize and disorganize the
outer membrane and thereby promote their own access
to the inner cellular layers. As we saw in Chapter 2, an-tiseptics and polymyxins also disrupt the barrier func-tion of the cytoplasmic membrane and finally pene-trate the cytoplasm. Lipophilic agents, including
sulfonamides, rifamcyins and macrolides, diffuse pas-sively across the cytoplasmic membranes of Gram-negative and Gram-positive bacteria. Hydrophilic
compounds, however, are unlikely to achieve in-hibitory intracellular concentrations unless there is
129
Drug transport
TABLE 7.3 Features of the bacterial cell
Envelope structure
envelope that mfluence the uptake of antibacterial agents
Effects on drug uptake
Lipid bilayer of Gram-negative
outer niembrane
High-efficiency poriu channels of Gram-negative bacteria
Low-efficiency porin of Pseudomonas
aeruginosa
Teichoic acids of Gram-positive bacteria
Lipid bilayer of cytoplasmic membrane
Facilitated transport systems of
cytoplasmic membrane
Multidrug efflux systems
Retards diffusion of both water-soluble and lipophilic compounds
Facilitate diffusion of water-soluble molecules of molecular mass up to
-600 Da
Permits only slow diffusion of water-soluble antibiotics
Strongly anionic character could in principle affect uptake of ionized
molecules
Rates of passive diffusion depend on lipophilicity of permetmt. Permits little
or no passive diffusion of water-soluble or strongly ionized molecules
Markedly enhance rates of transfer of nutrients and structural analogues,
e.g. i3-cycloserine, fosfomycin. Energy coupling leads to accumulation
against concentration gradients
Reduce intracellular drug concentrations by effluxing into external medium.
Occur in both wild-type and drug-resistant mutant bacteria
some form of active or facilitated transport or intracel-lular sequestration of unbound drug to maintain a steep
inward concentration gradient. Examples of facilitated
drug transport into the bacterial cytoplasm are de-scribed below.
D-Cycloserine
Ttiis antibiotic, a structural analogue of D-alanine, is
transported across bacterial cytoplasmic membranes
by alanine permeases. D-Cycloserine enters Strepto-coccus faecalis using this transport system and com-petitively inhibits the uptake of both stereoisomers of
alanine. In Escherichia coli, D~ and L-alanine are trans-ported by separate isostere-specific permeases and D~
cycloserine uses only the D~isomer~specific system.
There are high- and low-affinity transport systems for
D-alanine, and at minimal antibacterial concentrations
(~4 \iM) D-cycloserine favors the high-affinity carrier.
The high-affinity D-alanine permease is energy cou-pled to the proton motive force, ensuring accumulation
of D-alanine and D~cycloserine against their concentra-tion gradients. The accumulated D-cycloserine effec-tively inhibits the intracellular target enzymes, L-ala-nine racemase and D-alanyl-D-alanine synthetase, in-volved in peptidoglycan biosynthesis (Chapter 2).
Fosfomycin (phosphonomycin)
This simple phosphorus-containing antibiotic uses two
different physiological transport systems to gain ac-cess to the bacterial cytoplasm:
1. The stractural resemblance of fosfomycin to
a~glycerophosphate enables it to use the per-mease for this important biochemical. As ex-pected, the uptake of fosfomycin is competi-tively inhibited by high concentrations of
a~glycerophosphate.
2. The permease for hexose 6-phosphates in
certain enterobacteria and staphylococci is
also exploited by fosfomycin.
Both transport systems are induced by their nor-mal permeants, although not by fosfomycin. The ther-apeutic efficacy of fosfomycin in experimental infec-tions can be enhanced by pretreatment of the infected
animals with glucose 6-phosphate, which induces the
130
7,3 Facilitated uptake of antimicrobial drugs
bacterial permease, resulting in a higher intracellular
concentration of fosfomycin. In Gram-negative bacte-ria, fosfomycin crosses the outer membrane via nutri-ent channels used by a-glycerophosphate and hexose
6-phosphates.
Tetracyclines
TetracycUnes cross the outer membrane of Gram-neg-ative bacteria through the OmpC and OmpF porin
channels, probably as a chelation complex with a
magnesium ion. Mutant cells with decreased OmpF
expression exhibit some resistance to tetracycline, al-though not to the more lipophilic derivative, minocy-cline, which is thought to diffuse in its uncomplexed
form across the lipid bilayer rather than through the
porin channels.
If has long been known that tetracyclines are ac-cumulated against their concentration gradients by
Gram-negative and Gram-positive bacteria. This active
process, which is energized by the proton motive force
across the cytoplasmic membrane, partially explains
the antibacterial specificity of tetracyclines. The en-ergy-dependent accumulation of tetracyclines against
a concentration gradient appears to have some of the
hallmarks of facilitated diffusion mediated by a carrier
system associated with the cytoplasmic membrane.
Flowever, no such canier for the inward transport of
tetracycline across the cytoplasmic membrane has ever
been identified. Furthermore, there is scant evidence
that the rate of tetracycline influx is saturable at high
concentrations of the drug—a defining characteristic
of canier-mediated facilitated transport. How, then, is
the energy-dependent accumulation of tetracyclines to
be explained? The answer probably is that it is at least
partly due to the physicochemical properties of the
tetracycline molecule.
The tetracycline-Mg^”’ chelate is positively
charged and probably accumulates in the bacterial
periplasm of Gram-negative cells, owing to the Don-nan potential across the outer membrane. Reversible
dissociation of the chelate rel esses the lipophilic, un-charged tetracycline molecules. Calculations based on
the use of microscopic dissociation constants, which
define the protonation of tetracycline, show that ap-proximately 7% of the molecules are uncharged at
physiological pH. The uncharged molecules, unlike
their charged counterparts, diffuse rapidly across the
cytoplasmic membrane into the cytoplasm. The proton
gradient across the cytoplasmic membrane maintains
the internal pFI about 1.7 pFI units higher than the pFI
of the external medium. The effect of the higher inter-nal plT is to significantly increase the fraction of nega-tively charged tetracycline molecules within the cells.
When equilibrium is reached by passive diffusion, the
concentration of unchai^ged molecules must be the
same on both sides of the membrane. The total concen-tration of tetracycline, i.e. uncharged plus charged
molecules, is therefore higher in the bacterial cyto-plasm than in the medium. Calculations based upon
these assumptions predict that the intracellular con-centration of tetracycline in noniially metabolizing
bacterial cells should be approximately four times that
in the external medium. Direct measurements in Es-cherichia coli, however, reveal a 15-fold difference be-tween the internal and external concentrations. The
higher internal concentration of magnesium ions may
also contribute to the sequestration of tetracycline. Up
to 30% of the intracellular tetracycline is bound to the
3()S subunit of the ribosomes, probably as an Mg-“^
complex (Chapter 5). The maintenance of the pH gra-dient across the membrane depends on the energy
metabolism of the cell. Compounds that collapse the
gradient directly or indirectly by inhibiting energy me-tabolism, prevent the intracellular accumulation of
tetracycline and promote the release of previously ac-cumulated drug into the external medium.
The model described here provides a probable ex-planation for the energy-dependent accumulation of
tetracycline by bacterial cells, at least in Gram-nega-tive organisms. Flowever, before the possibility of car-rier-mediated influx is rejected entirely, it should be
noted that tetracycline-specific, carrier-mediated ef-flux systems occur widely in tetracycline-resistant
bacteria (Chapter 9). Furthemiore, even in wild-type
Escherichia coli there is evidence of a low-efficiency
tetracycline-specific efflux system, probably involving
a canier system. The existence of these tetracycline ef-flux pumps therefore raises the possibility that tetracy-cline influx may also be carrier mediated.
131
Drug transport
Quinolones
Antibacterial quinolones, such as ciprofloxacin, have
two ionizable centers—the carboxyl group, pi^T^ 7.5,
and the ‘distal’ nitrogen of the piperazine ring, pK^
6.5. Calculations similar to those applied to the tetra-cyclines show that at pH 7.4 approximately 10% of the
molecular population is in the uncharged form. The
precise values of these parameters for individual com-pounds depend on the nature of the substituents on the
quinolone ring system.
Quinolone entry through the outer membranes
of Gram-negative bacteria occurs mainly as charged
molecules through the porin channels. Ionization of
the carboxyl group enables quinolones to chelate
with Mg^+ ions, and a substantial part of the influx
through the porin channels is probably in the fomi of
the magnesium complex. The chelates are likely to
dissociate in the more acid environment of the
periplasm, leading to the establishment of a Donnan
equilibrium across the outer membrane, with a higher
total drug concentration in the periplasm than in the
external medium. This effect could explain why some
quinolones are more effective against Gram-negative
than against Gram-positive bacteria, possibly be-cause of the absence of a defined periplasm in the lat-ter organisms.
Bacterial cells accumulate quinolones and it was
thought that this was an example of active drug uptake
into the cytoplasm. However, much of the drug asso-ciated with Gram-negative bacteria is probably bound
to surface components via magnesium chelation. Con-sideration of the ionization equilibria of quinolones
suggests that their cytoplasmic concentration may ac-tually be less than that in the external medium, owing
to the pH gradient across the cytoplasmic membrane.
When equilibrium of the uncharged molecules is
reached by passive diffusion across the cytoplasmic
membrane, the total internal concentration (un-charged plus chai-ged molecules) is calculated to be
less than the external concentration. It is interesting
that carbonyl cyanide /n-chlorophenylhydrazone, an
agent used experimentally to collapse the proton gra-dient across cell membranes in bacteria, actually in-creases the uptake of certain quinolones into the cyto-plasm. Abolition of the pH difference between the cy-toplasm and the exterior is believed to establish a new
equilibrium position by promoting a transient-in-creased influx of quinolone that equalizes the total
drug concentrations on each side of the membrane. In
summary, there is no definitive evidence for carrier-mediated accumulation of quinolones by bacteria, and
the uptake phenomena observed can be explained
largely by the physicochemical properties of the com-pounds and compartmental differences in pH within
bacterial cells.
Aminoglycosides
These polycationic water-soluble molecules approach
the molecular mass exclusion limit of 600 Da for
porin-mediated transport through the Gram-negative
outer membrane. There is uncertainty, therefore, as to
the contribution of this mode of transport to the uptake
of aminoglycosides by Gram-negative bacteria. As
mentioned previously, aminoglycosides promote their
own penetration by competitive displacement of the
stabilizing Mg^”*” and Ca^”” ions from the LPS. The
large molecular size of the aminoglycoside cations in
comparison with the metal cations probably causes the
subsequent disorganization and disruption of the bar-rier function of the outer membrane. The initial uptake
of dihydrostreptomycin by Escherichia coli is charac-terized by an electrostatic interaction between the pos-itively charged guanidlno centres of the antibiotic and
the anionic groups of LPS. There may be some accu-mulation in the periplasm, followed by a slow, energy-dependent penetration into the cytoplasm. After 15-30
min, a third phase of rapid, energy-dependent intracel-lular accumulation of dihydrostreptomycin begins.
This final phase appears to be Irreversible in Es-cherichia coli, and the antibiotic can only be released
from the cells by damaging the cell membranes with
organic solvents such as toluene. The molecular mech-anisms Involved in the energy-dependent phases of
aminoglycoside uptake are obscure. Although evi-dence for an uptake carrier system across the cytoplas-mic membrane for aminoglycosides is lacking, the re-cent discovery of aminoglycoside-speciflc efflux
pumps In several species of bacteria (Table 7.2) sug-132
7,3 Facilitated uptake of antimicrobial drugs
gests that carrier-dependent influx could also exist,
perhaps mediated by a physiological nutrient carrier
system. The binding of aminoglycosides to 7()S ribo-sornes enhances accumulation within bacteria. The in-trinsic resistance of anaerobic bacteria to the amino-glycosides may be due to their limited ability to
accumulate these antibiotics.
7.3.2 Uptake of antimicrobial drugs by
eukaryotic pathogens
The mechanisms involved in the transport of drugs into
fungal and protozoal pathogens have received less at-tention than their counterparts in bacteria. In fungi and
protozoa, the cytoplasmic membranes are likely to be
the major penneability barriers against drug influx.
The complex outer walls of fungi could conceivably
hinder the access of some larger molecules, but in gen-eral the coarseness of the chitin and mamian mesh-works probably offers little resistance to drug influx.
5-Fluorocytosine
This antifungal drag provides an example of facilitated
transport into fungal cells. As a close analogue of cy-tosine, 5~fluorocytosine is transported across the cyto-plasmic membrane by the cytosine permease. Rapid
intracellular metabolism of 5-fluorocytosine to several
toxic pyrimidine nucleotides contributes to the mainte-nance of a downward concentration gradient of un-changed drug into fungal cells. A disadvantage of
reliance on cytosine permease for the uptake of 5-flu-orocytosine is that mutational inactivation of tiie trans-port system results in resistance to the dmg.
Polyoxins and nikkomycins
The inhibitory activities of these peptidonucleoside
antibiotics depend on their transport into fungal cells
by a peptide permease system normally intended for
the accumulation of nutrient dipeptides. However, an-tibiotic transport on the pemiease system is subject to
competitive inhibition by dipeptides, whicii commonly
occur in the blood and tissues of the infected host. As
in the case of .5~fluorocytosine, resistance to polyoxins
and nikkomycins also readily from mutations
that inactivate the permease system.
Azoles
The lipophilic character of the azole antifungal drags
probably ensures their penetration of cytoplasmic
membranes by passive diffusion.
Antiprotozoal drugs
With the exception of the polycationic, antitrypanoso-mal drug suramin, which enters the parasite coniplexed
with senim proteins by a process of endocytosis, most
antiprotozoal drugs probably dilfuse passively across
the cytoplasmic membranes of their target pathogens.
Positively charged antimalarial compounds, such as
chloroquine, subsetiuently accumulate within the
acidic environment of the digestive vacuoles of the par-asites. The binding of certain antimalarial drugs to
heme released by the digestion of hemoglobin, and to
the heme crystalline polymer hemozoin (Chapter 6)
also contributes to the persistence of favourable chem-ical gradients into the vacuoles.
The antitrypanosomal drug eflornithine may
enter trypanosomes by a permease that normally fa-cilitates the uptake of ornithine and other polyaniines.
However, as yet there is no experimental evidence for
this.
FurtliBr rBsdiiiQ
Balkis, M. M. et at. (2002). Mechanisms of iiingal resist-ance. Drugs 62, 1025.
Denyer, S. P. and Maillard, J.-Y., (2002). Ceiliilai” imperme-ability and uptake of biocides and antibiotics in Gram-negative bacteria. /. Appl. Microbiol. 92, 35S.
Faller, M. et al. (2004). The structure of a mycobacterial
outer-membrane channel. Science 303. 1189.
Hancock, R. E. W. (1997). The bacterial outer membrane as
a drug barrier. Trends Microbiol. 5, 37.
Lambert, P. A. (2002). Cellular impermeability and uptake of
biocides and antibiotics in Gram-positive bacteria and
mycobacteria,/. Appl. Microbiol. 92, 46S.
133
Drug transport
Mao, W. et al. (2001). MexXY-Opr efflux pump is required Schulz. G. (2002). The structure of bacterial outer membrane
for antagonism of aminoglycosides by divalent cations proteins. Biochem. Biophys. Acta 1565, 308.
in Pseudomonas aeruginosa. Antimicrob. Agents and Tute, M. S. (1972). Principles and practice of Hansch analy-Chemother. 45, 2001. sis: a guide to the structure-activity relationships for
McKeegan, K. S. et al. (2002). Microbial and viral drug re- the medicinal chemist. Adv. Drug. Res. 6, 1.
sistance mechanisms. Trends Microbiol. 10 SuppL, S8. Yu, E. W. et al. (2003) . Structural basis of multiple drug-Niederweis. M. (2003). Mycobacterial porins—new channel binding capacity of the AcrB multidrug efflux pump.
proteins in unique outer membranes. Moke. Microbiol. Science 300, 976.
49, 1167. Zgurskaya, H. I. (2002). Molecular analysis of efflux pump-Nikaido, H. (2003). Molecultir basis of bacterial outer mem- based antibiotic resistance. Int. J. Med. Microbiol.
brane permeability revisited. Microbiol. Molec. Biol. 292, 95.
Rev. 67, 593.
134
CliBptcr cicilit
^ ^ ^
The development of safe, elfective antimicrobial drugs
has revolutionized medicioe in the past 70 years. Mor-bidity and mortality from microbial disease have been
drastically reduced by modem chemotherapy. Unfor-tunately, micro-organisms are nothing if not versatile,
and the brilliance of the chemotherapeutic achieve-ment has been dimmed by the emergence of microbial
strains presenting a formidable array of defences
against our most valuable drugs. This should not sur-prise us, since the evolutionary history of living organ-isms demonstrates their adaptation to the environment.
The adaptation of micro-organisms to the toxic haz-ards of antimicrobial drugs is therefore probably in-evitable. In Chapter 7 we saw that in many species of
bacteria a degree of intrinsic resistance to toxic chem-icals is conferred by cellular permeability barriers and
low levels of expression of a range of multidrug efflux
pumps. Both elements of defence can be enhanced in
acquired drug resistance by genetic changes in re-sponse to higher levels of drug challenge. The extraor-dinary speed with which antibiotic resistance has
spread among bacteria and certain viruses such as HIV
during the era of chemotherapy is due, in large meas-ure, to the remarkable genetic flexibility of these or-ganisms. Figure 8.1 provides an example in the alarm-ing rise of a drug-resistant pathogen, methicillin (or
multidrug)-resistant Staphylococcus aureus, in part of
the United Kingdom in just nine years.
The first account of microbial drug resistance
was given by Paul Ehrlich in 1907, when he encoun-tered the problem soon after the development of ar-senical chemotherapy against trypanosomiasis. As the
sulfonamides and antibiotics were brought into med-ical and veterinary practice, resistance against these
agents began to emerge. Resistance to antibacterial
and antimalarial drugs is now widespread, and in-creasing resistance to antifungal and antiviral drugs is
also a major concern. Our intention in this chapter is
to give an outline of the genetic background to the
problem of drug resistance; in Chapter 9 we describe
the major biochemical mechanisms that give rise to
resistance.
The tremendous advances made in the science of
bacterial genetics over the past 60 years have found a
most important practical application in ftirthering our
understanding of the problem of drug resistance. As a
result, we now have a fairly complete picture of the ge-netic factors underlying the emergence of drug-resist-ant bacterial populations. Although the study of the ge-netics of resistance in pathogenic fungi and protozoa is
less developed, advances in DNA sequencing technol-ogy should bring about significant improvements in
our understanding of these organisms. The depress-ingly rapid emergence of drug-resistant variants of the
human immunodeficiency virus during the chemother-apy of AIDS has given a powerful impetus to the study
135
Genetic basis of drug resistance
45
40
<
^ 35 -m 30 -V)
0)
Q. 25
E
« 20 -o 15
s? „
10
5 -n  I
~\
f l
Year
FIGURE 8.1 The recent rise in
the incidence of multidnig-resist-ant Staphylococcus aureus in
England and Wales. The vertical
axis indicates MRSA as a per-centage of ail samples of Staphy-lococcus aureus examined in
clinical laboratories. (Source:
Health Protection Agency.)
of the genetic and biochemical basis of resistance to
antiviral drugs.
The early studies on the genetics of drag resist-ance were bedeviled by an exhausting controversy. On
the one hand were those who believed that the devel-opment of a resistant cell population could be ex-plained by the phenotypic adaptation of the ceils to an
inhibitory compound without significant modification
in their genotype. The opposing faction tooli the view
that any large population of ceils which was sensitive
overall to a drug was liliely to contain a few genotypi-cally resistant cells. The continued presence of the
drag resulted in the expansion of the numbers of resist-ant cells by a process of selection.
Evidence gathered over the years strongly sup-ports the second of these two theories. As we shall see,
there are examples of phenotypic adaptation to antimi-crobial drugs, but such cells are usually genotypically
different from wild-type cells. When the selective
pressure applied by an antimicrobial drug is removed,
the resistant microbial population may revert to drag
sensitivity if the resistant cells are at a selective disad-vantage compared with drug-sensitive cells in a drug-free environment and could therefore eventually be
outnumbered by the sensitive cells.
8.1 Mutations and the origins of drug-resistance genes
Once it was accepted that drag-resistant organisms are
genetically different from the wild types, it was natu-ral to consider how such differences might arise. One
obvious possibility is that of spontaneous mutations.
These can arise in several ways:
1. Damage to the genome caused by adverse en-vironmental factors, including ionizing radi-ation and chemical mutagens.
2. Base-pahing eiTors during genomic replica-tion.
3. Frameshift mutations caused by the deletion
of segments of DNA which frequently occur
at short DNA repeat sequences.
4. Frameshift mutations caused by the intra-genic insertion of mobilizable genetic mate-rial, such as transposons, which coiTupt the
correct flow of information from the wild-type genome.
Spontaneous mutations are relatively rare—on
the order of one mutation per lO^-lO” cells per gener-ation—although in organisms lacking a proofreading
136
8.1 AAutations and the origins of drug-resistance genes
mechanism during genomic replication, as in HIV, the
mutation rate is much higher. There is also a mutator
phenotype in some bacteria which ensm’es a much
higher mutation frequency thannomial. An interesting
example is that of Helicobacter pylori, the bacterium
associated with peptic ulcer disease and gastric can-cers. As many as 33% of Helicobacter pylori strains
show an abnomially high rate of mutation to antibiotic
resistance. The nature of this high mutation frequency
is not known at present, but it is of potential relevance
to the clinical challenge of eliminating Helicobacter
pylori from the stomach by treatment with antibiotics.
When the vast numbers of organisms in microbial pop-ulations are considered, the probability of even low
mutation rates causing drug resistance is quite high.
The simple and elegant technique of replica plating
convincingly demonstrates that spontaneous mutations
to drug resistance can occur in drug-sensitive bacterial
populations in the absence of drags (Figure 8.2). A
spontaneous mutation may occasionally cause a large
increase in resistance, but resistance often develops as
a result of numerous mutations, each giving rise to a
small increase in resistance. In this situation, highly re-sistant organisms emerge only after prolonged or re-peated exposure of the microbial population to the
drug.
of starting treatment with the reverse transcriptase in-hibitor lamivudine, spontaneous mutation results in
the replacement in the reverse transcriptase of the cir-culating viruses of methionine-184 by valine, a change
associated with high-level resistance to lamivudine.
Resistance to some other drugs, such as azi-dothymidine, develop through successive mutations
which progressively reduce the drug sensitivity of the
target enzyme. The loss of sensitivity to an inhibitor
can be associated with reduced catalytic efficiency,
which places the virus particles at a competitive disad-vantage compared with viruses with unimpaired en-zyme. However, the reduction in enzymic efficiency
may be compensated by further mutations which pro-gressively restore enzymic activity. A recent alarming
discovery is that under laboratory conditions, AZT and
lamivudine adversely affect the fidelity of reverse tran-scriptase, thus further increasing the frequency of mu-tations. The clinical significance of this finding has yet
to be explored. As discussed in Chapter 4, the clinical
approach to coping with the rapid acquisition of drug
resistance by HIV is to treat patients with a combina-tion of drugs. In this way, the emergence of resistant
viruses can be delayed by months or even years. Even
so, the eventual emergence of viral populations resist-ant to multidmg therapy may be inevitable.
8.1.1 Spontaneous mutations and drug
resistance in HIV
8.1.2 Origin of clinically important resistance
genes in bacteria
A major challenge to the effective treatment of AIDS
is the unique and alarming speed with which HIV be-comes resistant to every drug deployed against it, in-cluding inhibitors of viral reverse transcriptase and
HIV protease. The origins of drug resistance in HIV lie
in the high rate of viral replication and the ease with
which spontaneous mutations arise in its RNA
genome. As a single-stranded RNA virus, HIV lacks a
proofreading mechanism to eliminate sequence eiTors
resulting from the low fidelity of HIV reverse tran-scriptase. As a result, mutations occur with high fre-quency. During the course of an infection, the combi-nation of high replication and mutation rates pemiits
rapid and extensive evolution of the viral population in
response to immunological and chemotherapeutic
challenges to its survival. For example, within weeks
Originally it was believed that spontaneous mutations
followed by the selection of resistant organisms in the
presence of a drug explained the emergence of drug-resistant populations. However, while this appears to
be true in the case of HIV, the discovery that bacteria
can acquire additional genetic material by conjugation,
transformation and transduction led to the conclusion
that spontaneous mutations make an important but not
exlusive contribution to the emergence and spread of
drug resistance in bacteria. Mutations underlie the up-regulation of drug efflux pumps and the reduction or
loss of porin function in many bacteria as well as the
increased expression of constitutive p~lactamases in
pathogens such as Enterobacter cloacae and Citrobac-ter freiindii. Spontaneous mutations also lead to the
progressive modification of p-lactamases, enabling
137
Genetic basis of drug resistance
Plain agar plate
heavily seeded
with drug-sensitive
culture
®
Area corresponding to location of drug-resistant mutant is picked off and Inoculated
\ into drug-free liquid medium
•• ^ -^ .
,-s^ Replica
w plating
Plain agar plate is
seeded with celis
from culture enriched
witii resistant mutants
Agar plates
containing anti-microbial drug
Drug-resistant
colonies
FIGURE 8.2 The teclinique of replica plating reveals the existence of drug-resistant cells in a population that is overall
drug-sensitive. A plain agar plate is heavily seeded with cells from the drug-sensitive culture and incubated until growth oc-curs. Cells ai’e transferred by a velvet pad to a plate containing the antibacterial drug; this plate is then incubated and the po-sition of any colonies noted. The area on the drug-free plate cori’esponding to the location of the resistant colony on the drug
plate is picked off and cultured in drug-free medium. Although still contaminated with sensitive cells, this culture will con-tain many more resistant cells than the original culture. Plating out of the ‘ enriched’ culture on a plain plate followed by repli-cation to a drug plate therefore reveals a higher number of drug-resistant colonies. The experiment shows that drug-resistant
mutants occur in a bacterial population not previously exposed to the drug.
them to cope with the many novel chemical variants of
p-lactams. Strains of Mycobacterium tuberculosis re-sistant to isoniazid, rifampin, pyrizinamide, ethambu-toi and streptomycin can all be explained by mutations
in the genes encoding the target sites for these dnigs.
Resistance to the ciuinolones regularly arises through
mutations in genes encoding the target DNA gyrase
enzyme.
As we shall see in the next chapter, the biochem-ical machinery conferring bacterial resistance to drugs
of major importance in medicine can be complex. Un-derstandably, therefore, there is considerable interest
138
8.1 AAutations and the origins of drug-resistance genes
in the origins of the genes that variously encode drug-inactivating enzymes, drug efflux pumps, enzymes
that depress dmg sensitivity by the covalent modifica-tion of drug targets and proteins that block the binding
of drugs to their targets. Antibiotic-producing bacteria,
such as the streptomycetes, protect themselves against
the toxic effects of their own antibiotics with enzymes
that inactivate aminoglycosides, chloramphenicol and
p-lactams. In addition, many streptomycetes express
p-lactamases even though they do not produce p-lac-tams, presumably as a protective measure against P-lactams synthesized by other organisms in the micro-environment. Genes encoding the pumped efflux of
tetracyclines, proteins that protect ribosomes against
tetracyclines, and the enzymic modification of riboso-mal RNA associated with resistance to erythromycin,
have ail been identified in streptomycetes. Nucleic
acid and protein sequence data support the suggestion
that genes for aminoglycoside-inactivating enzymes
found in aminoglycoside-resistant clinical isolates
may have originated from streptomycetes.
Mosaic genes
Although bacterial genes which encode antibiotic-inactivating enzymes and drug efflux pumps almost
certainly evolved in the very distant past, antibacterial
drug resistance mediated by mutations is generally
thought to have emerged during the modern era of
chemotherapy. In addition to point mutations, dele-tions and insertions, there is also the remarkable phe-nomenon of mosaic genes which arise by interspecies
genetic recombination. By far the most common
mechanism of resistance to p-lactam antibiotics is that
of antibiotic hydrolysis by p~lactamases, which are
probably of ancient origin. However, p-lactam re-sistance in several important pathogens, including
Haemophilus influenzae, Neisseria gonorrhoeae,
Streptococcus pneumoniae, Staphylococcus aureus
and Staphylococcus epidermidis, can also be caused
by penicillin-binding proteins (Chapter 2) with re-duced affinity for p-lactams. This type of resistance is
relatively rare because the killing action of p-lactams
depends on drug interactions with several high-molec-ular weight PBPs, and resistance therefore necessitates
reductions in p-lactam affinity in each PBP. Although
it is conceivable that such reductions in affinity could
have arisen gradually from incremental changes in
protein structure that were due to the accumulation of
mutations in the PBP genes, it is clear that recombina-tion amongst PBP genes from different species is a
major cause of the low-affmity PBP phenotype in
bacteria.
Analysis of the sequences of genes for FBP2
from penicillin-sensitive and penicillin-resistant men-ingococci and gonococci reveals that whereas the se-quences from penicillin-sensitive bacteria are unifomi,
the resistant gene sequences have a mosaic structure.
The mosaics are created when regions essentially iden-tical with those from penicillin-sensitive bacteria re-combine with regions that have significantly divergent
sequences. The mosaic genes encode PBP2 variants
with decreased affinity for penicillin. Sequence infor-mation obtained from bacterial DNA databases show
that the divergent regions in the mosaic genes originate
from Neisseria flavescens and Neisseria cinerea.
PBF2 prepared from specimens of Neisseria flaves-cens presewed from the preantibiotic era has a much
lower affinity for penicillin than PBP2 from either
Neisseria gonorrhoeae or Neisseria meningitidis. The
mosaic genes are thought to have arisen by inter-species recombination following transfoixnation by
DNA released from lysed cells (see later discussion)
amongst these bacteria. Mosaic genes encoding low-affinity PBPs la, 2x, 2b and 2a have been isolated from
Streptococcus pneumoniae resistant to both penicillins
and cephalosporins. Pneumococci are also readily
transformable, and the divergent regions of the mosaic
genes appear to have originated from several other
bacterial species. However, not all low-affmity PBPs
are the result of mosaic gene formation. An important
example is that of PBP2a, which is encoded by the
rnecA gene responsible for the notorious methicillin-resistant Staphylococcus aureus. The mecA gene is lo-cated on a large (~50 kilobases) DNA element inserted
into the bacterial chromosome. This so-called resist-ance island encodes proteins that are homologous with
transposases and integrases (see later discussion)
which probably catalyze both excision and integration
of the mecA gene. Staphylococcus aureus may have
acquired mecA by gene transfer from another
organism.
Interspecies recombination amongst meningo-cocci also resulted in mosaic genes that encode sulfon-139
Genetic basis of drug resistance
amide-resistant dihydropteroate synthase (Chapter 4).
Allelic variations in the tetM gene, which detemiines
the ribosome protection form of resistance to tetracy-cline (Chapter 9), are due to recombination amongst
the distinct tetM alleles found in Staphylococcus au-reus and Streptococcus pneumoniae. TetM genes are
widely distrbuted in both Gram-positive and Gram-negative bacteria. Finally, it should be noted that the
generation of mosaic genes by interspecies recombina-tion in bacteria is not limited to resistance genes. The
phenomenon is widespread in bacteria and underlies,
for example, the highly divergent genes that encode
the proteins of the outer membranes of Neisseria spp.
8.2 Gene mobility and transfer in bacterial
drug resistance
The spread of dmg resistance amongst bacterial patho-gens owes much to the remarkable ability of bacteria
to mobilize genes in both chromosomal and plasmid
DNA and to transfer and exchange genetic infomia-tion. Evidence that drug resistance could be trans-ferred from resistant to sensitive bacteria came from
combined epidemiological and bacterial genetic stud-ies many years ago in Japan. The first clue was pro-vided by the isolation, from patients suffering from
dysentery, of strains of shigella resistant to several
drags, including sulfonamides, streptomycin, chlo-ramphenicol and tetracycline. Even more striking was
the discovery that both sensitive and multiresistant
strains of shigella could occasionally be isolated from
the same patient during the same epidemic. Most pa-tients harbouring multiresistant shigella also had mul-tiresistant Escherichia coli in their intestinal tracts.
This suggested that drug resistance markers might be
transferred from Escherichia coli to shigella and vice
versa. Subsequently it was confirmed that Gram-nega-tive bacteria can indeed transfer drug resistance not
only to cells of the same species but also to bacteria of
different species and genera. As we shall see, the phe-nomenon of horizontal gene transfer, as it is now
termed, is not confined to Gram-negative bacteria but
also occurs in Gram-positive organisms. However, be-fore we describe the transfer of drug-resistance genes
between bacterial cells, we must first consider the
movement of genes within the bacterial genome itself.
8.2.1 Transposons and integrons
For many years the movement of genes among plas-mids and chromosomes was believed to result from
classic recombination dependent on the product of the
bacterial recA gene and the reciprocal exchange of
DNA in regions of considerable genetic homology.
This permits the exchange of genetic information only
between closely related genomes. However, such a re-stricted phenomenon seemed unlikely to explain the
widespread distribution of specific resistance determi-nants. It is now clear that the acquisition of genetic
material by plasmids and chromosomes in both Gram-negative and Gram-positive bacteria is not limited by
classic recA-dependent recombination. Replicons,
known as transposons, can insert themselves into a va-riety of genomic sites that often have little or no ho-mology with the inserting sequence, although such
transposition events are rare, one in 10^^-10^ cells per
generation. Because there are many possible transpo-son, insertion sites in the bacterial genome, a higher
frequency of insertion would probably result in too
great a rate of gene disruption and mutation, in the
simplest transposons, the whole of the genetic infor-mation is concerned with the insertion function. Inser-tion sequences (IS elements) are sequences of approx-imately 750-1600 base pairs encoding a specific
endonuclease called a transposase. The IS elements
are flanked by inverted repeats of 15-20 base pairs that
are characteristic of individual transposons. Immedi-ately adjacent to the inverted repeats are short direct
repeats (5-11 base pairs) whose sequences depend on
the target site where the transposon is inserted.
The genes for drug resistance are carried by com-posite transposons designated by the prefix Tn. In the
class 1 transposon, Tn9, the gene encoding the enzyme
that confers resistance to chloramphenicol, chloram-phenicol acetyl transferase (Chapter 9), is flanked by
two IS elements. These genes are again bounded by in-verted repeats which in turn are flanked by short direct
repeats (Figure 8.3a). Tn3 (Figure 8.3b) is a class 2,
complex transposon which contains the genes for the
transposase and for resolvase, an enzyme that cat-alyzes recombination between the insertion sequences.
These genes, together with the gene for p-lactamase,
are flanked by the inverted repeats. Transposons car-rying arrays of drag-resistance genes have been
140
8.2 Gene mobility and transfer in bacterial drug resistance
(a)
Inverted repeat Qene for chloramphenicol ‘””^’*^’^ ‘^^^^^
A/-acetyl transferase
IS  IS
Direct repeat  Direct repeat
(b)
Inverted repeat Inverted repeat
Resolvase
Transposase  Beta-lactamase
FIGURE 8.3 The structure of traiisposoiis: (a) Class I
trausposou Tn9, which includes the gene for bacterial resist-ance to clilorampheiiicol. IS, insertion sequence, (b) This
class 2 complex transposon, Tn3, confers resistance to (i-lac-tarti antibiotics. The gene dimensions are not drawn to scale.
identified in both Grarn-positive and Gram-negative
bacteria.
In some trarisposoris the drug-resistance genes
are aiTanged within structures called integrons. These
consist of an iiit gene that encodes for a site-specific
recombination enzyme or integrase, an integron recep-tor site, atti, and one or more gene cassettes. Usually
each gene cassette contains a single drug-resistance
gene and a specific recombination site, called a 59-base-pair element, located downstream of the gene.
The association of the integrase function with the spe-cific recombination site confers mobility on gene cas-settes and the ability of integrons to capture and inte-grate whole arrays of cassettes. Cassette excision is the
reverse of integration and generates a circularized
form of the cassette which may exist independently for
extended periods. There are multiresistance integrons
that confer various combinations of resistance to (3-lactams, aminoglycosides, trimethoprim, chloram-phenicol, antiseptics and disinfectants. More than 40
gene cassettes and three classes of integrons ttre
known, and the reader is referred to reviews listed
under “Further reading’ for detailed descriptions of
this complex field. Although integrons are found in
transposons, they also occur frequently as independent
entities.
Mobilization of class 1 transposons along with
their complement of drug-resistance genes occurs by
nonreplicative trtmsposition; that is, the transposon
copy number is not increased during transposition. The
trttnsposon is excised from its original site ttnd reinserts
into a new site virtually jinywhere within the bacterial
genome, including chromosomal and plasmid sites.
Class 2 transposons, on the other hand, are mobilized by
a replicative process. The replicated copy of the trans-poson inserts into a new site. In both cases the trans-posase first introduces staggered cuts, nine base pairs
apart, at the donor site in the transposon and at the in-tended recipient site. The recipient site, 4-12 base pairs
in length, is then replicated to form noninverted, or di-rect repeats on either side of the inserted transposon. In
contrast, as described earlier, gene cassette excision and
capture in integrons is accomplished by site-specific re-combination, although there are rare examples of cas-settes that integrate into nonspecific sites.
To summarize, therefore, drug-resistance genes
in bacteria are subject to two major modes of intra-genic mobilization that promote a continual flux of re-sistance determinants around bacterial DNA:
1. Resistance genes associated with transposons,
whether or not as cassettes, are mobilized
along with the rest of the transposon and can
be inserted essentially anywhere in the bacte-rial genome, either cliroinosome or plasmid.
2. Both transposon-associated and independent
integrons containing resistance gene cas-settes exchange and capture cassettes by site-specific recombination.
Conjugative transposons
The transposons described so far are by themselves un-able to promote gene transfer by conjugation between
141
Genetic basis of drug resistance
bacterial cells, although they participate as passengers
during R-plasmid transfers. However, there is another
type of transposon, refeiTed to as conjugative trans-posons. These are discrete DNA elements normally in-tegrated into bacterial chromosomes which encode
proteins that enable excision of the transposon from
the chromosome and its transfer to recipient bacteria
by intercellular conjugation. Conjugative transposons
occur widely in Gram-positive bacteria and contribute
to the spread of drag resistance among major
pathogens such as Streptococcus spp. and Entewcoc-cus spp. In Gram-negative bacteria, conjugative trans-posons were first identified in the genus Bactewides,
which accounts for 25-30% of the microbial flora of
the human intestinal tract. Subsequently, conjugative
transposons carrying drug-resistance genes have been
found in other Gram-negative species, including Sal-monella, Vibrio and Proteus.
The potential for conjugative transposons to
spread drag resistance was highlighted when the first
conjugative transposon to be discovered, Tn916, was
found to carry resistance to tetracycline. Originally
Tn916 was detected on the chromosome of a multire-sistant isolate of Enterococcus (previously called
Streptococcus) faecalis, and it was also observed to in-tegrate readily into coresident plasmids and into many
sites of the chromosomes of bacterial recipients of
Tn916. A closely related conjugative transposon,
Tnl545, found in Streptococcus pneumoniae, also me-diates tetracycline resistance as well as resistance to
erythromycin and kanamycin.
The mobilization of conjugative transposons
from bacterial chromosomes involves the following
steps, although details of the initial signals for mobi-lization are not fully defined:
1. Staggered cuts are introduced at each end of
the transposon, leaving 6-nucleotide, single-stranded stretches of DNA, known as cou-pling sequences.
2. The noncomplementary coupling sequences
are then ligated to generate covalently closed,
double-stranded circular inteixnediates.
3. During the insertion stage, the coupling se-quences foixn temporary non-base-pairing
interactions with the target site, which can be
in either a coresident plasmid or the chromo-some of a recipient bacterium after conjuga-tion. It is not yet clear how correct base pair-ing is subsequently established, but it may in-volve either replication through the insertion
region or repair of a mismatch. The insertion
process of conjugative transposons differs
from that of ‘true’ transposons in that the re-cipient or target site is not replicated.
The intercellular conjugation process promoted
by conjugative transposons is not well understood. Un-like the process mediated by R-plasmids in Gram-neg-ative bacteria (see next section), surface pili do not ap-pear- to be involved. It is clear that only single-stranded
copies of the transposons are transfen-ed to recipient
cells during conjugation.
The intercellular- traffic of conjugative trans-posons is highly regulated. Many Bacteroides trans-posons caiTy the tetQ gene for tetracycline resistance,
which is dependent upon libosomal protection. Re-markably, tetracycline is a highly effective stimulant of
conjugative transposon-mediated mating in these
species. A suite of transposon genes activated by tetra-cycline promotes transposon mobilization and self-transfer as well as mobilization of coresident plasmids
sharing the same donor cells. Tetracyclines can there-fore stimulate the spread of the resistance genes
throughout the bacterial population of the intestinal
tract, including many other bacterial species.
Conjugative transposons comprise a highly vari-able group of mobilizable DNA elements, and the
reader is directed to references under ‘Further reading’
for more detailed descriptions.
8.2.2 R-plasmids
Cellular conjugation mediated by R-plasmids is the
major mechanism for the spread of drug resistance
through Gram-negative bacterial populations. R-plas-mids usually exist sepai-ately from the bacterial chro-mosome. They consist of two distinct but frequently
linked entities:
1. The genes that initiate and control the conju-gation process, and
2. A series of one or more linked genes, often
found within transposon-integron com-142
8.2 Gene mobility and transfer in bacterial drug resistance
plexes, which confer resistance to antibacter-ial agents.
The conjugative region is closely related to the F-plas-mid, which also confers on Gram-negative bacteria the
ability to conjugate with cells lacking an F-plasmid. A
complete R-plasmid resembles the F-prime plasmid
(F’) in carrying genetic material additional to that
which controls conjugation.
A great variety of R-plasmids have been de-scribed which carry various combinations of drug-resistance genes. Other phenotypic characteristics
conferred by R-plasmids cao be used in systems of
classification. These include the ability (fi’*) or uiabil-ity (fi”) to repress the fertility properties of an F~plas-niid coresident in the same cell; the type of sex pilus
(see later discussion) that the R~plasmid detemiines;
the inability of R-plasmids to coexist in a bacterium
with certain other plasmids, which permits the division
of R-plasmids into several incompatibility groups; and
finally, the presence of genes in the R-plasmid that
specify DNA restriction and modification enzymes. R~
Plasmids are usually defined by a combination of these
properties.
Molecular properties of R-plasmids
R-Plasmids can be isolated from host bacteria as circu-lar DNA (Figure 8.4) in both closed and nicked fonns,
and both forms coexist in the cell. The closed circular
structure is probably adopted by R-plasmids not en-gaged in replication. The contour lengths and thus mo-lecular weights of isolated R-plasmids depend very
much on the host bacterium and the culture conditions
prevailing immediately belbre the isolation procedure.
The R-plasmid may sometimes dissociate into its con-jugative and resistance detenninants. This is more
common in some host species, e.g. Proteus mirabilis
and Salmonella typhimurium, than in Escherichia coU,
where dissociation is rare. Dissociation seems to de-pend on the activity of a simple transposon that may be
inserted at the junction of the two regions. The molec-FIGURE 8.4 Electron micro-graph of R-plasmid DNA isolated
from Proteus mirabilis hai’bour-ing an R-plasmid with resistance
markers to streptomycin, sulfon-amides and chloramphenicol.
The circulai’ DNA has a total
length of 28..5 [iru. [This photo-graph is reproduced from / . Bac-teriol. 97, 383 (1969) by kind
peniiission of Dr. Royston
Clowes and the American Society
for Microbiology.]
143
Genetic basis of drug resistance
ulai- masses of between 50 x 10^ and 60 x 10^ kDa of
the conjugative regions from R-plasmids are much
greater than those of the drug-resistance genes. For ex-ample, the genes for chloramphenicol, streptomycin,
spectinomycin and sulfonamide resistance have a
combined molecular mass of only 12 x 10^ kDa.
The copy number of R-plasmids harboured by in-dividual bacteria is deteimined by the properties of the
plasmid and its hosts as well as the culture conditions.
As a general rule (to which there are exceptions), the
lai-ger R-plasmids ai^e present in only a limited number
of copies (between one and fouiO per chromosome in
Escherichia coli, whereas in Proteus mirabilis the
number is much more variable and even varies during
the growth cycle. Conditions that give rise to an in-creased number of R-plasmid copies aix sometimes as-sociated with enhanced resistance. However, the level
of cellular resistance does not always reflect the num-ber of resistance gene copies. For example, although
the number of R-plasmid copies is frequently greater
in Proteus mirabilis than in Escherichia coli, the level
of resistance to several drugs expressed in the fonner
organism is usually lower than in Escherichia coli.
Cellular conjugation and R-plasmid transfer
Cells bearing an R-plasmid (R””) are characterized by
their ability to produce surface appendages known as
sex pili. The sex pili of R^ bacteria resemble those pro-duced by F”*” organisms. When R^ cells are mixed with
sensitive R” cells, mating paks are immediately
formed by surface interaction involving the sex pili.
The transfer of a copy of the R-plasmid from the R^ to
the R” cell begins, and the acquisition of the R-plasmid
by the recipient cell converts it to a fertile, drug-resist-ant cell that can in turn conjugate with other R” cells.
In this way drag resistance spreads rapidly tlirough the
bacterial population. Uncovering the details of bacter-ial conjugation and the transfer of DNA has challenged
investigators for many yeai’s. Although a wealth of in-formation has emerged, several critical steps in the
process remain to be defined. What may appear super-ficially to be a fairly simple phenomenon is in fact
highly complex, and here we provide only an outline
of the process.
The conjugal pair is brought into close surface
contact by the attachment of the pilus of the donor cell
to the recipient and its subsequent retraction by a
process of ‘reeling in’. The interaction between the
cells triggers cleavage of a specific strand of the donor
R-plasmid in the origin-of-transfer site (oriT) within a
protein-DNA complex called the relaxosome, which
contains the DNA strand-cleaving, or relaxase, en-zyme. Only one strand, the T-strand, which is un-wound following plasmid cleavage at the oriT, is trans-ferred in a 5′ to 3′ direction from the donor to the
recipient cell. Deteimining how the T-strand leaves the
donor cell and penetrates the recipient has been a
major research challenge. The extrusion of DNA from
donor cells has some features in common with the se-cretion of toxins and virulence proteins referred to as
the type IV secretory process. So-called coupling pro-teins are involved in transferring the exported proteins
across the complex cell envelope of Gram-negative
bacteria into the external environment. A recent sug-gestion is that in a typical plasmid such as R388, after
generation of the T-strand of DNA, the relaxase pro-tein, TrwC, serves as a pilot to guide the T-strand into
the type IV secretory pore. A coupling protein, TrwB,
then ‘pumps’ the DNA strand through the transporter-pore system, which perhaps involves ATP hydrolysis
as an energy source. The details of the final transfer
into the recepient cell remain shrouded in uncertainty,
although the model suggests a possible role for the sur-face pili in breaching the peixneability baniers of the
recipient cells. It must be emphasized, however, that
this proposal is speculative and is supported mainly by
circumstantial evidence on the nature of the coupling
and pilot proteins and the effects of loss-of-function
mutations in these proteins on the DNA transfer
process. Once inside the recipient cell, the ends of the
transfen-ed strand are ligated to produce covalently
closed circular DNA. Finally, DNA replication, cat-alyzed by DNA polymerase III, generates double-stranded plasmid DNA from the single-stranded mole-cules in both donor and recipient cells.
Fortunately, the frequency of R-plasmid transfer
is much lower than that of F transfer. Following the in-fection of an R cell with an R-plasmid, a repressor
protein accumulates which eventually inhibits sex
pilus formation. The ability to conjugate is therefore
restricted to a short period immediately after acquisi-tion of the R-plasmid. Sex pilus production in F+ cells,
in contrast, is not under repressor control and conjugal
144
8.2 Gene mobility and transfer in bacterial drug resistance
activity is therefore unrestricted. Mutant R-piasmids
without the ability to restrict sex pilus formation ex-hibit a much higher frequency of R~plasmid transfer.
ft is also worth noting that certain R~plasrnids,
and other self-mobihzing plasmids without drug-resistance genes, can promote the intercellular transfer
of coresident plasmids that lack the genetic informa-tion for conjugation and transfer. Such mobilizable
plasmids achieve transfer either by using the conjugal
apparatus furnished by the self-mobilizing plasmids
(trans mobilization) or by integration with these plas-mids (cis mobilization). Cooperative interactions
amongst plasmids add significantly to the genetic flex-ibility of bacteria and to their ability to spread drug re-sistance through microbial populations.
Clinical importance of R-plasmids
It is generally agreed that R~plasmids existed before
the development of modern antibacterial drugs.
Clearly though, the widespread use and abuse of these
drugs led to a vast increase in drug resistance caused
by R~plasmids. This has been especially noticeable in
farm animals, which in many countries receive clini-cally valuable antibacterial drugs, or compounds
chemically closely related to them, in their foodstuffs
as growth enhancers. The animals act as a reservoir for
Gram-negative bacteria, such as Escherichia coli and
Salmonella typhimurium, harbouring R-plasmids po-tentially transferable to man. Fortunately, some coun-tries have restricted the growth-enhancer application
of clinically valuable antibiotics, although contraven-tion of the regulations is not unknown.
The adverse contribution of R-plasmid~mediated
dmg resistance to human morbidity and mortality is
undeniable. For example, the major requirement in the
treatment of neonatal dianliea caused by certain path-ogenic strains of Escherichia coli (a potentially dan-gerous condition) is the prevention of fatal dehydra-tion. Even so, elimination of the pathogenic organisms
may also be important, but this is often difficult in the
face of multiple resistance to commonly used antibac-terial agents. In one notorious outbreak, the children
were infected with a pathogenic strain of Escherichia
coli resistant to P-lactams, streptomycin, neomycin,
chloramphenicol and tetracyclines. The infection
eventually responded to gentamicin, which was the
only drug of those tested to which the pathogenic bac-teria were sensitive. Another potentially alamiing de-velopment has been the appeaiimce of the typhoid or-ganism, Salmonella typhi, carrying an R-plasmid with
genes for resistance to chloramphenicol and cotrimox-azole, the dnigs most commonly used to treat this
disease.
Certain ecological factors probably limit the clin-ical threat posed by R-plasmids. In the environment of
the gastrointestinal tract, the conjugal activity of R”^
bacteria may be less than that in the ideal culture con-ditions of the laboratory. The emergence of an R’*’ pop-ulation of bacteria during antibiotic therapy is more
likely to result from selection of resistant cells than
from extensive conjugal transfer of resistance. After
cessation of antibiotic treatment, the numbers of R”^
bacteria in the feces fall, although usually not to zero.
Low-level antibiotic contamination of the environment
and/or a previously unsuspected persistence of drug-resistance and resistance-transfer genes in bacterial
populations may contribute to this potentially serious
situation.
8.2.3 Conjugative plasmids in Gram-positive
bacteria
Although the existence of conjugative plasmids
which carry drug-resistance genes in Gram-positive
bacteria has been recognized for some time, their
role in the dissemination of drug resistance is only
now being more thoroughly investigated. As de-scribed earlier, the physical contact between Gram-negative bacteria necessary for the conjugal transfer
of genes is largely attributable to the sex pili. A sim-ilar mechanism has not been identified in major
Ch^am-positive pathogens. Major differences in the
cell envelopes of Gram-positive and Gram-negative
bacteria suggest that the modes of intercellular DNA
transfer may also differ substantially. However, se-quencing studies on several Gram-positive conjuga-tive plasmids reveal homologies with proteins of the
type TV secretion system involved in Gram-negative
R-plasmid transfer. Furthermore, the relaxosome of
Ch^am-positive conjugative plasmids is similar to that
of the R-plasmids.
145
Genetic basis of drug resistance
8.2.4 Nonconjugal transfer of resistance
genes
Transduction
During the two distinct processes of phage transduc-tion, which occurs in both Gram-positive and Gram-negative bacteria, genetic infomiation is transferred by
phage particles from one bacterium to a related phage-susceptible cell.
Generalized transduction may occur during the
lytic phases of both viralent and temperate phages.
Fragments of degraded host chromosomal and plasmid
DNA, which may carry drug-resistance determinants,
can become packaged into newly generated phage par-ticles, leaving behind some or all of the phage DNA.
Lytic release of the phages enables them to inject both
phage and donor host DNA into other bacteria, some
of which is integrated into the recipient genome, al-though between 70 and 90% of the transferred DNA is
not integrated in this way. Nevertheless, nonintegrated
DNA may also survive in the recipient and replicate as
a plasmid. In abortive transduction, none of the trans-ferred DNA is integrated into the recipient genome,
but again, the nonintegrated DNA survives and repli-cates as a plasmid. The drug-resistant phenotype in
recipient bacteria is maintained in both types of
transduction.
The process of specialized transduction depends
on an error in the lysogenic cycle. Excision of the
phage DNA from the host genome during induction of
the lytic phase is insufficiently precise and canies
some of the bacterial DNA along with phage DNA.
The resulting phage genome contains up to 10% of the
bacterial DNA next to the phage integration site in the
bacterial genome. Clearly this process has the poten-tial for generating infectious phage particles that carry
bacterial genes for drag resistance. Although recombi-nant or defective phages arising from specialized
transduction are able to inject their DNA into new-hosts, they cannot reproduce independently, nor are
they lysogenic. Specialized transduction has been
most thoroughly studied with lambda phage, and the
reader is referred to a relevant text on bacterial genet-ics for a detailed account of the mechanisms involved
in lambda phage transduction. In general terms, rela-tively little of the injected recombinant phage DNA is
integrated into the bacterial genome unless the phage
population contains nomial phages as well as the de-fective ones. The nomial phages insert into the bacter-ial genome at a specific att site that resembles the
phage att site. The insertion process generates two hy-brid bacterial-phage att sites where the defective re-combinant phage DNA can insert. The presence of the
normal phage renders the bacteria susceptible to the
induction of phage-mediated lysis. The resulting
lysate, containing roughly equal amounts of defective
recombinant phages and nomial phages, is highly effi-cient in transduction.
Although transduction of drug-resistance deter-minants is readily demonstrated under laboratory con-ditions, its contribution to the spread of drug resistance
in natural and clinical settings is difficult to quantify.
Transformation
Under certain conditions most genera of bacteria can
absorb, integrate and express fragments of ‘naked’
DNA containing intact genetic information, including
that for drag resistance. The phenomenon of transfor-mation of bacteria by DNA is more complex than it
may appear at first glance, it has been most thoroughly
investigated in the species in which it was first discov-ered more than 60 years ago. Streptococcus pneumo-niae. Only bacteria in a state of competence are able to
absorb and integrate exogenous DNA into their own
genome. Streptococcus pneumoniae becomes compe-tent during exponential growth when the population
density exceeds 10^-10*^ cells m\r^. Under these con-ditions the bacteria secrete a competence factor that
stimulates the synthesis of up to 10 other proteins es-sential for transfomiation. Competent cells bind dou-ble-stranded DNA, provided that its molecular mass is
at least 500 kDa. One strand of the DNA is hydrolyzed
by an exonuclease associated with the cell envelope
and the remaining strand enters the cell while bound to
competence-specific proteins, integration into a ho-mologous region of the recipient genome probably oc-curs by nonreciprocal general recombination.
While competent Streptococcus pneumoniae can
take up VmA from a range of bacterial species, the
Gram-negative opportunist pathogen//oemop/yf/Mj in-fluenzae is more fastidious and only accepts DNA
from closely related species. Furthermore, Haemo-146
8.4 Genetic basis of resistance to antifunial drugs
philus influenzae does not produce a competence fac-tor but absorbs double-stranded DNA encapsulated in
membrane vesicles. Although transformation is a
widespread phenomenon, it is not surprising to find
important differences in the details of the actual mech-anism among the bacterial species. The complexity
and diversity of the transformation system indicates its
evolutionary importance in the exchange of genetic in-formation in the bacterial world. The frequency of
transformation of genetic markers can be as high as
10″”””-^ under laboratory conditions when artificially high
levels of DNA are added, i.e. one cell in every thou-sand takes up and integrates a particular gene. Trans-formation is probably therefore a significant contribu-tor to the spread of drug-resistance genes. A specific
example of the relevance of transfomiation to drug re-sistance is illustrated by the existence of the mosaic
genes for PBPs with diminished affinity for p-lactam
antibiotics, for sulfonamide-resistant dihydropteroate
synthase and for the tetM form of tetracycline resist-ance refen-ed to previously. Unfortunately, the extent
to which transfomiation occurs in relevant environ-ments, such as hospital wards and the intestinal tract,
cannot be quantified with any certainty.
s%^ %M B «# KJ$ tPi 9 S %?%j IMi B CS s>%^ I «ij^ ly . S %J S ml %j | S %?«iJ^S «iJ^%S’l! 9 9 9.j> %? I 9 9
G B’^SS’W^ 9% jf% jffi’S i T 9 S Jf df% STi *] i tf^ w £3i 9″ B ^ idii i in^llclliwu iJcll.Blcl id
We have seen how the capture of several detemiinants
for resistance to individual drugs by mobile and trans-ferable genetic elements can result in bacteria acquir-ing the multidmg-resistant phenotype. However,
Gram-negative bacteria have yet another means of
achieving a similar end. Genes called regulons exert
transcriptional control over several chromosomal
genes that confer resistance to many antibiotics by re-stricting access to their molecular targets. The marA
locus in Escherichia coli contains an operon, marRAB,
whose expression is inducible by at least two antibi-otics, tetracycline and chloramphenicol, and by uncou-plers of oxidative phosphorylation. Resistance to these
and to many other drugs is increased by the induction
process. The MarA protein encoded in the operon be-longs to a family of transcriptional regulators and con-trols the expression of numerous other genes, probably
in concert with MarR, a regulator protein, and the
MarB protein, whose function is currently unknown.
The mar operon, when induced, upregulates the mul-tidrug efflux pump AcrB and its linked outer mem-brane component, TolC (Chapter 7). The mar operon
also increases the expression of niicF, an RNA mole-cule which slows the expression of the OmpF porin
channel protein. Enhanced drug efflux combined with
diminished outer membrane permeability caused by a
reduction in the expression of OmpF underlies the in-crease in resistance to a range of structurally unrelated
drags that is mediated by the inarA locus.
Another global regulator of drug resistance,
raniA, has been found in the Gram-negative pathogen
Klebsiella pneumoniae. The ramA gene encodes a
transcriptional activator protein, RamA, that is dis-tantly related to the MarA protein of Escherichia coli.
Like MarA, RamA confers resistance to a wide range
of structurally unrelated drugs by upregulating the ex-pression of several genes. RamA-mediated resistance
also appears to depend upon a combination of drug ef-flux and a reduction in the level of the OmpF protein
in the outer membrane.
Genetic loci resembling marA and ramA are
widespread among Gram-negative bacteria, and the
regulation of multidmg resistance by these operons is
likely to be a significant contributor to the overall
problem of dmg resistance in Gram-negative bacteria.
sntifunosl druiis
The yeast pathogen Candida albicans exists exclu-sively as a diploid organism, i.e. it has two copies of
every gene. Allelic differences between the two gene
copies are commonly encountered among climcal
isolates of Candida albicans. Resistance to the azole
antifungal drugs is often caused by mutations in the
target enzyme 14{x-demethylase (Chapter 3) encoded
by ergll. For example, there is a point mutation
(R467K) which replaces lysine with arginine at posi-tion 467. Analysis of azole-resistant clinical isolates
of Candida albicans showed that resistance was high
when both gene copies canied the R467K mutation
compared with heterozygotes in which only one al-lele was mutated. Resistance to the nucleoside ana-logue, 5-fluorocytosine in Candida albicans is also
147
Genetic basis of drug resistance
modulated by allelic differences. As described in
Chapter 4, 5~FC is first converted in the cells to 5-flu~
orouracil by the enzyme cytidine deaminase. A muta-tion in one allele for this enzyme results in partial re-sistance to 5-FC because the unaffected allele
continues to express the wild-type enzyme. The gen-eration of cells homozygous for the disabled gene via
mitotic recombination causes high-level resistance to
5-FC. In contrast, the haploid yeast Candida neofor-mans can acquire high-level resistance to 5-FC in a
single step because there is only one copy of the gene
for cytidine deaminase. This situation is reflected in
the clinic, where high-level resistance to 5~FC is
more commonly detected in Candida neoformans
than in Candida albicans.
Transposable genes and plasmids occur widely in
fungi, especially in yeasts. However, at present there is
no evidence that these elements contribute to the
spread of drug resistance among fungal pathogens
such as Candida spp., and there is no phenomenon in
these organisms comparable with that of mobilizable
and transmissable drug-resistance genes in bacteria.
8.5 Genetic basis of drug resistance to
antimalarial drugs
As described in Chapter 9, the biochemical basis of re-sistance to antimalaiial drugs can be due either to re-duced drug uptake by the parasite, as in the case of
chloroquine and possibly other quinoline drugs, or to
the loss of dnig sensitivity in target enzymes, includ-ing dihydrofolate reductase and dihydropteroate syn-thase. Spontaneous mutations resulting in the selection
of drug-resistant parasites in response to sustained
drug challenge appear to be a common pattern
throughout the malarious regions of the world. Sexual
reproduction and genetic recombination in the malar-ial parasite provide additional opportunities for the
spread of drug resistance.
Further reading
Balis, M. M. et al. (2002). Mechanisms of fungal resistance.
Drugs 62, 1025.
Chopra, I. and Roberts, M. (2001). Tetracycline antibiotics:
mode of action, applications, molecular biology and
epidemiology of bacterial resistance. Microbiol.
Molec. Biol. Rev. 65, 232.
Courvaiin, P. (1994). Transfer of antibiotic resistant genes
between Gram-positive and Gram-negative bacteria.
Antimicrob. Agents Cliemotlier. 38, 1447.
Errington, J. et al. (2001). DNA trtmsport in bacteria. Nat.
Rev. Mol. Cell. Biol. 2, 538.
George, A. M., Hall, R. M. and Stokes. H. W. (1995). Mul-tidrug resistance in Klebsiella pneumoniae: a novel
gene ramA confers a multidrug resistance phenotype in
Escherichia coli. Microbiol. 141, 1909.
Gomis-Ruth, F. X. et al. (2002). Structure and role of cou-pling proteins in conjugal DNA transfer. i?<?i’. Micro-biol. 153, 199.
Grohmann, E. et al. (2003). Conjugative plasmid transfer in
Gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67,
277.
Flenriques-Normark, B. and Nomiark, S. (2002). Evolution
and spread of antibiotic resistance. / . Int. Med. 252,91.
Le Bras, J. L. and Durand, R. (2003). The mechanisms of re-sistance to antimalarial drugs in Plasmodium falci-parum. Fundam. Clin. Pharmacol. 17, 147.
Llosa, M. et al. (2002). Bacterial conjugation: a two-step
mechanism for DNA transport. Molec. Microbiol.
45,1 .
Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, R, Balti-more, D. and Darnell, J. (2000). Molecular Cell Biol-ogy. 4th edn. W. H. Freeman & Co., New York.
Pembroke, J. T. et al. (2002). The role of conjugative trans-posons in the Enterobacteriaceae. Cell. Mol. Life Sci.
59, 2055.
Pillay, D. (2001). The emergence and epidemiology of resist-ance in the nucleoside-experienced HIV-infected pop-ulation. Antivir. Titer. 6 Suppl. 3, 15.
Prescott. L. M., Harley, J. R and Klein, D. A. (2004). Micro-biology, 5th edn, William C. Brown, Dubuque, lA.
Recchia. G. D. and Hall, R. M. (1995). Gene cassettes: a new
class of mobile element. Microbiol. 141, 3015.
148
C himtpr ninp
Although the individual modes of resistance to antimi-crobial drugs are diverse, they can be grouped into a
set of general mechanisms that account for most types
of resistance encountered in medical practice.
1.
4.
5.
Conversion of the active drag to an Inactive
derivative by enzyme(s) synthesized by the
resistant cells;
Loss or downregulation of an enzymic mech-anism required to convert an inactive drug
precursor to an active antimicrobial agent;
Loss of sensitivity of the drag target site as a
result of: (a) modification of the target site by
enzyme activity in the resistant cells, (b) mu-tation(s) in the microbial chromosome aftect-ing the target, (c) horizontal acquisition of
genetic information encoding a drug-resist-ant form of the target enzyme, overproduc-tion of the drag-sensitive enzyme, or pro-tein(s) that protect the target site from
inhibition;
Removal of the drug from the cell by drug ef-flux pumps.
Reduction in cellular permeability to drags
caused by changes in the cell envelope.
The actual level of cellular resistance observed may be
due to a combination of factors. In Giram-negative bac-teria, for example, resistance often results from the low
pemieability of the outer membrane combined with
drug efflux and other mechanisms. In addition there are
unique modes of resistance not included in this broad
classification but which are nevertheless of major med-ical importance, e.g. vancomycin resistance.
The rest of this chapter is devoted to examples of
the biochemical processes involved in resistance to
clinically important drugs used in the treatment of
infections caused by bacteria, ftmgi, viruses and
protozoa.
9 1 EiizMnnic iiiacti¥ation of druos
9.1.1 P” Lactams
The destraction of penicillins, cephalosporins and
carbapenems by bacteria that produce f3-lactamases is
one of the most widespread and serious forms of mi-crobial resistance. The general inactivation reactions
are shown in Figure 9.1. The P-lactam bonds of peni-cillins and cephalosporins are cleaved to yield the
149
Biochemical mechanisms of drug resistance
(a)  R.CO.HN-H
-c
H S – ,CH3
O
N C COOH
H
R.CO.HN-H
• c
(b) R.CO.HN^
H
C-H
-C ‘
HO  O
C “
H
.CH3
CH3
-COOH
CH2
//
o
c
COOH
C.CHj.R^
R.CO.HN
H
HO O
H
-C -i
N-H
•CH,
C
COOH
C.CH2.R
Unstable
FIGURE 9.1 Inactivation of (a)
penicillins and (b) cephalo-sporins by P-lactamase. Whereas
penicilloic acid is relatively sta-ble, the corresponding cephalo-sporin product is highly unstable
and decomposes spontaneously
to a complex mixture. R and R’
represent a wide variety of side
chains that may substantially af-fect the efficiency of P-lactamase
attack.
biologically inactive derivatives penicilloic acid and
cephalosporanoic acid, respectively. Penicilloic acid is
a stable end product, but cephalosporanoic acid spon-taneously degrades to several other compounds. As we
shall see, the nature of the R-substituent in the arnide
side chain of p-lactams is important in detennining the
susceptibility of compounds to p-lactamase attack.
The number of p-lactamases produced by different
bacteria is astonishing—more than 340 such enzymes
have been identified so far, with a wide range of sub-strate preferences for penicillins, cephalosporins and
cai^bapenems. Clinical isolates of bacteria commonly
express several different p-lactamases, thus providing
a fonnidable aiTay of defences against many different
antibiotics.
Classification of P-lactamases
The numbers and diversity of p-lactamases are re-markable; as mentioned previously, at least 340 bacte-rial P-lactamases have been described. The classifica-tion of this wealth of enzymes is a major challenge and
there are two current schemes: the Ambler classifica-tion based on similarities in amino acid sequences, and
the Bush-Jacoby-Medeiros system based on substrate
and inhibitor profiling. In the Ambler scheme there are
four classes:
Class A: penicillinases and cephalosporinases
usually found on plasmids or transposons.
Class B: metallo p-lactamases.
Class C: chromosomal cephalosporinases
Class D: oxacillinases
The Bush-Jacoby-Medeiros scheme also has four
classes or groups:
Group 1: chromosomally encoded cephalospori-nases that are poorly inhibited by clavulanic
acid.
150
9,1 Enzymic inactiwation of drugs
Group 2: penicillinases, cephalosporinases and
broad-specificity enzymes, including carba-penemases, both chromosomally and pias-mid encoded that are inhibited by clavulanic
acid and other active-site-directed inhibitors
of p-lactamases
Group 3: metaiio-p-lactamases unaffected by all
the conventional p~lactamase inhibitors
Group 4: a limited number of uncharacterized
penicilMnases that are not inhibited by clavu-lanic acid
Group 2 includes an extended and varied set of en-zymes that are further subdivided into eight subclasses
according to theh substrate and inhibitor profiles. As-suming that novel forms of p-lactamases continue to
be discovered, further revision of the existing schemes
of classification may eventually be needed.
Gram-positive fi-lactamases
The most important B-lactamase in Gram-positive bac-teria is thai produced by Sfap,y!ocoLs aureus.
which was responsible for the rise in the resistance of
this pathogen to penicillin first observed in the late
194()s and 1950s. In many hospitals today more than
90% of the Staphylococcus aureus isolates are resist-ant to the simpler penicillins because of P-lactamase.
The p-lactamase of Staphylococcus aureus is an in-ducible enzyme. Enzyme production is very low in the
absence of penicillin or cephalosporin. The addition of
minute quantities of antibiotic (as little as ().()()24 jig
ml”””‘- of medium) increases enzyme production enor-mously, and the p-lactamase may account for more
than 3% of the total protein synthesized by the bac-terium. The enzyme, which preferentially attacks peni-cillins, is released from the bacterial cell and inacti-vates antibiotic in the surrounding medium. The
resulting dilution of p-lactamase is the basis of the
well-known ‘inoculum effect’. A small inoculum of
Staphylococcus aureus cells may not destroy all the
antibiotic in the medium, but the much greater quantity
of enzyme produced by a heavy inoculum of cells is
able to overcome the challenge. Staphylococcal resist-ance to penicillin is therefore dependent upon the size
of the inoculum.
The regulatory genes which control the expres-sion of the P-lactamase protein, BlaP (BlaZ in Staphy-lococcus aureus), are blal, blaRl and blaR2. Blal, the
protein encoded by blal, is a repressor that binds to op-erator sites between blal and blaP’Z and prevents the
expression of p-lactamase. BlaRl is a large mem-brane-spanning protein of 601 amino acid residues
with a 261-amino acid extracellular domain that inter-acts with P-lactams in the external environment. The
recognition site is located mainly in the region around
serine-402 (serine-389 in Staphylococcus aureus),
which has close homology with the domains flanking
the active-site serine residue of p-lactamase. Recent
work with Sfaphyloccocus aureus shows that p-lactam
antibiotics 0-acylate the hydroxyl group of serine-389,
which is activated by the nearby presence of the car-boxylated side chain of lysine-392. A similar type of
serine activation is also observed at the active center of
p-lactamase. The acylation of BlaRl by p-lactams ini-tiates a conformational change in the extracellular do-main of BlaRl which transmits to a cytoplasmic region
of the protein via the transmembrane domains. The cy-toplasmic region of BlaRl has a zinc-dependent pro-tease domain which is activated by the conformational
change and then cleaves the repressor protein Blal.
The repressor function of Blal is eliminated by the pro-teoh’tic cleavage, thus pemiitting expression of the
structural blaP gene and production of p-lactamase.
The function of the BlaR2 protein at present is not
known, but it is nevertheless essential for the regula-tion of p-lactamase synthesis. In Staphylococcus au-reus, the genes encoding the synthesis and regulation
of p-lactamase are frequently found on plasmids.
Gram-negative p-lactamases
The complex outer envelope of Gram-negative cells
makes them intrinsically less sensitive to many p-lac-tams. However, after the introduction of penicillin de-rivatives such as ampicillin with good activity against
Gram-negative bacteria, p-lactamase-mediated resist-ance amongst these pathogens soon began to emerge.
Many p-lactamases in Gram-negative bacteria are
not inducible and are expressed constitutively. In Els-cherichia coli, p-lactamase is encoded by the chromo-somal ampC gene, which is expressed constitutively at
low levels. However, ampC mutations which confer
overproduction of p-lactamase are observed at the low
frequency of approximately 10″””””. The genetic changes
151
Biochemical mechanisms of drug resistance
responsible are often point mutations or insertions that
increase the transcription of the ampC gene. There are
also attenuator mutations that allow increased tran-scriptional readthrough into ampC and examples of
amplification of the ampC gene. In some Gram-nega-tive species, including Enterobacter cloacae, Cit~
robacter freundii and Pseiidomonas aeruginosa, the
enzyme is inducible, although the mechanism of in-duction differs from that in Gram-positive bacteria.
Whereas p-iactamase in these species is nomially ex-pressed at low constitutive levels, mutations causing
high levels of constitutive enzyme synthesis occur at
much higher frequencies than in Escherichia coli:
10 *. The expression of the ampC gene in Enterobac-ter cloacae and Citrobacter freundii is controlled by an
adjacent ampR gene. Unlike the system in Gram-posi-tive bacteria, p-lactams are not directly involved in the
induction process in Gram-negative species. Under
normal growth conditions, the ampC gene-activating
protein, AmpR, interacts with a cytosolic intermediate
in the biosynthesis of peptidoglycan, UDP-mu-ramylpentapeptide (Chapter 2), which blocks the acti-vating function of AmpR.
Inhibition of peptidoglycan cross-linking by p-lactams causes an increase in the normal turnover of
peptidoglycan in Gram-negative bacteria. The turn-over process involves the transport of peptidoglycan
fragments generated in the cell wall across the cyto-plasmic membrane into the cytoplasm. The transport
process is effected by a transmembrane protein en-coded by the anipG gene that is also essential for the
induction of p-lactamase. The peptidoglycan frag-ments generated by the action of p-lactams displace
UDP-muramylpentapeptide from its interaction with
AmpR, thus allowing AmpR to exert its activating
function and enhance the expression of arnpC. Another
gene, ampD, encodes an amidase, AmpD, that hy-drolyzes the activating peptidoglycan fragment shown
in Figure 9.2. The tripeptide released in this reaction is
normally directly reutilized in peptidoglycan synthe-sis. Mutations that inactivate ampD result in high-level
constitutive p-lactamase biosynthesis, owing to accu-mulation of the activating fragment. The negative reg-ulatory activity of the amidase specified by wild-type
ampD is presumably overwhelmed by the sudden in-flux of peptidoglycan fragments when the cell is under
attack by p-lactams.
Catalytic mechanisms of P-lactamases
Although there is considerable diversity in amino acid
sequences among the many known p-lactamases, the
majority are acyl serine transferases. A smaller group
includes zinc-dependent enzymes. The carbonyl car-bon of the p-lactam amide bond transfers to a serine
residue at the active center of the acyl serine trans-ferase to form a serine ester-linked p-lactamoyl en-zyme complex (Figure 9.3). This reaction is facilitated
by activation of the serine y-hydroxyl group by the
carboxylated side chain of a nearby lysine residue in a
manner comparable to the interaction of p-lactams
with the BlaR protein described previously. The pro-ton of the serine y-hydroxyl group is abstracted, en-abling the y-oxygen atom to attack the carbonyl group
of the p-lactam molecule. The abstracted proton is
transferred to the adjacent nitrogen atom. In the next
stage a proton is abstracted from a water molecule at
the active center and the activated hydroxyl group at-tacks the serine-lactamoyl ester bond. The ensuing hy-drolysis releases free enzyme and a biologically inac-tive derivative, either penicilloic or cephalosporanoic
acid. This reaction is comparable to that between p-lactams and the penicillin-binding proteins involved
in peptidoglycan biosynthesis (Chapter 2). In the lat-ter case, the p-lactamoyl-PBP complexes, unlike the
corresponding complexes in p-lactamases, are rela-tively resistant to attack by water molecules, resulting
in long-lasting inactivation of the PBPs. The analo-gous interactions of the active-site serine residues of
p-lactamases and PBPs with p-lactams, and similari-ties in the active-site sequences and the secondary
structures of both proteins, suggest that p-lactamases
and PBPs may have evolved from the same ancestral
protein.
Several species of pathogenic bacteria, including
Klebsiella pneumonia, Pseudomonas spp., Bacter-oides spp., Serratia marcescens and Acinetobacter
spp. produce chromosomally mediated p-lactamases
with a zinc ion at the active center. Until relatively re-cently these metallo-p-lactamases were considered to
be little more than interesting biochemical curiosities.
However, their ability to degrade penicillins, cephalo-sporins and carbapenems, together with their lack of
susceptibility to inhibitors of the acyl serine trans-ferase p-lactamases, has highlighted the threat of
152
9,1 Enzymic inactiwation of drugs
1,6 Anhydromuramic acid
L-Alanine
NHCOCH3
Bond cleaved by the AmpD
amidase
NH
OHOOOrf
D-Glutamic acid
meso-Diaminopimelic acid
2
NH
I
COOH NH,,
FIGURE 9.2 Structure of the
molecule arising from the turn-over of peptidoglycan that partic-ipates in the induction of the
ampC P~lactamase of Grani~neg-ative bacteria. Cleavage of the
compound by the AmpD amidase
inactivates the inducing activity.
[Adapted with permission from
C. .lacobs e! al. Molec. Microbiol.
15, 55 (1995). Published by
Blackweil Science, London.]
metallo-P-lactamases to the treatment of serious
infections.
In the metaJlo-P-lactamases the zinc ion at the ac-tive center activates a bound water molecule as a nU”
cleophile. X-Ray crystallographic analysis of the met-allo-P”lactaniase from Bacillus cere us suggests that
the adjacent aspartate~90 residue also contributes to
the activation process by acting as a general base to re-move a proton from the water molecule. The ab-stracted proton is donated to the nitrogen atom of the
p-lactam bond and cleavage of the bond ensues. Al-though most, if not all, of the recognized metallo-P-lactamases are believed to involve a zinc ion in the cat-alytic process, significant differences in the amino acid
sequences and tertiary structures among the enzymes
suggest that there may also be differences in the mo-lecular details of the catalytic process.
Approaches to the P-lactamase problem
P-Lactamase-stable compounds . The advent of the
semisynthetic P-lactams during the 19.5()s offered an
apparent escape from the problem of staphylococcal
resistance caused by p-lactamase. Compounds such as
methicillin and cloxacillin (Chapter 2), with bulky
substituents in the penicillin side chain, were found to
be poor substrates for p-lactamase. The affinity of me-thicillin for staphylococcal p-lactamase is much lower
than that of benzylpenicillin, and the maximum rate of
hydrolysis of methicillin by this enzyme is only one-153
Biochemical mechanisms of drug resistance
RH N
O
1 p-CH3
.^j^ N ‘—Co o H
X
E-serine-OH  y
0= C N L
RHN -C
I
O
I
\i 1 — N-\
CH3
CH3
COOH
E-serine
+ H2O
OH H
+ E-serine- serine
COOH
FIGURE 9.3 The essential reactions at tfie
active centre of tfie serine P-lactamases. I: The
proton of the 7-OH of the active-centre serine is
abstracted and the resulting activated y-oxygen
atom attaclis the P-!actam carbony! group to
fonn an ester link. The proton is then back-do-nated to the adjacent niti’ogen atom. II: Ab-sti’action of a proton from a water molecule at
the active centre results in an attack of the acti-vated water OH group on the serine-lactamoyl
ester bond to release the degraded P-lactam and
regenerated enzyme (III). E represents the rest
of the enzyme molecule.
thirtieth of that of benzylpenicillin. Until relatively re-cently, methicillin was effective against infections
caused by p-lactamase-producing staphylococci, even
though its intrinsic antibacterial activity is substan-tially lower than that of benzylpenicillin. Although it is
only slowly degraded by Gram-negative p-lactamases,
methicillin is ineffective against Gram-negative infec-tions because its physical characteristics limit its abil-ity to penetrate the outer membrane. To combat the
menace of Gram-negative p-lactamases, therefore,
compounds were needed that both resisted p-lacta-mase attack and penetrated effectively to the PBPs in
the cytoplasmic membrane.
An extensive range of novel p~lactam derivatives
has been developed, with good activity against & -cherichia coli strains producing the most commonly
encountered p-lactamase of Gram-negative bacteria,
TEM-1. This enzyme, whose name is derived from that
of a patient treated for a p-lactam-resistant infection, is
encoded on an R-plasmid that transmits readily to
other enterobacteria. The examples in Table 9.1 show-that £jc/!er/r/-H”a coli cells producing TEM~1 are effec-tively inhibited by p-lactamase-resistant compounds.
Extended spectrum P-lactamases. Unfoitu-nately, following the introduction of novel p~lactams,
the plasmid-borne enzymes in Escherichia coli, TEM-1 and TEM~2, underwent mutations near the active
center that markedly increased their ability to hy-drolyze several of these valuable agents. A third p-lac-tamase, SHV-l, which originated in Klebsiella spp.
and conferred resistance to ampicillin, eventually
transferred to Escherichia coli and evolved to hy-drolyze novel p-lactams. There are now more than 90
of these extended-spectrum p-lactamases (ESBLs),
with more than 70 in the TEM family and 20 or more
in the SHV group. The ESBLs include both serine-active site and metallo-p-lactamases. Many stractural
variants of p-lactams are hydrolyzed, although the in-dividual enzymes exhibit significant substrate speci-ficities. It is interesting that ESBLs are frequently
more sensitive to p~lactamase inhibitors, although in-hibitor-resistant variants have appeared in both the
United States and Europe. Two examples of ESBLs—
TEM-12 and TEM-26—-are listed in Table 9.1. TEM-26 has two critical amino acid replacements: serine-for-arginine at position 164 and lysine-for-glutamate
at position 104. These changes dramatically enhance
the hydrolytic efficiency of the enzyme against drags
of major importance such as ceftazidime and cefurox-ime, and bacteria equipped with this enzyme are
markedly more resistant to these drags (Table 9.1).
The carbapenems, in which the sulfur atom of the p-lactam fused ring system is replaced by a carbon atom,
e.g. thienamycin and meropenem (Chapter 2), gener-ally have good stability to the serine-active-site p-lac-tamases and are highly active against bacteria produc-ing the various forms of TEM (see imipenem in Table
9.1). However, it is now apparent that carbapenems are
hydrolyzed by some ESBLs, including both metallo-p-lactamases, and by some unusual serine-active-site
enzymes.
Some bacteria, such as Enterobacter cloacae,
have evolved a different strategy to combat p-lacta-154
9,1 Enzymic inactiwation of drugs
TABLE 9.1 The effects of TEM P-lactamase and two mutant variants on the hydrolysis
of and bacterial sensitivity to P-lactams
Enzyme
Relative rates of hydrolysis
(benzylpenicillin = 100)
MIC values for Escherichia, coli
(/.ig/inl)
AMP CTAX CTAZ  IMP  AMP  CTAX CTAZ IPM
R-TEM-i
TEM-12
TEM-26
-110
14
-0.07
2.4
7.5
-0.01
3.8
170
-<0.01
> 1
4
>256
>256
>256
0.125
0.125
0.5
64
0.25 0.25
0.125 0.25
64 0.25
128 0.25
Notes: AMP, ampicillin; CTAX, celotaxime; CTAZ, ceftazidime; iPM, imipenem, ,’\JEhough no value for (he rate of
iiydi’olysis or.’\MP t)y TI-iM-26 is preseEited, the hi,di mininiai inhibitory conceEitratioEi (MIC) ^gaAnsl Escheiickia
coli expressing tills enzyme indicates rapid destruction of the antibiotic. R” indicates bacteria witiiout a resistance
phismid.
mase–resisla:t]t antibiotics. .As discussed previously,
the chromosomal ampC gene encoding p-iactamase in
this organism is normally an inducible gene that is in-dh-ectly regulated via the action of p-lactams on pepti-doglycan metabolism. In the modifred strategy, the
challenge of p-lactamase-stable dnigs is countered by
mutations in the regulatory cascade that marliedly in-crease the production of enzyme. Large quantities of a
p-lactaniase with wea.k activity degrade enough antibi-otic to allow the bacteria to sui-vive and proliferate.
Inhibitors of P-lactamase. Some of the early p-lactamase-stable p-lactams, like cloxacillin, have a de-gree of inhibitory activity against p-lactamases. How-ever, it was the discovery of a naturally occuning
inhibitor of these enzymes, clavulanic acid (Chapter
2), that opened the way for effective synergism with P-lactamase-susceptible drugs such as ampicillin and
amoxycillin. Although clavulanic acid has little intrin-sic antibiotic activity, it is a remarkably effective in-hibitor of many p~lactamases of Gram-positive and
Gram-negative bacteria. Clavulanic acid and other
clinically useful compounds, including sulbactam and
tazobactam, react irreversibly with the active-site ser-ine of p~lactamase to fomi stable, enzymically inactive
complexes. However, bacterial evolution is again prov-ing equal to the challenge of inhibitors of P-lactamase.
Mutant forms of the enzyme have emerged which are
highly resistant to inhibition. Numerous inhibitor-re-sistant fomis of the TEM p-lactamases have been iden-tified in clinical isolates of Escherichia coli, Klebsiella
pneumoniae and Proteus mirabilis. Replacement of
methionine at position 69 by the aliphatic amino acids
isoleucine, leucine or valine, and of arginine-244 by
serine or cysteine, cause marked increases in resist-ance to inhibitors. Other bacteria resistant to combina-tions of p-lactamase inhibitors with susceptible p-lac-tams are characterized by overproduction of
p-lactamase, which overwhelms the protective capac-ity of the inhibitors.
The clinical tlireat posed by the metallo-p-lacta-inases continues to challenge the ingenuity of medici-nal chemists to devise inliibitors of these enzymes. Un-fortunately, at present there are no clinically useful
inhibitors of metallo-p-lactamases, although there ai’e
some promising developments at the laboratory stage.
The three-dimensional structures of both serine-active-site and metallo-P-lactamases have been solved
by X-ray crystallography. Two examples are illustrated
in Figures 9.4(a) and 9.4(b). It is hoped that the infor-mation provided by the X-ray data together with syn-thetic organic chemistry will result in a continuing
flow of novel agents to contain the tlireat of drug-resistant, p-lactamase-producing bacteria.
9.1.2 Chloramphenicol
The potential toxicity of chloramphenicol limits its use
mainly to the treatment of life-threatening infections,
such as typhoid and meningitis, where bacterial resist-ance to the drug may have serious consequences. Re-sistance to chloramphenicol is primarily due to the en-zyme chloramphenicol acetyl transferase (CAT) which
is widespread amongst most genera of Gram-positive
155
Biochemical mechanisms of drug resistance
FIGURE 9.4 Tliree-dimeiisional sti’uctures, as revealed by X-ray crystallograpliic analysis, (a) The TEM-1 serine-active-center P-lactamase from Escherichia coli. [Reproduced with pennission from C. Jelsch et al. Proteins: Structure, Function
and Genetics 16, 364 (1993). Published by Wiley-Liss, Inc., a subsidiary of John Wiley & Sons.] (b) The zinc P-lactamase
fmra Bacilhis cereus. [Reproduced with permission from A. Carfi et al. EMBO J. 14, 4914 (1995). Published by Oxford Uni-versity Press.] In both diagrams the spiral ribbons represent a-helices; the arrowed ribbons, P-pleated sheets; and the strings,
looped regions of the proteins. The zinc atoms at the active centre of the metalloenzyme are represented by the two silvered
spheres.
and Gram-negative bacteria. Genes encoding many
variants of the enzyme are either chromosomaUy or
plasmid-located. A major subtype in Gram-negative
bacteria is found on transposon Tn9. CATs normally
exist in solution as trimers with subunit molecular
masses of between 24 and 26 kDa.
Catalytic process of chloramphenicol
acetyl transferase
CAT metabolizes chloramphenicol in a two-stage
process to a 1,3~diacetoxy derivative (Figure 9.5). The
antibiotic is first converted to the 3-acetoxy compound
using acetyl coenzyme A as an essential cofactor. A
slow, nonenzymic rearrangement then transfers the
acetoxy group to the 1 position. A second round of en-zymic acetylation at the 3 position generates the final
1,3-diacetoxy product, although this reaction is much
slower than the first step, owing to the impaired fit of
the 1-acetoxychloramphenicol into the active site of
the enzyme. Since both the mono- and diacetoxy de-rivatives are inactive as antibiotics, the two-stage
acetylation sequence is biologically inefficient, but it
is nevertheless a consecjuence of the spontaneous shift
of the acetyl group from the 1 to the 3 position. Al-though the chloramphenicol molecule is potentially
vulnerable to various forms of metabolic inactivation,
including dehalogenation, reduction of the nitro group
156
9,1 Enzymic inactiwation of drugs
O
II H
HN
H I
c-c-I I
N CI
•CI
O
II H c-c-ci I I
O , N—( \ />—C-C-C-O H
‘ ^ ^ i. H H,
OH
Chloramphenicol
Acetyl CoA
/^T N HN CI
– * O M—<\ )>—C-C-C-0-C-CH ,
»- ‘ \_!7 I H H, II ^
CoA ^ OH 2 o
3-Acetoxychloramphenlcol
Molecular rearrangement
O^N  W //
O
II H
C-C-C I
I I
HN CI
H I
C-C-C-0-C-CH „ •*
A H H„ N ‘ -O ” O CoA  Acetyl CoA
0=C-CH ,
O
II H
C-C-C I
I I
HN CI
H I
C-C-C-O H i ^ ^^ I
0=C-CH ,
1,3-Diacetoxychloramphenlool  1-Acetoxychloramphenicol
FIGURE 9.5 Inactivatioii of chloramphenicol by chloramphenicol acetyl transferase. 3-Acetoxychloramphemcol is formed
first, followed by a nonenzymic shift of the acetyl group to the 1 position. A second enzymically catalyzed acetylation at the
3 position yields the 1,3-diacetoxy derivative.
and hydrolysis of the amide bond, the acetylation
mechanism is the overwhelmingly significant contrib-utor to resistance among bacterial pathogens.
Of the various fomis of CAT defined by their
stractural and biochemical properties, the type III en-zyme (CAT|j|) has been most thoroughly studied. The
determination of the structure of CATu, by X-ray crys-tallography was a major contributor to the understand-ing of the catalytic mechanism (Figure 9.6). The
trimeric holoenzyme has three identical active sites in
the interfacial clefts between the monomers. Histidine-195 in one face of each cleft acts as a general base to ab-stract a proton from the 3-hydroxyl group of chloram-phenicol. The resulting oxyanion attacks the 2-carbonyl
carbon atom of acetyl coenzyme A to yield a tetraliedral
intemiediate. The oxygen atom of the intermediate is
hydrogen bonded to the hydroxyl group of serine-148.
The intemiediate may also form hydrogen bonds with a
water molecule linked to threonine-174. Finally, the
tetrahedral intemiediate collapses to yield 3-<9-acetyl-chloramphenicol and free coenz3’me A. The first-stage
acetylation by CATfj, is an extremely efficient reaction,
with a turnover rate of 600 molecules s”K
Physiology of chloramphenicol acetyl
transferase synthesis
Grani–positive bacteria. The genes encoding CATs
in Gram-positive bacteria such as Staphylococcus spp.
and Bacillus spp. are inducible by chloramphenicol.
However, the mechanism of induction does not in-volve increased transcriptional activity but rather an
activation of the translation of the niRNA for the en-zyme. Investigation of the inducible cat gene from
Bacillus pumilus revealed that there is an 86-base pair
region immediately 5′ to the coding sequence for the
enzyme. Within the 86-base pair region, there are two
distinct domains: domain A contains a ribosome-bind-ing site (RBS-2), a translation initiation codon (GTG)
and an open reading frame of nine codons ending in an
upstream inverted-repeat sequence within domain B.
The latter domain has two 14-base pair inverted repeat
157
Biochemical mechanisms of drug resistance
OjN
Ci^CHCONH
^ \ >
-=^–”’ H OH
Histidine~195 t” f^
Hfi…
•:N. , ,N H
,SCoA
CLCHCONH
Hydrogen bond
Histidine’195
H Nc:
SCoA
Senne-148
FIGURE 9.6 The key events in the catalytic
mechanism of chloramphenicol acetyl trans-ferase. Histidine-195 at each of the three active
centres acts as a general base to abstract a pro-ton from the 3-OH group of the antibiotic. The
resulting activated oxygen attacks the 2-car-bonyl group of acetyl coenzyme A to generate
a tetrahedral intermediate that is hydrogen
bonded to the y-OH group of serine-148 of the
enzyme. Subsequently the intermediate col-lapses to release 3-acetoxychloramphenicol,
coenzyme A and free enzyme. [Reaction mech-anism adapted with kind permission of L A.
Murray and W. V. Shaw and the American So-ciety of Microbiology; Antimicrob. Agents
Chemother. 41, 1 (1997).]
sequences separated by 12 base pairs. The downstream
inverted repeat spans the specific ribosome-binding
site (RBS-3) for the cat gene transcript.
The secjuence of domain B suggests that its tran-script contains a stable stem loop whose secondary
stnicture hinders translation of the cat gene transcripts.
A detailed analysis of the transcriptional mechanism
shows that while the cat coding sequence and the up-stream 86-base pair region can be transcribed into a
single mRNA molecule, about 50% of the observed
transcripts terminate immediately after the regulatory
region. This suggests that the mRNA stem loop also
acts as a weak transcription termination signal. In the
absence of chloramphenicol, even the full-length tran-scripts are not efficiently translated into enzyme pro-tein because RBS~3 is hidden within the stem loop
stnicture.
Addition of the antibiotic causes a ribosome en-gaged in translating the leading sequence of domain A
to stall (remember that chloramphenicol inhibits pro-tein synthesis, Chapter 5). The stalled, chlorampheni-col-bound ribosome masks sequences in the mRNA,
leading to destabilization of the stem loop secondary
stnicture of domain B. The previously hidden RBS-3
is now made available to chloramphenicol-free ribo-somes to initiate translation of the cat gene transcripts.
Remarkably, therefore, the ability of chloramphenicol
to inhibit protein biosynthesis facilitates the efficient
translation of the mRNA and synthesis of the enzyme
that inactivates the antibiotic. The principal result of
the destabilization of the stem loop is to enhance the
efficiency of translation of the cat gene transcripts,
with only a small effect in relieving transcription ter-mination. This mechanism of induction is referred to
as transiational attenuation, and we shall see it in ac-tion again in inducible resistance to erythromycin and
tetracyclines.
Gram-negative bacteria. CAT synthesis is con-stitutive in most Gram-negative bacteria. However,
CAT synthesis in Escherichia coli is subject to catabo-lite repression. Synthesis is faster in cultures grown on
glycerol than in glucose-supported cultures. Cyclic
AMP complexed with the catabolite activator protein
(CAP) is required for optimal CAT synthesis. During
glucose-supported growth, the intracellular levels of
cyclic AMP are low and the uncomplexed CAP cannot
bind to the promoter region that regulates the cat gene.
158
9,1 Enzymic inactiwation of drugs
Transcription of the cat gene is therefore inefficient.
Conversely, when cyclic AMP levels rise, the CAP-cyclic AMP complex binds to the promoter region, fa-cihtating the interaction of DNA~dependent RNA poly-merase with the structural gene and its transcription.
9.1.3 Aminoglycosides
Bacterial resistance to aminoglycosides can result
from mutations affecting the ribosomes and from
changes in cellular permeability, but the most impor-tant cause of resistance is enzymically catalyzed inac-tivation of the antibiotics. Although more than 50
aminoglycoside-inactivating enzymes have been re-ported, they catalyze only three major types of
reactions:
1. A’-Acetylation of vulnerable amino groups
using acetyl coenzyme A as the acetyl donor.
The A’~acetyl transferases comprise the
largest group of aminoglycoside-inactivating
enzymes.
2. 0-Adenylylation involving the transfer of an
AMP residue from AFP to certain hydroxyl
groups. The <9-adeny!yl transferases form the
smallest group of inactivating enzymes.
3. 0-Phosphorylation of hydroxyl groups with
ATP acting as the phosphate donor.
The enzymes are fiirther divided into subgroups ac-cording to the molecular position on the drugs at which
these reactions occur. This results in a very complex
array of bacterial defences against the aminoglyco-sides. There are now examples of X-ray-determined
three-dimensional structures trom each of the three
groups of aminoglycoside-inactivating enzymes.
Typical reactions involving streptomycin and
kanamycin A are shown in Figure 9.7. Streptomycin is
subject to both adenylylation and phosphorylation but
Phosphorylation
ATP
ADP
CHjOH^ -ATP ^ ^
Adenylation
O  \^ NHCH3
\\,
NH,
HO /
OHC” ‘^’^
C- NH Streptomycin
>0 H
N-C-NH ,
n
H NH
CoA Acetyl CoA
T
A/-Acetylation
Kanamycin A
FIGURE 9.7 Tliree modes of enzymic inacti-valion of aminoglycoside antibiotics. Unlike
kanamycm A, streptomycin is not subject to N-acetylation, while kanamycin A is also inacti-vated by 0-adenylylation tmd 0-phosphoryla-tion. An extensive array of bacterial enzymes is
involved in the inactivation of aminoglyco-sides. A review listed at the end of the chapter
provides further details.
159
Biochemical mechanisms of drug resistance
it is not a substrate for the iV~acetyl transferases. The
A/^-acetyl transferases are specific for the amino groups
of other aminoglycosides in four different positions (1,
3, 6′ and 2′). The 0-adenylyl transferases attack OH
groups in 2″, 3″ and 4 positions and the 0-phosphoryi
transferases target OH groups in the 3′, 3″ and 4 posi-tions. Isozymes exist for many of these enzymes. A bi-functional enzyme that catalyzes both W-acetyl and 0~
phosphoryl transferase activities appears to be the
result of gene fusion, with protein domains responsible
for each enzymic activity being derived from different
genes. For a comprehensive account of the many en-zymes that metabolize aminoglycosides, the reader is
referred to a review in ‘Further reading.’
The cellular location of the aminoglycoside-modifying enzymes is somewhat uncertain. Because
the target of aminoglycoside action is at the ribosome,
the cytoplasm was thought to be the most likely loca-tion for the inactivating enzymes. However, a cyto-plasmic location might be relatively inefficient in pro-tecting the ribosomes because the enzymes are only
synthesized in small amounts. It would make more bi-ological ‘sense’, therefore, if the enzymes were se-creted into the periplasm where aminoglycoside inac-tivation would occur before entering the cytoplasm.
However, it not clear how the essential cosubstrates
ATP and acetyl coenzyme A could gain access to the
periplasm. Nevertheless, there is evidence for the loca-tion of at least one 0-adenylyl transferase in the
periplasm. Signal sequences of 20-30 amino acids at
the N-terminals of proteins ensure the export of pro-teins across the cytoplasmic membrane into the
periplasm. Many members of the A/^-acetyl transferase
family have these sequences, although they are not
found among the 0-phosphoryl transferases. Thus al-though the periplasm appears to be an optimum loca-tion for aminoglycoside-modifying enzymes, this may
not always be the case. In most bacteria the expression
of genes encoding aminoglycoside-modifying en-zymes is not subject to regulation. However, the genes
for A^’-acetyltransferases in Serratia marceseens and
Providencia stuarti appear to be tightly regulated, al-though in some clinical isolates of aminoglycoside-re-sistant strains the control of expression is relaxed.
Surveys of aminoglycoside resistance among
bacterial pathogens from countries around the world
reveal a complex pattern in which many organisms
harbour a combination of several genes, either chro-mosomally or plasmid-located, expressing different
modes of aminoglycoside metabolism. Resistance to a
range of aminoglycosides is especially marked among
Citrobacter, Enterobacter and Klebsiella species, al-though other Gram-negative bacteria also exhibit re-sistance. Several semisynthetic aminoglycosides have
been designed to counter the action of the inactivating
enzymes. Tobramycin, netilmicin and amikacin were
all developed to resist 0-phosphotransferases but
were nevertheless found to be susceptible to iV-acetyl
transferases. The range and diversity of aminoglyco-side-inactivating enzymes will probably continue to
frustrate efforts to devise compounds that resist deac-tivation. An alternative possibility lies with enzyme
inhibitors, although the discovery of agents with
broad specificity would be challenging. Specific inhi-bition of the 0-phosphotransferases is potentially at-tractive because of some structural similarity between
these enzymes and the eukaryotic protein phosphory-lating kinases. The latter enzymes are of great interest
in mammalian phamiacology and many inhibitors are
already available, some of which show a degree of ac-tivity against the aminoglycoside 0-phosphotrans-ferases.
9.1.4 Streptogramins
The ever-growing threat of bacterial resistance to the
most commonly used antibiotics has encouraged the
introduction of a limited number of more unusual
drugs into human clinical medicine, including the
streptogramins. As described in Chapter 5, group A
compounds such as dalfopristin are given in combina-tion with group B compounds like quinupristin to en-sure a synergistic antibacterial effect. This combina-tion is effective against dangerous pathogens,
including multidrug resistant Staphylococcus aureus
and vancomycin-resistant Enterococcus faecium. A
commonly encountered form of resistance to the group
A compounds is due to 0-acetylation of the lone hy-droxyl group on these dmgs (e.g. dalfopristin. Figure
5.14). The reaction is catalyzed by an 0-acetyl trans-ferase encoded by a family of plasmid-borne vat genes
in Gram-positive cocci. This enzyme shares some
amino acid sequence similarity with acetyl trans-liO
9.2 Loss or downregulation of drug activation
ferases that confer low-level resistance to chloram-phenicol. However, the latter enzymes should not be
confused with the ‘classic’ CAT||, 0-acetyl transferase
described previously, which is responsible for high-level resistance to chloramphenicol. X-Ray analysis of
crystals of the dalfopristin <9-acetyl transferase reveals
it as a homotrimeric enzyme with the active center lo-cated between two adjacent subunits. The acetyl donor
is acetyl coenzyme A. Details of the complex structure
of the enzyme and its interactions with the antibiotic
and acetyl coenzyme A ms available in a reference
listed in ‘Further reading.’
The appearance of human pathogenic cocci re-sistance to group A streptogramins may be linked to
the widespread use of the group A compound virgini-amycin as a growth promoter in livestock since the
vatA gene mediates resistance to both dalfopristin and
virginiamycin. A detailed understanding of the struc-ture of the 0-acetyl transferase and its interactions
with antibiotic substrates and the cofactor may assist in
the design of novel group A compounds which are im-pervious to <9-acetylation.
A lyase enzyme has been isolated from Staphylo-coccus aureus that inactivates group B streptogramins
by cleaving the hexadepsipeptide chain of the
antibiotics.
downregulation of drui
as^f iwisif iosn
9.2.1 Metronidazole
As described in Chapter 6, this dnig has important ap-plications in the treatment of infections caused by
strictly anaerobic bacterial pathogens and certain pro-tozoal parasites and also in the elimination of the
causative organism of peptic ulcer disease, the micro-aerophilic bacterium Helicobacter pylori. The activity
of metronidazole depends upon its metabolic reduc-tion by the target pathogens to various short-lived,
highly reactive radicals which attack DNA and suscep-tible proteins. Resistance to metronidazole occurs in
all of the target species and generally appears to be due
to loss or downregulation of the enzymes involved in
the activation of the drug. In susceptible Helicobacter
pylori, activation is carried out by a nitro reductase en-coded by the rdxA gene. Metronidazole resistance in
most clinical isolates is associated with null mutations
in the rdxA gene which prevent the reductive activation
of the drug. Resistance in the parasitic protozoa Giar-clia diioclenalis and Trichomonas vaginalis is due to
downregulation of the pyruvaterferredoxin oxido re-ductase required for the activation of metronidazole in
these species.
9.2.2 Isoniazid
The efficacy of isoniazid against Mycobacterium tu-berculosis depends initially on the intracellular activa-tion of the compound by a bacterial catalase-peroxi-dase encoded by the katG gene (Chapter 2). While the
identity of the active metabolite of isoniazid is still a
matter of some debate, it is clear- that mutations ad-versely affecting katG confer bacterial resistance. Mu-tations in the katG gene are the major cause of high-level resistance to isoniazid in clinical isolates of
Mycobacterium tuberculosis from around the world.
Of these mutations, the replacement of serine at posi-tion 315 of the catalase-peroxidase by tteeonine is one
of the most frequently encountered. This mutation re-sults in a markedly reduced enzyme affinity for isoni-azid, so that there is little catalase-peroxidase-medi-ated conversion of the drug to the active molecular
species. Although the mutant enzyme has somewhat
lower catalytic activity for its physiological substrates
than the wild-type enzyme, the virulence of Mycobac-terium tuberculosis expressing the mutant enzyme is
apparently unaffected. An understanding of the nature
of the binding interaction between isoniazid and the
wild-type enzyme and how this is impaired by the thre-onine-for-serine mutation may useliil in the design of
isoniazid derivatives that retain substrate activity for
the mutant enzyme.
9.2.3 Pyrazinamide
This antitubercular drug also requires activation from
the inactive parent compound to the inhibitory pyTazi-noic acid by a mycobacterial enzyme referred to as
pyrazinamidase (Chapter 2). Some clinical isolates of
161
Biochemical mechanisms of drug resistance
Mycobacterium tuberculosis lack this enzyme and are
resistant to pyraziamide as a result.
9.3 Modification of drug targets
9.3.1 P-Lactams
Wtiile the most common mechanism of resistance to p-lactam antibiotics is that of inactivation by p-lacta~
mases, resistance can result from amino acid changes
in penicilHn-binding proteins that depress their affinity
for p~lactams. Mosaic genes encoding hybrid PBPs
with reduced affinity for p-lactams were described in
Chapter 8. Such genes confer resistance to both peni-cillins and cephalosporins in meningococci, gonococci
and pneumococci. In this section we concentrate on
another example of p-lactam resistance caused by a
target change that is causing grave concern: the resist-ance of Staphylococcus aureus to methicillin.
This organism was rightly regarded as a particu-larly dangerous pathogen in the preantibiotic era. The
introduction of benzylpenicillin created a fortunate in-terlude during which staphylococcal infections re-sponded readily to the new drug. The subsequent rise
of p-lactamase-mediated resistance in staphylococci
was initially countered by the introduction of the semi-synthetic, p-lactamase-stable methicillin. However,
the 1980s saw the emergence of methicillin-resistant
Staphylococcus aureus (MRSA), which is also resist-ant to all other p-lactams. Resistance is caused by the
acquisition of a transposon-located novel gene, mecA,
that encodes a novel PBP (PBP2′, otherwise desig-nated as PBP2a) with very low affinity for all p-lac-tams. The transposon integrates into the chromosome
of Staphylococcus aureus to create a 50-kb resistance
island which is widely distributed among staphylococ-cal species. Remarkably, the PBP2′ protein takes over
the function of all the other PBPs, thus rendering the
growth of rnecA’^ bacteria resistant to methicillin and
other p-lactams.
Some bacterial strains have an upstream regula-tory region, mecRl-mecl, that negatively controls the
expression of mecA. In the absence of methicillin, syn-thesis of the MecRl protein is repressed. Resistance is
slowly induced to low levels by methicillin. Mutations
in, or complete loss of, the mecl region permit the syn-thesis of both MecRl and PBP2′, giving rise to high
levels of methicillin resistance. There is marked se-quence homology of mecRI and mecl with the blaRI
and blal genes that regulate P-lactamase synthesis in
Gram-positive bacteria. Consequently the mecA gene
is also regulated by blaRI and blal and PBF2′ expres-sion is induced by P-lactams that are recognized by the
BlaRI protein.
Although PBP2′ replaces the function of the other
PBPs in Staphylococcus aureus, its precise function in
peptidoglycan synthesis is not clear and its activity
leads to peptidoglycan with a lower than noimal de-gree of cross-linking. The level of PBP2′ synthesis in
mecA’*’ bacteria does not correlate closely with the
level of resistance, and it is known that other genes
contribute to methicillin resistance, possibly even in-cluding genes that encode ‘super’ penicillinases capa-ble of degrading methicillin.
9.3.2 Macrolides
The inhibition of protein synthesis by erythromycin
and other macrolides depends upon their interaction
with domain V of 23S rRNA (Chapter 5). While there
are bacterial strains that enzymically inactivate eryth-romycin and other macrolide antibiotics, macrolide
metabolism does not contribute significantly to the
problem of clinical resistance. Most macrolide-resist-ant Gram-positive pathogens, including Staphylococ-cus aureus and Streptococcus spp., harbour plasmid-borne genes (erm) that encode a family of A*’-methyl
transferases, or methylases as they are also known.
The substrates for these enzymes are specific adenine
residues in 23 S rRNA in the domain involved in the in-teraction of erythromycin with the ribosome. There are
no iV^-methylated adenine residues in the 23S rRNA of
wild-type, erythi’omycin-sensitive bacteria. In con-trast, A’^-dimethylated adenine appears in the 23S
rRNA of resistant ermA’^ Staphylococcus aureus cells
grown in medium containing erythromycin. Evidence
that the modified 23S rRNA confers ribosomal resist-ance is provided by the observation that 70S ribosomes
reconstituted with 23 S rRNA from resistant Bacillus
subtilis and ribosomal protein from erythromycin-sus-ceptible cells ai’e resistant to erythromycin. The criti-cal adenine residues targeted in the peptidyl trans-1i2
9.3 AAodification of drug targets
ferase domain by the ernHiiediated methylase are A-2058 in Escherichia coli and A~2086 in Bacillus
stearothermophilus. Ditnethylation prevents the nec-essary hydrogen bonding between these adenine
residues and the 2′ hydroxyl group of the desosamine
ring of the macrolides and sterically hinders antibiotic
access to the inhibitory binding site.
The A?-methylase products of the erm gene family
use 5-adenosylmethione as the methyl donor. Some of
the enzymes transfer a single methyl group to the 7V^’
position of adenine, whereas others donate two groups.
Dimethylases may confer a higher level of resistance
to a broader range of macroiide antibiotics than the
monomethylases. The 23S iRNA substrate is pre-sented to the niethylases as a component of nascent ri-bosomes rather than as part of the mature, functioning
particles.
Regulation of erm gene expression
The expression of erm genes is inducible by erythro-mycin. The inducing activity of erythromycin is
closely linked to its ability to inhibit ribosornal func-tion, and erythromycin derivatives devoid of inhibitory
activity cannot induce expression of the A/-rnethylases.
The mechanism of erythromycin induction of the A’-methylases is analogous to that of chloramphenicol
acetyl transferase by chloramphenicol, i.e. induction is
achieved by translational attenuation rather than in-creased gene transcription. Translation of the erm
mRNA is slow and inefficient in the absence of eryth-romycin because of the unfavourable conformation of
a 141-nucleotide leader sequence upstream of the open
reading frame for the enzyme. The secondary structure
of the leader sequence is believed to mask the first two
codons of the orf as well as the ribosornal binding site
for the mRNA, resulting in very low constitutive syn-thesis of AZ-methylase. However, when translation of
the leader secjuence is inhibited by binding of erythro-mycin to ribosomes, the efficiency of subsequent
translation is increased, probably because the second-ary stmcture of the leader sequence undergoes a con-formational rearrangement to a new state that is con-sistent with a more rapid readthrough of the orf and
increased enzyme synthesis. Mutations in the erm
genes that destabilize the secondary stracture of the
leader sequences result in higher levels of constitutive
A’-methylase biosynthesis.
inolones
The role of the quinolones in the treatment of bacterial
infections has grown steadily with the introduction of
novel derivatives with broader spectra of action than
the progenitor compound, nalidixic acid. The emer-gence of resistance accompanying this increasing use
of quinolones is due lai^gely to mutations in the A
(GyrA) subunit of the target enzyme, topoisomerase 13,
or DNA gyrase (Chapter 4), although drug efflux may
also contribute to the problem. Enzymic inactivation of
quinolones has not so far been detected as a mecha-nism of bacterial resistance.
The GyrA subunits of the tetrameric enzyme
(GyrA2GyrB-,) are responsible for introducing double-stranded breaks in DNA and for subsequent resealing
of the breaks during the negative supercoiling process.
A region described as the quinolone-resistance-deter-mining region (QRDR) is located between amino acid
residues 67 and 107 of GyrA in Escherichia coli. This
sequence of amino acids gives rise to a positively
charged molecular surface to which the DNA is be-lieved to bind. Furthermore, the region around serine-83 is critical for the enz3’me-quinolone interaction and
in several quinolone-resistant strains of Escherichia
coli this amino acid is substituted by nonpolar, bulkier
amino acids such as leucine, alanine or tryptophan, hi
Staphylococcus aureus, the homologous position is
serine-84, which is replaced by leucine in many
quinolone-resistant clinical isolates. Quinolone-resist-ant Staphylococcus aureus and other Gram-positive
pathogens may also have mutations in the ParC subunit
of topoisomerase IV (Chapter 5), strengthening the
supposition that this enzyme is also a significant target
for quinolones in Gram-positive species. In the GyrA
subunit of (Campylobacter jejuni, Klebsiella pneumo-niae and Pscudomonas aeruginosa, threonine is the
normal amino acid at position 83 instead of serine and
these organisms are intrinsically much more resistant
to quinolones. The greater bulk of threonine at position
83 probably hinders the optimal binding of Cjuinolone
to the target site.
163
Biochemical mechanisms of drug resistance
9.3.4 Streptomycin
In addition to inactivation by aminoglycoside-modify-ing enzymes, resistance to streptomycin also arises
from mutations affecting the target site in the 30S ribo-somal subunit (Chapter 5). Streptomycin still finds
some application in the treatment of tuberculosis.
DNA analysis of clinical isolates of resistant Mvcodoc-terium tuberculosis shows that about 70% have muta-tions affecting the rpsL gene that codes for protein S12
which contributes to the binding of streptomycin to the
ribosomal subunit. The mutations cause replacement
of lysine~43 or lysine~88 by arginine and a consequent
loss of binding of streptomycin to the ribosome. Muta-tions affecting the highly consei-ved position 904 of
16S rRNA also cause streptomycin resistance in some
Mycobacterium tuberculosis isolates. Ribosomal re-sistance to streptomycin is found in clinical isolates of
other bacteria, including Neisseria gonorrhoeae,
Staphylococcus aureus and Streptococcus faecalis.
A recently discovered novel mechanism for the
protection of ribosomes against several aminoglyco-side antibiotics, except streptomycin, is a plasmid-me-diated enzyme which methylates a residue in the A site
of 16S rRNA, probably guanine-1405. This form of ri-bosomal protection against valuable dmgs such as to-bramycin and amikacin has appeared in clinical iso-lates of Pseudomonas aeruginosa and Serratia
marcescens.
9.3.5 Rifampicin
Resistance to rifampicin is proving to be a consider-able threat to the successful treatment of tuberculosis.
Originally this drug was highly effective against My-cobacterium tuberculosis, but mutations affecting the
P subunit of the target enzyme, DNA-dependent RNA
polymerase (Chapter 4), are often responsible for the
loss of bacterial sensitivity to rifampicin. Most resist-ant clinical isolates of Mycobacterium tuberculosis
have replacements at serine-531 or histidine-526.
These changes, caused by mutations near the center of
the rpoB gene which encodes the p subunit, are read-ily detected by DNA analysis and provide a rapid test
for drag resistance. A detailed analysis of a range of ri-fampicin-resistance mutations in Escherichia coli and
Mycobacterium tuberculosis indicates that the amino
acid substitutions either directly or indirectly ad-versely affect the interaction of rifampicin with its
binding site, thus reducing the affinity of the antibiotic
for its target enzyme. Mutations conferring rifampicin
resistance do not affect the growth of Mycobacterium
tuberculosis, whereas rifampicin resistance in & -cherichia coli causes slower growth. The reason for
this difference is not known, but it may be associated
with the naturally slower growth of Mycobacterium tu-berculosis compared with that of Escherichia coli.
9.3.6 Inhibitors of dihydrofelate reductase
Trimettioprim
The basis of one major form of resistance to trimetho-prim is analogous to that of methicillin resistance;
namely, resistant cells acquire additional genetic infor-mation for a molecular target with reduced susceptibil-ity to the drug. In Gram-negative bacteria, 16 different
genes for trimethoprim-resistant DHFRs have been
identified. These genes are mostly plasmid-bome, al-though they may temporarily reside on the chromo-some because of their association with transposons.
The enzymes fall into two families. Family 1 has five
members with polypeptide chains sharing 64-88% se-quence identity. The enzymes of family 1 are homod-imeric proteins with IC,,, values between 1 and 100
}iM, compared with an IC^,, for the wild-type DHFR of
In M. Family 2 is larger, with 11 members more
closely related than those of family I, with sequence
identities of 78-86%. The enzymes of family 2 are all
homotetramers and are highly resistant to trimetho-prim, with ICjQ values greater than I mM. The most
widely distributed trimethoprim-resistant DHFR
amongst Gram-negative bacteria is encoded by the
d.hfrl gene and belongs to family 1. The dhfrl gene is
located within a highly mobile cassette associated with
a promiscuous transposon (Tn7) that inserts into the
chromosomes of many bacteria.
Some trimethoprim-resistant bacteria overpro-duce modified DHFRs. For example, a highly resistant
strain of Escherichia coli overproduces by about 100-fold an enzyme that is about threefold more resistant to
trimethoprim than the wild-type enzyme. Trimetho-1i4
9.3 AAodification of drug targets
prim-resistant isolates of Haemophilus influenzae (an
important clinical target) also overproduce DHFR, al-though in this case the enzyme is 100- to 300-fold
more resistant to the drug. Enzyme ovei”production in
both species of bacteria is associated with mutations in
the promoter sequences that control expression of the
stractural genes.
It is interesting that the kinetic parameters of
trimethoprim-resistant enzymes for the normal sub-strates, dihydrofolate and NADPH, are essentially un-changed. The mutant enzymes are therefore competent
to take over the metabolic functions of the drug-sensi-tive enzyme.
Sulfonamides
Trimethoprim is usually administered in combination
with a sulfonamide such as sulfamethoxazole. Unfortu-nateh’, bacterial resistance to sulfonamides often
coexists with trimethoprim resistance. Sulfonamide re-sistance can be due either to mutations in the chromo-somal gene, dhps, that mediates dihydropteroate syn-thase, or to the acquisition of plasmid-borne genes
coding for sulfonamide-resistant fomis of the enzyme,
in Neisseria meningitidis there are two variants of a
chromosomaily mediated resistant dihydropteroate
synthase, one of which may be the result of recombina-tion between two related dhps genes. At least two types
of plasmid-bome dhps genes code for sulfonamide-re-sistant enzymes in Gram-negative bacteria. In all these
resistant enzymes the Michaeiis constants {K^^) for the
natural substrate, p-aminobenzoic acid, are similar to
those for the dmg-sensitive enzyme. Sulfonamide-re-sistant forms of dihydropteroate synthase have also
been found in strains of the malarial parasite Plasmod-ium falciparum in regions of the world where the sulfa
drug sulfadoxime is used in combination with in-hibitors of dihydrofolate reductase.
Pyrimethamine and cycloguanil
Inhibitors of DHFR, including pyrimethamine and the
liver metabolite of proguanil, cycloguanil (Chapter 6),
have been central to the prophylaxis and treatment of
malaiia for more than 60 years. The relentless increase
in resistance to these drugs in many parts of the world
is therefore a major threat to the containment of one of
the most prevalent infections on Earth. Because of the
many technical difficulties in working with protozoal
parasites, the definition of the mechanisms of resist-ance in naturally occurring infections has been ex-tremely difficult. Much of the available biochemical
infomiation has therefore been obtained with drug-re-sistant malarial protozoa developed in the laboratory.
Nevertheless it is believed that this information gives a
reasonable indication of the nature of dmg resistance
in the ‘field’. Furthermore, by using technologies such
as the polymerase chain reaction for genetic analysis,
it is now possible to correlate laboratory and field stud-ies of drug-resistant protozoa.
Resistance to pyrimethamine in the most danger-ous malarial parasite, Plasmodium falciparum, is com-monly due to a single-point mutation in DHFR that re-places serine-108 with asparagine. This mutation has
been detected in laboratory isolates and from patients,
but suiprisingly it does not confer cross-resistance to
the structurally similar cycloguanil. However, when
serine-108 is replaced by threonine together with a va-line-for-alanine replacement at position 16, the para-site becomes resistant to cycloguanil but not to
pyrimethamine. Cross-resistance to both drugs occurs
in strains harboring a combination of mutations at po-sitions 51, 57, 108 and 164 which arise from genetic
recombination during the sexual reproduction phase of
the parasite’s life cycle.
A three-dimensional model of the DHFR from
Plasmodium falciparum based on its homology witii
other DHFRs was used to explore the effects of the
mutations at positions 16 and 108 by virtual ‘docking’
experiments in an attempt to understand the lack of
cross-resistance to cycloguanil and pyrimethamine in
such mutants. Pyrimethamine was found to dock
equally well with both the double-mutant enzyme and
the wild-type enzyme, whereas the binding of cy-cloguanil was sterically hindered by the bulky valine
residue at position 16 in the mutant enzyme. While the
binding of inhibitors was progressively weakened by
the gradual accumulation of mutations in DHFR, the
catalytic efficiency of the enzyme remained unaffected
in viralent strains of the parasite. Certain strains of
Plasmodium falciparum and another protozoal patho-gen, Leishmania major, resist pyrimethamine by over-producing DHFR, either by gene duplication or by in-creasing expression levels.
165
Biochemical mechanisms of drug resistance
9.3.7 Inhibitors of HIV reverse transcriptase
Nucleoside analogues
The introduction of AZT (3’~azido~3′-deoxythymi-dine, Chapter 4) was a therapeutic landmark in the
treatment of AIDS. AZT has been followed by other
nucleoside analogue inhibitors of the reverse tran-scriptase (RT) of HIV and by non~nucleoside in-hibitors such as nevirapine. Unfortunately, the re-markable propensity of HIV for mutation (Chapter 8),
combined with the inability of RT inhibitors to sup-press viral replication by more than 90%, leads to the
rapid emergence of drug-resistant strains of the virus.
Resistance to nucleoside inhibitors of RT is often due
to mutations close to the nucleotide binding site which
map to the p3-p4 loop in the fingers of the p66 sub-unit (Chapter 4). High-level resistance to lamivudine
is caused by a valine-for-methionine substitution at
position 184, which is close to the ribose ring of the
nucleoside triphosphate substrate. The increased bulk
of the valine residue stericially hinders the oxathi-olane ring of lamivudine, thus reducing the binding
affinity of the enzyme for the triphosphate metabolite
of the drag. A group of mutations referred to as the
QISIM complex causes progressively increasing re-sistance to the nucleoside inhibitors of RT. Usually
the first mutation replaces glutamine (Q) at position
151 with methionine (M), which is situated very close
to the nucleotide binding site. This mutation is fol-lowed by a series of secondary mutations which grad-ually increase the activity of the enzyme and enhance
viral drug resistance.
Another interesting resistance mutation of RT re-sults in the removal of the incorporated terminal nucle-oside analogue from the viral DNA chain, thereby al-lowing the normal extension and completion of the
viral genome. Apparently the mutated enzyme pro-motes the incoiporation of either ATP or pyrophos-phate into the DNA chain adjacent to the incorporated
analogue. In this position the ATP or pyrophosphate
can attack the phosphodiester bond linking the ana-logue to the DNA chain, resulting in the expulsion of
the drag from its terminal position. Mutations of this
type occur at residues distant from the nucleotide bind-ing site, e.g. leucine-for-methionine at position 41 and
tryptophan-for-leucine at position 210.
Non-nucleoside inliibitors of HIV reverse
transcriptase
In contrast with the nucleoside analogues, the
NNRTIs, including such compounds as nevkapine and
efavkenz (Chapter 4), ai^e noncompetitive inhibitors of
RT. The NNRTIs bind to a hydrophobic pocket some
lOA from the catalytic center of the enzyme. Muta-tions causing resistance to nevirapine and efavirenz ai^e
located within two p-strands in the NNRTI-binding
pocket between residues 100-110 and 180-190 and re-duce the affinity between enzyme and drug. Fortun-tately, therefore, these mutations do not cause cross-resistance to the nucleoside analogues, although
cross-resistance can occur amongst different NNRTIs.
9.3.8 Inhibitors of HIV protease
These drags (Chapter 6) fomi part of the standard
triple therapy for AIDS in combination with two dif-ferent inhibitors of RT. Triple therapy is considered es-sential because mutant vimses with drag-resistant pro-teases emerge readily when protease inhibitors are
given as a single therapy. It may be recalled that the an-tiviral effectiveness of protease inhibitors is due to
their suppression of the cleavage of the precursor viral
polyproteins, which is an essential stage in the cycle of
viral replication. The development of resistance during
monotherapy with the protease inhibitor ritonavir
(Chapter 4) has been documented in considerable de-tail. The gradual loss of effectiveness of the drag in
AIDS patients is associated with a sequential accumu-lation of mutations in the target enzyme. The rate at
which mutations appear is inversely related to the con-centration of drug in the patient’s blood, strongly sug-gesting that blood levels of the drag should be main-tained as high as possible to minimize viral replication.
Mutations at single loci in HIV protease are associated
with low-level viral resistance to ritonavir: a 7- to 10-fold increase in resistance requires the accumulation of
three to four mutations, and high-level resistance
(> 20-fold) requires four to five mutations. Of the nine
amino acid changes contributing significantly to resist-ance, replacement of valine-82 is probably the most
important. X-Ray analysis of the susceptible enzyme
shows that this residue interacts directly with ritonavir.
lie
9.4 Drug efflux pumps
Ritonavir-resistant viruses are only partial!}’ cross-re-sistant to otiier protease inhibitors, indicating tiiat
there are significant and clinically important differ-ences in the molecular interactions of the various in-hibitors with the mutant enzymes.
Because mutations corifening resistance to in-hibitors of HIV protease are usually located in the sub-strate binding pocket, they may also reduce the cat-alytic efficiency of the enzyme. However, these
adverse effects are often compensated by mutations el-swhere in the enzyme which enhance its stability or
catalytic activity, thus helping to restore the prolifera-tive activity of the vims. Resistance to protease in-hibitors is also associated with mutations in the viral
polyprotein substrate for the protease which increase
its susceptibility to cleavage by the enzyme.
9.3.9 Acyclovir
Infections caused by the various hei-pes viruses range
from the relatively trivial to severely disabling or even
life-threatening. One of the most important applica-tions of acyclovir (Chapter 4) is the treatment of her-pes infections in immunosuppressed patients, in whom
drug-resistant fomis of the viruses develop most read-ily. Acyclovir is a prodmg that is first converted to the
monophosphate derivative by thymidine kinase (TK)
encoded in the viral genome, and subsequently to the
inhibitory triphosphate by enzymes of the infected
host cell. One relatively uncommon form of resistance
to acyclovir results from mutations affecting the viral
DNA polymerase which is the ultimate target for the
drug. The modified enzyme has a diminished affinity
for acyclovir triphosphate while retaining a relatively
unchanged ability to bind the four nucleoside triphos-phates required for viral DNA synthesis. However, by
far the most common mechanism of resistance to acy-clovir in immunosuppressed patients depends on mu-tations in the viral TK. The acquisition of an inappro-priately placed stop codon results in a truncated
polypeptide with little or no enzymic activity. Viruses
lacking TK activity cannot phosphorylate acyclovir
and lose the characteristic ability of herpes viruses to
reactivate from a dormant state in neuronal cells. Mis-sense mutations in viral TK, on the other hand, allow
the enzyme to retain its affinity for thymidine while
maiiedly depressing that for acyclovir and, further-more, do not affect the reactivational capability of the
virus.
9.3.10 Antiinfluenza inhibitors of viral
neuraminidase
Since the introduction of zanamivir and oseltamivir
between 1999 and 2002 for the treatment of influenza
(Chapter 6), there has been extensive surveillance of
clinical isolates in order to assess the likelihood of the
emergence of drag-resistant viruses. A laboratory-de-rived mutation with a valine-for-glutarnic acid replace-ment at position 119 in viral neuraminidase, which
causes the loss of enzyme affinity for both zanamivir
and oseltamivir, has also been observed in viruses iso-lated from patients treated with oseltamivir. However,
although resistant viruses with mutations in the en-zyme target are readily generated in the laboratory by
repeated passage of cultured virus in the presence of
drugs, the overall incidence of comparable resistant
strains from clinical isolates has so far been very low.
9.4 Druci efflux PUITIDS
The various types of drug efflux pumps in prokaryotes
and eukaryotes and their role in the intrinsic resistance
of micro-organisms to drugs and other toxic chemicals
were discussed in Chapter 7. This section is concerned
with the part that the pumps play in acquired resistance
to medically important antimicrobial drags.
9.4.1 Tetracyclines
The clinical value of tetracyclines, which are among
the cheapest and mostly widely used antibacterial
drugs, has been severely compromised by the emer-gence of resistant bacteria in both Gram-positive and
Gram-negative groups. There are two major mecha-nisms of resistance to tetracyclines: drug efflux and ri-bosomal protection. This account is concerned with
tetracycline efflux; ribosomal protection is described
in a later section.
The genes for tetracycline-specific, efflux-medi-ated resistance are almost exclusively plasmid and
167
Biochemical mechanisms of drug resistance
transposon-located, although there is also a chromoso-mal tetracycline efflux system in Escherichia coli as-sociated with the global regulator locus, mar A, that en-hances intrinsic resistance to many drugs (Chapter 8).
High-level expression of marA boosts the production
of the multidrug efflux pump found in many wild-type
Gram-negative bacteria, which includes tetracycline
among its many substrates.
Some 25 genes currently identified among Gram-positive and Gram-negative bacteria encode tetracy-cline efflux pumps. Eighteen of them are designated as
tet genes and seven as otr (oxytetracycline resistance),
although there is no functional difference between tet
and otr genes. New tetracycline efflux genes continue
to be discovered. A review listed at the end of this
chapter provides a comprehensive analysis of this
complex topic. Most of the efflux pumps confer resist-ance to all tetracyclines except minocycline and gly-cylcyclines like tigicycline (Chapter 5).
The tetracycline efflux genes encode membrane-bound proteins of approximately 46 kDa, all belonging
to the MPS family of efflux pumps (Chapter 7). The ef-flux proteins can be assigned to six groups according to
the degree of shared amino acid sequence identity. In
general, the Gram-negative proteins have 12 hydropho-bic membrane-spanning domains and the Gram-posi-tive proteins have 14. Regions of hydrophilic amino
acids loop out into both the periplasmic and cytoplas-mic regions. The tetracycline efflux proteins (Tet),
which probably exist as multimers within the mem-brane, extrude a tetracycline molecule complexed with
a divalent ion (probably Mg-+) in exchange for a proton.
Tetracycline is pumped out of the cytoplasm against its
concentration gradient and the energy required for this
is derived from the proton motive force. Mutational
studies suggest that the cytoplasmic loops interact with
the proton motive force, although the structural details
are not known. There is no direct link between tetracy-cline efflux and ATP hydrolysis. Gram-negative Tet
proteins have functional a and p domains, correspon-ding to the N- and C-terminal halves of the proteins.
Genetic evidence indicates that amino acids distributed
across both domains participate in the efflux function.
Although the Tet proteins of Gram-negative bacteria are
more closely related to each other than to the larger 14-transmembrane-domain proteins of Gram-positive bac-teria, the consei-vation of certain sequence motifs across
the species suggests that the mechanism of all tetracy-cline pumps driven by proton motive force is basically
similar. The binding site for the transported tetracy-clines appears to be located in helix 4 of the transmem-brane domain. The tetracycline efflux proteins have
both amino acid sequence and stractural similarities
with efflux pumps that extrude other drags, including
chloramphenicol and the quinolones as well as quater-nary ammonium antiseptics.
Regulation of tetracycline resistance. It was
discovered many years ago that efflux-dependent tetra-cycline resistance is inducible by tetracyclines in both
Gram-negative and Gram-positive bacteria. A low
level of resistance is evident when the cells are first ex-posed to antibiotic, followed by a rapid shift to high-level resistance. The mechanism of induction is differ-ent in Gram-negative and Gram-positive bacteria. The
tetracycline efflux system in Gram-negative bacteria is
mediated by a stractural gene for the Tet protein and by
a gene coding for a repressor protein. The two genes
are aiTanged in opposite directions and share a central
regulatory region with overlapping promoters and op-erators. In the absence of tetracycline, a-helices in the
N-terminal domain of the repressor protein bind to the
operator regions of the repressor and structural genes,
thereby blocking the transcription of both. The intro-duction of tetracycline complexed with Mg-+ leads to
binding of the drag to the repressor protein. This
causes a confoixnational change in the repressor
(which has been revealed by X-ray crystallography)
that eliminates its binding to the operator region and
permits transcription of the repressor and stractural
genes. The shift from low-level to high-level resistance
takes place within minutes of exposure of the bacteria
to tetracycline. The process is reversible and a down-shift of resistance follows the removal of tetracycline
from the bacterial environment.
In contrast to Gram-negative bacteria, there is no
Tet repressor protein in Gram-positive cells. The regu-lation of tetracycline resistance in the Gram-positive
bacteria examined so far involves another example of
translational attenuation. The mRNA for the Tet pro-tein has two ribosomal binding sites, RBS1 and RBS2.
In the uninduced state, the ribosome binds to RBSl
and a short leader peptide sequence is translated before
the RBS2 site, which precedes the start of the struc-1i8
9.4 Drug efflux pumps
tural gene proper. At this stage the RBS2 site is
thought to be inaccessible within the secondary struc-ture of the mRNA, thus preventing translation of the
stracturai gene. The addition of tetracycline causes a
confoniiational change in the mRNA, perhaps as a re-sult of slowing translation of the leader seciuence,
which uncovers the RBS2 site. Translation of the
stracturai region follows, leading to the expression of
the tetracycline efflux pump.
9.4.2 Quinolones
Although the major forms of resistance to the
quinolones depend on the mutations in DNA gyrase
and topoisomerase IV previously described, an en-ergy-dependent efflux of quinolones is increasingly
found in Gram-positive and Gram-negative bacteria. A
pump-mediated efflux of fluoroquinolones has also
been reported in the urogenital pathogen Mycoplasma
homiiiis. In Staphylococcus aureus, the iiorA gene en-codes a multidrug efflux pump protein NorA belong-ing to the MFS family, which extrudes quinolones
from resistant cells. Increased expression of mutant
forms of nor A, which may be associated with an in-creased half-life of the norA mRNA, is responsible for
the higher levels of resistance compared with wild-type bacteria. It is interesting that inhibitors of the
function of the NorA efflux protein, such as reserpine
and omeprazole (an inhibitor of acid secretion in the
stomach), dramatically improve the activities of
ciprofloxacin and norfloxacin against strains of
Staphylococcus aureus which overproduce NorA.
More hydrophobic quinolones, such as trovafloxacin
and moxifloxacin, are relatively poor substrates for
NorA, which may enhance their antibacterial activity
against pump-mediated resistant organisms. Homolo-gous MFS-type quinolone effluxing pumps also occur
in Streptococcus pneumoniae, Enterococcus faecium
and Enterococcus faecalis.
compromised individuals. Oropharyngeal candidiasis
caused mainly by Candida albicans is a particularly
common and distressing infection in AIDS patients.
Although azole antifungal agents, such as fluconazole
(Chapter 3), are generally effective against Candida
infections, their ever-increasing use has led to the
emergence of azole-resistant strains of this pathogen.
There are several modes of resistance, including a re-duction in the affinity of the cytochrome P,^^^J compo-nent of the 14-a-demethylase target and a substantial
increase in the cellular content of this enzyme. How-ever, there is little doubt that efflux pump activity
makes a substantial contribution to the problem of re-sistance to azoles. Clinical isolates of resistant strains
of Candida that fail to accumulate radiolabelled flu-conazole may overexpress several genes that encode
multidrug efflux systems, including the cdrl and cdr2
genes (signifying Candida drug resistance) which en-code members of the ABC transporter family, and
camdrl, which belongs to the PMF-driven MFS super-family. Both cdrl and cdr2 cloned from Candida into
Saccharomyces cerevisiae confer resistance to flu-conazole, ketoconazole and itraconazole. The camdrl
gene confers resistance to benomyl and fluconazole.
The level of expression of these genes in Candida al-bicans, as indicated by mRNA measurements, may in-crease dramatically during prolonged fluconazole
treatment of persistent infections in immunocompro-mised patients The observed dual overexpression of
cdrl and cdr2 suggests that these genes share a com-mon transcriptional regulator. However, the mecha-nisms underlying the overexpression of efflux pump
genes in azole-resistant Candida albicans could also
include gene amplification, mutations in the promoter
regions, ft-an.s’-acting factors, or simply a greater stabil-ity of the mRNAs. It should be emphasized that the
phenomenon of acquired cellular resistance to azoles
almost certainly results from a combination of in-creased efflux pump activity with the other mecha-nisms mentioned earlier.
9.4.3 Azole antifungal drugs  9.4.4 Chloroquine
Azole antifungal drags have become increasingly im-portant because the incidence of serious fungal infec-tions has risen shaiply, especially among immuno-The prevention and treatment of malaria is now in
jeopardy in many parts of the world because of the re-sistance of malarial parasites to chloroquine, a main-169
Biochemical mechanisms of drug resistance
stay against malaria for more than 50 years. It has been
clear for some time that the resistance of Plasmodium
falciparum to chloroquine is associated with reduced
accumulation of the drug within the parasite. The dis-covery of two genes (pfindr-l and pfmdr~2) for ABC-type transport proteins in chloroquine-resistant strains
of Plasmodium falciparum suggested that the reduced
accumulation of drug might be due to the activity of
broad-specificity efflux pumps in removing chloro-quine from the protozoal cytoplasm. However, several
lines of evidence cast doubt on this appealing model:
1. Drug resistance does not cosegregate with
the pfmdr genes in a genetic cross between
chloroquine-sensitive and chloroquine-re-sistant strains.
2. It had been expected that the transport protein
would be located in the cytoplasmic membrane
in order to fulfil its putative efflux function. In
fact, the protein is found mainly in the mem-brane of the intracytoplasmic food vacuole of
Plasmodium falciparum. The position of the
protein in the membrane suggests that it prob-ably transports substi^ates, including chloro-quine, into the vacuole rather than out of it.
3. Some laboratory strains of chloroquine-re-sistant Plasmodium falciparum have de-creased levels of expression of pfmdr-1
rather than the increased levels that might
have been expected. However, if pfmdr-1 en-codes a transport protein that promotes the
uptake of chloroquine into the digestive vac-uole, it can be ai-gued that decreased expres-sion of pfmdr~l would give rise to resistance.
4. Increased expression of pfmdr-1 in clinical
isolates from Thailand, a region of highly re-sistant malaiia, is associated with resistance
to two other antimalarial drugs, mefloquine
and halofantrine, but increased sensitivity to
chloroquine.
It seems unlikely, therefore, that increased ex-pression of pfmdr-tncoded proteins is involved in
malarial resistance to chloroquine. More recent inves-tigations have defined another gene, cg2, that is closely
linked to the chloroquine-resistant phenotype in Plas-modium falciparum. A genetic cross between a drag-sensitive strain from Honduras and a resistant strain
from Southeast Asia showed that the cg2 gene derived
from the resistant parasite has a complex set of poly-morphisms, i.e. mutations, that are closely associated
with the resistant phenotype. These specific polymor-phisms were also detected in chloroquine-resistant
parasites from various locations in Southeast Asia and
from Africa, thus strengthening the proposal that mu-tations in cg2 are responsible for resistance to the drug.
A different set of cg2 mutations was identified in
chloroquine-resistant Plasmodium, falciparum isolates
from South America.
The precise location of the CG2 protein encoded
by cg2 within the malarial parasite has been a matter of
some debate, but it is now believed to be close to, but
not actually within, the membrane of the digestive vac-uole. Although the CG2 protein has no amino acid ho-mology with known drag efflux or ion transport pro-teins, mutant forms of the protein may nevertheless
have some role in limiting the intravacuolar concentra-tion of chloroquine. The discovery of yet another gene,
Pfcrt, apparently tightly linked to the chloroquine-re-sistant phenotype, adds further complexity to the chal-lenge of unravelling the mechanism of chloroquine re-sistance. The function of Pfcrt is not known at present,
but the deduced amino acid sequence of the encoded
protein has many of the features of a membrane pro-tein, suggestive perhaps of a transmembrane channel
or pump. It is possible that mutations in Pfcrt associ-ated with chloroquine resistance may also in some way
limit the access of the drag to its site of action in the di-gestive vacuole (Chapter 6), either by restricting influx
to the vacuole or by drug extrusion. Clearly there is
some way to go before a complete explanation of
chloroquine resistance is available, and indeed of re-sistance to other aminoquinoline antimalarial drugs. It
seems possible that the products of several genes, in-cluding pfmdr-1, pfmcIr-2, cg-2 and Pfcrt may all con-tribute to the resistant phenotype.
9.5 Other mechanisms of resistance
9.5.1 Ribosomal protection against
tetracyclines
Ribosomal protection against macrolides and certain
aminoglycosides by enzymically catalyzed covalent
170
9.5 Other mechanisms of resistance
modification of ribosomal RNA has already been de-scribed. The mechanism of resistance to tetracyclines
afforded by ribosomal protection proteins is quite dif-ferent. Nine ribosomal protection proteins encoded by
a series of tetracycline resistance genes, including
tet(M), tet(O) and tet(Q’) among others, are widely dis-tributed among Gram-positive and Gram-negative bac-teria, although they appear to be absent from some
Gram-negative enteric species. In contrast to efflux-mediated resistance, the degree of resistance to tetracy-clines confen-ed by ribosomal protection proteins is
relatively modest although broader in scope, and in-cludes minocycline. The ribosomal protection proteins
have significant amino acid sequence homology with
the elongation factors EF-Tu and EF-G which are in-volved in protein biosynthesis (Chapter 5). The highest
degree of homology is found with the GTP-binding do-main of the elongation factors. One of the few riboso-mal protection proteins so far studied in detail, Tet(M),
allows aminoacyl tRNA to bind to the ribosomal A site
in the presence of concentrations of tetracycline that
would nomially inhibit this process. Tet(M) apppears
to displace the antibiotic from the ribosome. The fa-vored explanation for the action of the ribosomal pro-tection proteins is that they bind to the ribosome and
induce a conformational change which disrupts the in-teraction between the tetracycline molecule and its
binding pocket fomied by the head of the 30S subunit
at the A site (Chapter 5). GTP hydrolysis catalyzed by
the ribosomal protection proteins may energize the
conformational change. X-Ray crystallographic data
will be needed to substantiate this model of ribosomal
protection. There is some evidence that ribosomal pro-tection is inducible by tetracyclines.
9.5.2 Vancomycin
Vancomycin (Chapter 2) has been described as the
‘last chance’ antibiotic because it is the only drug ef-fective against methicillin-resistant Staphylococcus
aureus and p-lactam-resistant enterococci. Unfortu-nately, vancomycin-resistant enterococci are appear-ing in hospitals in many parts of the world. The vaiiA
vancomycin resistance gene cluster in Enterococcus
faecaUs transfers to Staphylococcus aureus by conju-gation under laboratory conditions and expresses high-level resistance, although as we shall see, vaw A-medi-ated resistance is probably not (yet) a major factor in
staphylococcal resistance to vancomycin. Details of
the mechanisms of vancomycin resistance are of con-siderable importance in planning appropriate counter-measures.
The antibacterial activity of vancomycin hinges
on its ability to bind to the D-alanyl-D-alanine temiinal
of the peptidoglycan precursor of the cell wall, thereby
blocking the activity of the transglycolase essential for
the synthesis of the peptidoglycan sacculus (Chapter
2). Enterococcal resistance to vancomycin rests on an
unusual strategy in which the terminal D-alanine of the
peptidoglycan precursor is replaced by an a~hydroxy
acid, D-lactate. The affinity of vancomycin for the D-alanyi-D-lactate terminal is 1000-fold less than for the
D-alanyl-D-alanine terminal of susceptible bacteria. A
gene cluster harboured by transposon Tnl546 confers
vancomycin resistance in enterococci and encodes five
proteins. Vantl is a dehydrogenase that converts pyru-vic acid to D-lactic acid. A ligase, VanA, catalyzes the
formation of an ester bond between the D-alanyl
residue and o-lactate; and a third enzyme, VanX, is a
DD-peptidase that hydrolyzes D-alanyl-D-alanine,
thereby virtually eliminating the synthesis of peptido-glycan precursors with D-alanine terminals. The DD-peptidase activity of a fourth protein, VanY, removes
the terminal D-alanine residue of any residual normal
peptidoglycan precursors that are produced despite the
attention of VanX. VanY enhances but is not essential
for vancomycin resistance. The fifth protein, VanZ,
confers resistance to the related antibiotic teicoplanin
by an unknown mechanism.
The drastically reduced affinity of vancomycin
for the terminal D-alanyl-D-lactate compared with that
for D-alanyl-D-alanine stems from the elimination of a
critical hydrogen bond. The amidic NH group of the D-alanyi-D-alanine linkage contributes one of five hydro-gen bonds involved in the binding of vancomycin to
the dipeptide. This bond is lost when the amide bond is
replaced by the oxygen-containing ester link in D-alanyi-D-lactate.
The sophisticated machinery for vancomycin re-sistance is regulated at the transcriptional level by a
two-component system. VanS is believed to be a sen-sor protein associated with the cytoplasmic membrane
that both detects the presence of vancomycin and
171
Biochemical mechanisms of drug resistance
controls the phosphorylation of an activator protein,
VanR, required for the transcription of an operon con-taining the vanH, vanA and vanX genes. Phosphoryla-tion of VanR reduces its affinity for the promoter DNA
of the vanHAX operon. A current model of the in-ducibility of vanocmycin resistance by vancomycin
and other glycopeptide antibiotics suggests that the
VanS sensor controls the phosphorylation level of
VanR. The presence of glycopeptide antibiotics in the
bacterial environment leads to increased phosphoryla-tion of VanR, which in turn pemiits a higher rate of
transcription of the vanHAX operon. Just how VanS
detects the presence of antibiotic is not known, but
possibly an accumulation of peptidoglycan precursors
caused by the antibiotic may be involved rather than a
direct interaction of the antibiotic with VanS. It is
thought that VanS is either a protein phosphatase or
protein kinase. The model proposes that in the pres-ence of inducing glycopeptides the activity of VanS is
either inhibited or stimulated, depending on whether it
turns out to be a phosphatase or kinase. In either event,
the phosphorylation level of VanR would be increased.
A semisynthetic derivative of vancomycin, orita-vancin (Chapter 2), has significant activity against
vancomycin-resistant enterococci. The presence of the
p-chlorobenzyl side chain in oritavancin, together with
the highly dimerized structure of this antibiotic, is
thought to enhance anchoring of the drug to the cyto-plasmic membrane, thereby increasing the effective
dmg concentration at the target site and partially off-setting the decreased affinity for the D-alanyl~D~lactate
terminals.
Antibiotic ‘trapping’ in vancomycin-resistant
Staphylococcus aureus
Contrary to earlier expectations, the mechanism of
vancomycin resistance in most clinical strains of
Staphylococcus aureus is not due to the VanA/VanB
phenotype but appears to be associated with a remark-able thickening of the peptidoglycan wall which, it is
suggested, effectively traps the antibiotic before it
reaches the cytoplasmic membrane. The meshwork of
the outer layers of thickened peptidoglycan is said to
be ‘clogged’ by the trapped vancomycin molecules
themselves, thus further hindering the inward diffusion
of antibiotic from the external medium. The cell walls
of many VRSA strains from several countries around
the world were found to have a mean thickness of 31.3
+ 2.6 nm compared with 23.4 + 1.9 nm in vancomycin-susceptible strains. Despite this striking association
between vancomycin resistance and the thickening of
the wall of Staphylococcus aureus, an actual reduction
in the access of the antibiotic to its target site has yet to
be convincingly demonstrated.
How does the increased thickness of peptidogly-can arise? Current evidence points to a reduction in the
turnover of peptidoglycan associated with reduced ac-tivity of the autolysin enzymes that normally remove
the older, outer layers of peptidoglycan as the bacterial
cell grows. This resembles the long-recognized phe-nomenon of tolerance in the pneumococci to inhibitors
of cell wall biosynthesis. In antibiotic-susceptible
pneumococci, the drugs indirectly deregulate the nor-mal control of autolysin activation. Increased autolysin
activity synergizes with inhibition of cell wall biosyn-thesis by causing more rapid dissolution of the pepti-doglycan. Mutations in the regulatory control of au-tolysin in antibiotic-tolerant pneumococci block the
signaling mechanism which triggers increased au-tolysin activity in antibiotic-challenged susceptible
bacteria. A range of such mutations has been identified
in clinical strains of vancomycin-tolerant pneumo-cocci. The genetic basis of vancomycin resistance in
Staphylococcus aureus is currently under intensive
study.
9.6 Drug resistance and the future of
antimicrobial chemotherapy
The development of eifective drugs against microbial
infections is undoubtedly one of the outstanding suc-cesses of twentieth-century science and medicine.
However, as we have seen, the whole therapeutic en-teiprise is threatened by the relentless rise of drug-re-sistant bacteria, fungi, viruses and protozoa. Mecha-nisms for eliminating naturally occurring antibiotics
by enzymic inactivation and efflux pump systems ex-isted long before the chemotherapeutic era. These, in
combination with the mutability of micro-organisms,
their high replication rates, and especially in bacteria,
their ability to exchange and acquire new genetic ma-terial, pose enormous practical and intellectual chal-172
9.6 Drug resistance and the future of antimicrobial chemotherapy
lenges to scientists and physicians. Some authorities
have raised the grim prospect of a time not too distant
when the chemotherapy of infectious disease may fail
completely in the face of dnig-resistant organisms.
However, while treatment failures already occur in
specific situations, the strategy detailed here may post-pone or even avert a global collapse of antimicrobial
chemotherapy.
1. The probability of infection itself can be re-duced by (a) insistence on high standards of
hygiene in hospitals and nursing homes; in
educational institutions and places of em-ployment, entertainment and hospitality; and
in the manufacture, preparation and cooking
of foods, and (b) the further development and
vigorous use of prophylactic vaccines to pre-vent infection.
2. Undoubtedly the trivial, sometimes irrespon-sible use of antimicrobial drugs in human and
veterinary medicine and in agriculture has
fostered the emergence and spread of resist-ant organisms. Even the medically re-spectable use of antimalarial drugs as pro-phylactic agents to protect people in
malarious zones has contributed to the emer-gence of dmg-resistant malarial parasites.
Publicity and a growing awareness of drug
resistance has encouraged progress towards a
more restrained use of antimicrobial drugs,
but their ready availability without medical
supervision in many parts of the world re-mains a serious cause for concern.
3. The therapy of infections should be designed
to ensure maximum effectiveness in terms of
the choice of drug or combinations of drugs,
the dose levels, dosing frequency and dm^a-tion and finally, patient compliance with the
agreed treatment regime. Whenever possible,
the objective must be to eliminate the infect-ing organisms by a combination of direct
chemotherapeutic attack and the activity of
the patient’s immune defences. The need to
minimize the numbers of infecting organisms
by chemotherapy is of overwhelming impor-tance in immunocompromised patients. Sen-sitive techniques for monitoring the viral
load in AIDS patients have a major role in
managing the use of combinations of anti-HIV drugs. Unfortunately, these techniques
are often not available in many developing
areas of the world where AIDS is now
endemic.
4. Despite the measures listed above, there will
certainly be a continuing need for new drugs
to combat drug-resistant infections. Where
the biochemical mechanisms of resistance
are understood, ingenious drug design and
skillful chemical synthesis may deliver fur-ther successes comparable with the develop-ment of novel p-lactams. The elucidation of
the biochemical systems essential for micro-bial survival provides new targets for
chemotherapeutic attack and will continue to
occupy many scientists in academia and the
phamiaceutical and biotechnology indus-tries. The ingenious application of molecular
genetics to micro-organisms capable of an-tibiotic synthesis holds the promise of creat-ing novel structures with improved anti-microbial activities. Knowledge of the
moleculai- genetic basis of drug resistance
may also be turned to good effect in devising
agents to hinder the emergence and spread of
resistant micro-organisms.
.5. The application of agents to boost the im-mune defences of patients during infections
has so far been relatively limited and re-stricted largely to immunocompromised indi-viduals. Such treatments are generally expen-sive compared with antimicrobial drugs, but
the ability of biotechnology to produce the
many proteins involved in immunocompe-tence may eventually be valuable in the man-agement of infectious disease.
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174
A23187
ionophore specific for divalent ions, 57
preferential binding of Ca^”*, 57
structure, 57
Abacavir
inhibitor of HIV reverse transcriptase,
73-75
structure, 73
Acceptor in peptidoglycan biosynthesis, 24
Acceptor site in protein biosynthesis, 89
Acetylation
of aminoglycosides, i59–160
of cliloramphenicoi, 156-159, 161
of streptogramins, 160-161
Acquired Immune Deficiency Syndrome
cun’ent epidemic, 1
drugs for, 72-75, 109,110
Acridines
binding to DNA, 81-83
inhibition of nucleic acid synthesis by,
81-83
medical history, 81
vital staining. 81
Acriflavine, 3
Activation of aminoacids
inhibition by amino acid analogues and
antibiotics, 91-92
Activation of antimicrobial compounds by
metabolism, 12, 149
Active transport of antimicrobial agents,
123,126-133
Acyclovir
activation by thymidylate kinase, 72
antiviral action, 72
selectivity for viral enzyme, 72
Adenosine triphosphatase
inhibition by A23187, 57
in membrane, 50
inhibition by chlorhexidine, 50
Adenosine triphosphate
inhibition of synthesis by antimicrobial
agents, 11
role in drug efflux, 127
.Adenylylation of iiminoglycosides,
159-160
AIDS xcc Acquired Tmniime Deficiency
Syndrome
D~ Alanine
antagonism of antibacterial action of cy-closerine, 31
liberation in cross-linking of peptidogly-can. 24
release in peptidoglycan cross-linking
inhibited by penicillin, 36
D-.AIanyl-D-alanine ligase
inhibition by cycloserine, 31
in peptidoglycan biosynthesis, 23
D-.AIanine carboxypeptidases
in bacterial membrane, 27
inhibition by P~lactams, 37
Alanine pemreases in accumulation of D-cycloserine, 130
Alanine raceniase
function, 31
inhibition by cycloserine, 31
Alcohols as antiseptics, 49
Allylamine antifungals
clinical use, 63
inhibition of fungal squalene epoxidase,
63
Amantadine
antiviral action. 111
interaction with Mj protein, i 11
structure, 111
Amikacin, 94
/7-Aminobenzoic acid
as a bacterial growth factor, 67
competitive antagonism of sulfonamide
action, 67
part of the structure of folic acid, 67
7-Aminoceplialosporanic acid, 35
Aminoglycoside antibiotics
acetylation, 159-160
action on protein synthesis, 92-96
adenylylation, 159-160
interaction vt’ith 16S rRNA, 93-94
killing action of, 94
phosphorylation, 159-160
possible inhibition of fran.^-translation,
96
resistance, 159-160
uptake by bacteria, 132-133
see also Gentiimicin: Kanamycin A;
Neomycin C: Spectinomycin;
Streptomycin
6-Aminopenicillamc acid. 33
Amoxycillin, 34, 35
Amphotericin B
action on cell membranes. 60 61
effect as an antifungal agent, 59
interaction with cholesterol, 60
structure, 60
toxicity, 59
Ampicillin
antibacterial spectrum, 35
structure, 34
Antifungal agents, 41 -44, 108-109
Antigenic groups in Gram-negative wall.
“20-21
Antimicrobial action
at molecular level, 13
determination of mechanism, 10-11
evidence from effect on uptake of nutri-ents, 11
relationship to chemical stracture, 10-11
selectivity towards micro-organisms, 13
Antimicrobial agents
distinction between primary and second-ary effects, 10
early remedies, 2-3
metabolism in the body, 12
pharmacological biochemistry, 13
selectivity through concentration in the
microbial cell, 13, 96
social and economic importance, i-2
structural analogies with biologically
important molecules, 10-11
.Antiprotozoal drugs, 9, 68-69, 113-i 19
Antisepsis, 3
Anhseptics
bacteriostatic action at low concentra-tions, 48
causing leakage of constituents, 48
factors in bactericidal action, 48
need for bactericidal action, 47
penetration into bacterial cells, 48
see also Antisepsis
Ara A see .Arabinosyl adenine
Arabinogalactan in cell walls of mycobac-teria, 39
.Arabinosyl adenine
antiviral action, 72
inhibition of D.N.A synthesis, 72
structure, 71
Arseniciils, 4
Artemisinin
antimalarial action, 115 116
inhibition of Ca'”” -dependent .ATPase,
116
interaction with heme, 115
structure, 115
Artesunate
antimalarial action, 115-116
structure, 115
Asepsis, 3
Atebrin see Mepacrine
175
Index
Atovaquone
antimalarial action, 116
inhibition of mitochondria, 116-117
specificity of action, 116-117
structure, 117
Atoxy!
efficacy against trypanosomes, 4
structure, 4
Auxotrophic bacteria, use in determining
site of antibacterial action, 111
Azidothymidine
antiviral action, 73
biochemical mechanism of action, 13-15
structure, 71
Azole antifungals
clinical use, 61
inhibition of C-14 demethylase, 61
structures, 63
toxicity. 63-64
AZT see Azidothymidine
Bacitracin
binding to polyprenylpyrophosphates, 30
structure, 30
topical application, 30
Bacterial cell wall, protective function of,
17-18
Bacteriostatic and bactericidal action com-pared, 47
Benznidazole
in treatment of Chagas’disease, 119
structure, 108
Benzyl penicillin
enzymic conversion to 6-aminopenicil-lanic acid. 33
instability to stomach acid, 33
structure, 34
susceptibility to P~lactamase. 1.5.5
Biocides, 47
Biofilms and resistance to antibiotics, 121
Bioinformatics, role in drug discovery, 14
Bone marrow toxic effects of chloram-phenicol on, 99
Bon-elidin, 91
Bronopol
interaction vi’ith thiol groups, 49
structure, 49
Calcium
binding to A23187, 57
in Gram-negative bacteria! outer mem-brane, 124
Carboxypeptidases
catalytic mechanism, 37
role in peptidoglycan synthesis, 27
targets for P-lactiims, 37
Caspofungin,
antifungal drug, 43
inhibitor of glucan biosynthesis, 43
structure, 43
CAT see Chloramphenicol acetyl transferase
Cationic antiseptics, 49-50
Cefotaxime, 34
Cefofefan
antibacterial spectrum, 36
structure. 34
Cefuroxime
stability to P-lactamases, 35 36
structure
Cephalexin, 34
Cephaloridine, 35
Cephalosporanoic acid, 152
Cephalosporin C
biogenesis, 35
structure, 34
Cephamycin C, 36
Cetrimide
chain length and antiseptic action, 49-50
structure, 49
Chagas’ disease, see South American try-panosomiasis
Chain, Ernst, 7
Chelation of cations
by tetracyclines, 98
by quinolones, 132
Chemotherapy
early history, 4-5
its debt to Ehrlich, 5-6
Chitin synthase
inhibition by polyoxins and
nikkomycins, 42^ 3
isoenzymes, 42
Chitin
biosynthesis, 42
role in fungal cell walls, 41-42
Chloramphenicol
binding to ribosomes, 99-100
cliniciil use, 99
effect on bone marrow, 99
inhibition of peptide bond formation,
99-100
inhibition of puromycin reaction, 99
resistance, 155-159
Chloramphenicol acetyl transferase
catabolite repression of, 158-159
catalytic mechanism, 156-158
induction mechanism, 157-159
structure, 157
types, 156
Chlorhexidine
antiseptic action, 50
‘blistering’ of cell wall. 50
physical properties, 50
precipitation of bacterial nucleic acid
and protein, 50
structure, 49
ChloTotiuine
inhibition of hemozoin formation,
114-115
interaction with DNA, 81
resistance, 113, 169-170
structure, 81
uptake into malarial pjirasite, 133
Cilastatin
inhibitor of renal dehydropeptidase, 36
structure, 36
Ciprofloxacin
inhibitor of DNA gyrase, 76-77
stracture, 76
Clavulanic acid
inhibitor of P-lactamase, 35, 155
stracture, 35
Clindamycin
action on ribosomes, 100
antibacterial spectrum, 100
structure, 100
Cloxacillin
resistance to P-lactamase, 35
structure, 34
Coccidiosis and drag treatment, 56
Codon misreadings induction by aminogly-cosides, 92-95
Conjugation
bacterial, 141-145
transfer of drug-resistance by, 142-145
Conjugative tnmsposons, 141-142
Cotrunoxazole, double block of tetrahydro-folate biosynthesis, 69
Coumarin anfibacterials, 78-79
Coupling proteins, role in DNA transfer,
144
Creuzfeldt-.Tacob syndrome, 2
Cross-linking of peptidoglycan, 24-28
Cycloguanil. 68
Cycloheximide
action on ribosomes, 104
action on translocation, 104
antimicrobial action, 104
structure, 104
Cycloserine
active uptake by bacteria, 31, 130
antagonism of antibacterial efi’ect by D-alanine, 31
inhibition of alanine racemase and D-alanyl-D-alanine synthetase, 31
significance of rigid structure, 31
structure, 31
Cyclothialidines
inhibition of bacterial DNA gyrase,
78-79
structure, 78
Cytoplasmic membranes
interaction with polyenes. 59-61
interaction with polypeptide antibiotics,
52-56, 58-59′
interaction with synthetic antiseptics,
48-49
permeability barrier to antimicrobial,
agents 121-123
Dapsone see 4.4′-diaminodiphenyl-sul-phone
Dalfopristin
176
I liClU A
inliibitor of protein biosynthesis, 101
structure, 102
synergism with quinupristin, 102
3,4-Deliydroproline incorporation into ab~
nonmJ proteins, 91
Delavirdine
inhibitor of reverse transcriptase, 74
structure, 74
3-Deoxy-D-raanno~octu]osonic acid
binding of Ca^”‘” and Mg^”*, 20
component of lipopolysaccaride core, 20
Diclobutrazole
inhibitor of ergosterol biosynthesis, 63
structure, 63
Didanosine
inhibitor of reverse transcriptase, 73
siTucture, 73
4,4′ -Diarn i nodipheny I sij I phone
biochemical action, 66-67
siTucture, 66
use in leprosy, 66
Diaminopimelic acid in peptidoglycan, 28
Diffusion
fccililaled, 123
passive, 122-123
Difhioroinethyloniithine see eflornithine
Dihydrofolate reductase inhibitors
biochemical effects, 68-70
resistance to, 164 165
specificity towards different organisms,
68
Dihydrofolate reductase
differences between bacterial and mam-malian enzymes, 68
drug-resistant forms, 164-165
X-ray analysis of structure, 69
Dihydropteroate synthase
inhibition by sulfonamides, 67
role in folic acid biosynthesis, 67
structure, 67
sulfonamide-resistant form, 165
Disinfectants
chemically reactive, 48
early history, 47
modern uses. 47
need for bactericidal action, 47
DNA
effects of intercalating drugs on physical
properties, 81, 82
polymerases, 65
DNA gyrase
inliibition by cyclothialidines, 78-79
inhibition by quinolones, 76-78
inhibition by novobiocin, 78-79
role in supercoiling, 75 76
DNA synthesis inhibition
by inhibitors of nucleotide biosynthesis,
66 71
by inhibitors of polymerization, 72-75
Donor site in protein biosynthesis, 89
Doxycycline, 97
Drug efflux
in bacteria, 126-127, 167-169
in resistant fungal cells, 169
Drug-resistimce
biochemical mechanisms, 149-174
genetic basis. 135-148
Ebola fever, 2
EchinocandinB
effect on glucan biosynthesis, 43-44
structure, 135-148
Eflornithine
inhibition of ornithine deciirboxylase, i i 9
permease-inedialed entry into cells, 119
specific antitrypanosomal action, 119
structure, 118
Ehrlich, Paul, 4-6
Emetine, 3
Energy metabolism inhibition by antiproto-zoal agents, 116-118
Ergosterol
biosynthetic pathway. 62
biosynthesis inhibition by antifungal
agents. 61-64
target for polyenes, 60-61
Erythromycin
antibacterial action, 100
blocks access to polypeptide exit tunnel,
100
effect of pH on antibacterial activity of,
122 123
interaction with 23S rRNA, 101
resistance, 162-163
structure. 101
Ethambutol
activity against mycobacteria, 40
inhibition of arabinosyl transferase, 40
structure, 39
Ethanol
disinfectant action, 49
effects on membranes, 49
Ethidium
action on trypanosomal DNA, 83
intercalation with DNA, 81-82
structure, 81
Ethionamide, inhibitor of niycolic acid
biosynthesis, 40
Ethionine incorporation into abnormal pro-teins, 91
;Y-Ethylglycine, 91
Ethylenediaminetetraacetic acid, disruption
of Gram-negative outer membrane,
124
Exit domain of ribosomes, 86
Extended spectrum p-lactamases, 154
Facilitated diffusion of antimicrobial
agents. 123, 129-133
Fict’s law of diffusion. 122
Filamentous fonns of E. coli caused by p-lactams, 37
Filipin, 61
Fleming, Alexander. 7
Florey, Howard, 7
5-Fluorocytosine
antifungal action, 70
facilitated transport into fungal cells, 70
metabolism of, 70
resistance, 70
structure, 70
Fluoroquinolones see Quinolone antibacte-rials
Folic acid
biosynthesis. 67-69
inhibition of biosynthesis, 67-69
structure, 67
Folic acid aniilogues, 68 69
Footprinting, application to antibiotic-ribo-some interactions, 93
.(V-Formylmethionyl tRNAp role in protein
biosynthesis, 87
Foscarnet
antiviral drug, 74-75
inhibitor of reverse transcriptase, 75
structure, 73
Fosfomycin
inhibition of MurA, 31
inhibition of peptidoglycan synthesis,
3(K51
structure. 30
Fungal cell walls
inhibition of biosynthesis, 42-44
structure. 41-42
Fungal liyphae affected by griseofulvin,
108-109
Furanomycin. 91
Furazolidone
antimicrobial action, 107
redox potential, 107
structure, 108
Fusidic acid
clinical use, 103
inhibition of translocation, 103-104
resistance of Gram-negative bacteria, 103
structure, 103
Ganciclovir
antiviral action, 72
structure. 71
Gemifloxacin
inhibitor of DNA gyrase, 76
structure, 76
Gene cassettes, role in bacterial drug resist-ance, 141
Genomics, importance in antimicrobial
drug research, 14
GentamicinCj.^
biochemical action, 94
structure, 95
Global regulators of expression of drug re-sistance genes, 147
Glucan
177
Index
in fungal cell walls, 41
inhibition of biosynthesis, 43-44
L-a~Glycerophosphate permease transports
fosfomycin, 130-131
Glycine-specific ti’ansfer RNA in peptido-glycan biosynthesis. 24
Giycopeptide antibiotics, 32-33
Glycopeptide see Peptidoglycan
Glycyltetracycline, see tigicycline
Gramicidin A
biochemical action, 58-59
effect on membrane conductivity, 58-59
pore formation in membranes, 59
structure, 58
Gramicidin D inhibits Plasmodium falci-parum, 59
Gramicidin S
antibacterial action, 52
conformation in solution, 53
structural requirements for activity, 53
structure, 52
Gram-negative bacteria, intrinsic resistance
to certain antibacterial agents,
123-124
Gram-negative bacterial walls, 19-22
Gram-positive bacteria
osmotic pressure, 18
cell wall, 18-19
Griseofulvin
antifungal activity, 108
inhibition of microtubule formation,
108-109
structure, 109
Guanine nucleotides, inhibition of biosyn-thesis by ribavirin, 71
HAART, combination therapy against HIV,
73
Halofantrine
antimalarial drug, 115
structure, 114
Halogens as biocides, 48
Halophilic bacteria, resistance to antibi-otics affecting peptidoglycan synthe-sis, 30
Hansch equation application to antibacter-ial agents, 122
Hansen’s disease, see leprosy
Haptophore, 5
Herpes treatment with nucleoside ana-logues, 72
Hexose-6-phosphate permeases, in accu-mulation of fosfomycin, 130-131
HIV protease
as a target in AIDS therapy, 109
involvement in viral replication, 109
inhibitors. 109-110
HIV see Hiunan Immunodeficiency Virus
Holmes, Oliver Wendell, 3
Human Immunodeficiency Virus
entry into cells, 110-111
mutations in, 137
resistance to HIV protease inhibitors,
166-167
resistance to reverse transcriprtase in-hibitors, 166
treatment with inhibitors of HIV pro-tease, 109-110
treatment with inhibitors of reverse tran-scriptase, 72-75
treatment with inhibitors of viral entry,
110-111
Hydroxynaphthoquinones as antiprotozoals,
116-117
Immunity enhancement by ribavirin, 71-72
Immunological control of infectious dis-ease, 2, 173
IMP-dehydrogenase inhibition by ribavirin,
71
Indinavir
inhibition of HIV protease, 109
structure, 110
Indolmycin, 91
Influenza prophylaxis and therapy,
Ill’-I12
Initiation of protein biosynthesis, 87-89
Integrons
role in bacterial drug resistance, 141
structure, 141
Intercalation of DNA by planar molecules,
77. 80-83
Interferon
antiviral action, 112
biological activities, 112
gene family, 113
receptor, 113
mechanism of antiviral action, 113
5-Iododeoxyuridine
interference with DNA synthesis and
function, 72
structure, 71
use in herpes infections, 72
Ionization of antibacterial agents, effect on
penetration into bacterial cells,
122-23
lonophoric antibiotics
iintiprotozoal activity, 54, 59
effects on cation transport, 54-56
Ipecacuanha root, 2
Isoniazid
activation by catalase-peroxidase, 40
inhibition of InhA, 40
resistance in mycobacteria, 40, 161
structure, 39
Isonicotinic acid, metabolite of isoniazid,
39, 40
Isoprenyl phosphate (C.,) see Unde-capreny! phosphate
Isopropanol, effects on membranes, 49
Kanamycin A
W-acetylation. 159
biochemical action, 94
stracture, 95
KDO see 3-Deoxy-D-manno-octulosonic
acid
Ketoconazole
clinical use. 61-62
inhibition of C-14 sterol demethylation,
61-62
structure, 63
toxicity. 62-63
Ketolides, 10(V101
Koch, Robert, 3
6-Lactamases
approaches to the problem of, 153-155
catalytic mechanisms, 152-153
classification of, 150-151
extended spectrum, 154
Gram-negative, 151-152
Gram-positive, 151
inhibitors of, 155
metallo-enzymes, 152-153
regulation of, 151-152
relationship to ciirboxypeptidases and
transpeptidases, 152
Lamivudine resistance in AIDS patients,
166
Lassa fever, 2
Leprosy, treatment with 4,4′-diamin-odiphenylsulphone, 66
Linezolid
antibacteriiil spectrum. 102
inhibitor of protein biosynthesis, 103
stracture, 102
Lipid A component of lipopolysaccharide,
21
Lipopolysacchiirides of outer membrane of
Gram-negative bacteria, 21
Lipoprotein
in Gram-negative bacteria, 21
structure, 21
Lister, Joseph, 3
Lopinavir, 110
L-phase enhanced sensitivity to antibacter-ial agents, 124
Magainins, 52, 54
Magnesium
displaced from cytoplasmic membrime
by polymyxin, 53
release from mitochondria by A23187,
57
significance in Gram-negative bacterial
wall, 20
quinolones and, 132
tetracyclines and, 98, 131
Malaria
178
I liClU A
drug resistance, 165, 169-170
incidence, 113
treatment, 9. 68, 113
Maphaisen
metabolite of salvarsan, 6
structure, 4
Meciliinain
specific effect on Grain-negative bacte-ria, 35
structure, 34
Mefloquine
mode of action, 115
resistance. 170
structure, 114
Melarsoprol
inhibition of glycolysis in African tiy-panosomes, 118
interaction with trypanofhione, 118
structure, 118
Mepacrine
antirmJiuial agent, 113, 115
structure, 114
Mercuric chloride
as disinfectimt, 4
failure as systemic antibacterial, 4
Mi;’,«<9-2,6-diaminopimelic acid in peptido-glycan, 28
Messenger RNA,
translational attenuation in induction of
drug resistance, 157-158. 163,
168-169
;r««.S”translation of, 96
Metabolic activation of antmiicrobial com-pounds, 12, 39-40, 41, 70, 72-75,
107-108
Methicillin
resistance, 162
stability to P-lactamase, 35. 154, 162
structure, 34
Methotrexate
cytotoxic action, 68
structure, 68
Methylene blue, 4
;V~Methyl transferases
in resistance to macrolides, 162 163
induction mechanism, 163
Metronidazole
activity due to reduction product,
107-108
antimicrobial spectrum, 107
resistance, 161
siTucture, 108
Miconazole
antifungal action, 61-63
structure, 63
Microiuxay expression technology, in stud-ies of drug action, 12
Microbial resistance
recognihon by Ehrlich, 6
genetics, 135-148
biochemical mechanisms, 149-174
Minocycline
low susceptibility to efflux pumps, 168
structure, 97
Misreading of genetic code, induction by
am inoglycosides, 92-96
Mitochondrial ribosomes, 85
Mode of action of antimicrobial agents
methods of study, 10-13
Monensin
coccidiostat, 56
preferential binding to sodium, 56
structure, 57
use to improve food conversion in rumi-nants, 57
Monobactams, 36
Morpholine antifungals, inhibition of sterol
biosynthesis, 63-64
Mosaic genes
origins, 139
role in drug resistance, 139-140
MrsaY, 24
MRSA
methicillin-resistant Siaphylococcus au-reus, 135, 162
progressive increase in incidence,
135-136
Mucopeptide see Peptidoglycan
Multidrug efflux systems, 126-129
Mupirocin
antibacterial spectrum, 91
inhibition of bacterial isoleucyl fRNA
synthestase, 91 92
structure, 91
Mur A, 22
Mur B, 22
Mur C, 23
Mux D. 23
Mur E, 23
Mur F, 23
Mux G. 24
Murein sec Peptidoglycan
Mutations
drug-resistance and, 136-140
types of, 136
Mutator phenotype, role in bacterial drug
resistance, 137
Mycobacteria
low permeability of cell wall, 39,
123-124
treatment of infections, 39-41
Mycolic acid linkage to peptidoglycan in
mycobacteria, 39
Mycoplasmas
resistance to antibiotics affecting pepti-doglycan synthesis, 30
variations in sensifivity to polyene an-tibiotics, 60
Naftifine
antifungal topical use, 63
inhibition of squalene epoxidase, 63
selectivity Ibr the fungal enzyme, 63
structure, 64
Nalidixic acid
clinical use, 76
inhibitor of DNA gyrase, 76-78
resistance, 77, 163, 169
structure, 76
Neomycin
biochemical action, 94
structure, 95
.Neuraminidase
inhibitors, 111-112
of influenza virus, 111 112
.Nevirapine
inhibition of reverse transcriptase, 73, 75
resistance, 166
structure, 74
Nikkomycin Z
inhibition of fungal chitin biosynthesis. 42
structure, 42
Nitrofurantoin
clinical uses, 107
structure, 108
Nitrofurazone, 108
Nitroheterocyclic drugs
antimicrobial action, 108
mutagenic action, 108
Nonactin, 55
Non-nucleoside inhibitors of reverse ti’an-scriptase. 75
Novobiocin
disappointing clinical history, 79
inhibition of DNA gyrase, 78-79
structure, 78
Nucleic acid polymerases as targets for
drug action, 65
Nucleic acid
disturbance of template function of.
80-83
inhibitors of biosynthesis of, 65-83
Nucleotides, inhibition of biosynthesis,
66-70
.Nystatin
clinical use, 59
disruption of membrane integrity, 60
interaction with sterols, 60-61
structure, 60
Oritavancin, 32, 33
Oseltamavir
action against influenza. 11 I -112
inhibitor of viral neuraminidase, 112
structure, 112
Outer membrane of Gram-negative bacteria
asymmetry, 21
phospholipid composition, 20-22
porins, 22, 125-126
selective permeability, 22, 125 126
Oxazolidinones
inhibitors of protein biosynthesis, 102-103
179
Index
novel antibiotics, 102
Oxytetracycline, 97
Ozone as sterilant, 48
Pasteur, Louis, 3
Paromomycin
interaction with ribosonie, 95
structure, 95
Penicillin binding proteins
differing functions, 27
interaction with P~lactams, 26-27
mosaic genes for, 139
Penicillin G inactivation by acid. 33
Penicillin V stability to acid, 33
Penicillin
accumulation of nucleotides in Staphy-lococcus aureus, 36
antibacterial action, 33-35
derivatives stable to penicillinases, 35
discovery, 7-8
inhibition of peptidoglycan cross-linking,
37-39
isolation and purification, 8
precursors in fennentation medium, 33
resistance, 149-155, 162
rigid structure, 38
structural resemblance to D-alanyl-D~
alanine end group, 38
Penicillinase see P-lactamases
PenicUlium notatum, source of penicillin,
7-8
Pentaglycine group in peptidoglycan
biosynthesis, 24
Pentamidine
anti-trypanosomal action, 118
structure, 118
Peptide chain tennination and release, 90
Peptidoglycan biosynthesis
amidation of carboxyl group of D-glutamic acid, 23
cross-linking, 24-27
inhibition by antibiotics, 29-39
Peptidoglycan
differences among bacterial strains,
27-28
flexibility, 18
function, 17,18
fragment in induction of Gram-negative
p-lactamase, 152
structure and biosynthesis, 22-27
turnover during cell growth, 18
Peptidyl transferase
inhibition by antibiotics, 98-103
in peptide bond formation, 89
role of 23S rRNA, 89
Periplasm, 19
Permeability of cytoplasmic membrane
caused by antiseptics, 48-52
Permeability of microbial cells to drugs,
121-134
Phage ti’ansduction of drug resistance, 146
Pharmacological biochemistry of antimi-crobial agents, 13
Phenols as antiseptics, 48^ 9
Phenoxymethylpenicillin (penicillin V), 33,
34
Phosphatidyiefhanolamine in Gram-nega-tive outer membrane, 20
Phosphatidylglycerol in Gram-negative
outer membrane, 20
Phospholipids in Gram-negative walls,
20-21
Phosphonomycin see Fosfomycin
Phosphorylation of aminoglycosides,
1.59-160
Pimaricin
polyene food preservative, 59
structure, 60
Plasmids in drug resistance, 142-145
Polyene antibiotics
action on fungal infections, 59
basis of selectivity, 60
effects on permeability of fungal mem-branes, 60 61
sti’uctures, 60
toxicity, 59, 60
Polymyxin
binding to bacteria, 52
clinical application, 52
effects on permeability of cytoplasmic
membrane, 52-53
nature of binding to cytoplasmic mem-brane, 53
structure, 52
Polyoxins
competition with UDP-iV-acetylglu-cosamine, 42
entry into fungiil cells, 42
inhibition of chitin synthesis, 42^ 3
structure, 42
Polypeptide iintibiotics, 52-56
Porins
permeability characteristics, 21.125
role in drug penetration, 125-126
structure, 125, 126
Pradimicin A
antifungal spectrum, 44
complex with mannan, 44
structure, 44
Prions, 2
Proflavine
disinfectant action, 81
intercalation with DNA, 81-83
sti’ucture. 81
Proguanil
activation by host metabolism, 68
resistance, 165
structure, 68
Prontosil rubram
activation by host metabolism, 66
discovery, 6
structure, 66
Protein binding
effects on activity of antimicrobial
agents, 13
promotion of surimiin uptake by, 117
Pi’otein biosynthesis
inhibition by drugs, 8.5-105
stages in, 87-90
Pi’oteomic analysis, in studies of drug ac-tion, 12-13
Proton motive force, energy source for
drug transport across membranes.
123″, 127
Protozoal diseases, chemotherapy,
9,107,113-120
Pseudomonic acid see mupirocin
Furomycin
effects on protein biosynthesis, 90
fragment reaction, 91
structural analogues, 91
structural resemblance to aminoacyl
IRNA, 90
Pyrazinamide
inhibitor of fatty acid synthase in my-cobacteria, 41
structure, 41
Pyrazinoic acid, metabolite of pyrazi-namide, 41
Pyrmiethamine
atfinity for dihydrofolate reductase from
malarial parasite, 69
structure, 68
Pyrophosphatase inhibition by bacitracin,
30
Qing hao su see artemisinin
Quinacrine see mepacrine
Quinine
discovery, 2
toxicity to malarial parasite, 115
Quinolone antibacterials
inhibition of topoiosmerase 11 (DNA gy-rase), 76-77
inhibition of topoisomerase IV, 78
resistance, 77, 163, 169
uptiike by bacteria, 132
Quinuprisfin
inhibitor of protein biosynthesis, 102
structure, 102
synergism with dalfopristin, 102
Receptors for drugs, Ehrlich’s theory, 5
Recombination in
integrons. 140-141
mosaic genes, 139-140
transposons, 140-141
Release factors, role in peptide chain termi-nation, 90
Replica plating technique, 137, 138
Resistance genes, origins of, 137-139
Resistance
approaches to the conti’ol of, 172-173
180
I liClU A
biochemical mechanisms, 135-174
genetic basis, 121–134
Reverse transcriptase
inliibihon by nucleoside analogs, 13-15
inhibition by non-nucleosides, 75
mutations in, 166
Ribavirin
antivual effects, 70-71
enhancement of host immunity by,
71 72
inhibition of IMP dehydrogenase, 71
inhibition of viral capping, 71
structure, 71
Ribosome
cycle, 88
protection and tetracycline resistance,
170-171
proteins, 85
RNA, 85
role in peptide bond synthesis, 86
target for for antibiotics, 85
types, 85 86
structure, 85-87
subunils, 85-86
X~ray analysis of stincture, 86-87
Rifampicin
clinical use, 79
inhibition of bacterial RNA polymerase,
79 80
resistance to, 164
structure, 110
Riftimpin see rifampicin
liifamyciiis see rifampicin
Ritonavir
induction of insulin resistance in pa-tients, 110
inhibition of HIV protease, 109-110
in combination therapy for AID,S, 109
resistance to, 166 167
structure, 110
RNA biosynthesis
effects of inhibition on protein biosyn-thesis, 65
inhibition by rifaraycins, 79-80
inhibition by streptolydigin, 80
inhibition by streptovaricins, 80
RNA polymerase
inhibition by antibiotics, 79 80
subunil structure, 80
R-plasmids
classification. 145
mechanism of transfer, 144-145
relationship to F-factors, 145
role in spread of drug-resistance, 145
structure, 142-143
Salvarsan
efficacy against ssiphilis, 4, 5
metabolism to niapharsen, 6
structure, 4
Saquinavir, 110
SARS, see ,Severe Acute Respiratory Syn-drome
,Severe Acute Respiratory Syndrome, 2
Screening
in drug discovery, 5, 114
high-throughput, 14
Paul Ehrlich, 5
targeted, 14-15
.Sedimentation coefficient of DNA, effect
of intercalating dyes, 81
Selectivity in antimicrobial action, 13
.Semmelweiss, Ignaz, 3
Septum formation, effect of P-laction an-tibiotics, 37
,Sex pili role in bacterial conjugation, 144
Sleeping sickness
aloxyl lieatment, 4
eflornithine treatment, 119
melarsoprol treatment, 118
see also Trypanosomiasis
Spectinornycin
biochemical action, 96
structure, 95
Sterilants. 47
Sterol biosynthesis inhibition
by ailylamines, 63
by azoles, 61-63
by morpholines, 63-64
Sterols in fungal membrane, binding of
polyene antibiotics, 60-61
Streptograrnins
inhibition of protein biosynthesis, 102
resistance to, 160-161
synergism between Types A and B,
“io 1-102
Streptolydigin, inhibitor of bacterial RNA
polymerase, 80
Streptomycin
adenylylation of, 159 160
antibacterial action, 92-94
binding to ribosomes, 93-94
clinical use, 92
discovery, 8
effects on protein biosynthesis, 92-93
induction of codon misreading, 93-94
mode of entry into bacterial cells.
132 133
phosphorylation, 159 160
possible inhibition of (‘rfvn.i’-lranslation
of messenger RNA, 96
resistance, 159-160
structure, 92
to.iiicity, 92
Streptovaricins
inhibitors of bacterial RNA polymerase,
80
structure, 79
Sulfazecin, 35, 36
Sulfamethoxazole, combination with
trimethoprim, 69
Sulfanikonide
biochemical action, 67
metabolic product from Prontosil
rubrum, 66
structure, 66
Sulfonamides
antibacterial activity, 66
competition with p-aminobenzoate, 67
inhibition of synthesis of dihydropteroic
acid, 67
resistance to, 165
Suramin
inhibition of trypanosomal enzymes, 117
structure. 7
uptake into Irypanosomes. 117
Syphilis treatment by salvarsan, 5
Teiclioic acid
labile linkage to peptidoglycan, 18,
28-29
possible function, 18
possible influence on drug penetration.
18
structure, 18, 28
Teichoplanin
inhibitor of bacterial cell wall biosynthe-sis, 32-33
structure, 32
Teichuronic acid, 18
Telithromycin
inhibitor of protein biosynthesis. 100-101
structure, 101
Tenofovir
antiviral drug, 73-74
inhibitor of reverse transcriptase, 74-75
structure, 73
Terbinafiiie
antifungal use, 63
inhibition of squalene epoxidation in
fungi, 63
structure, 64
Termination in protein biosynthesis, 90
Terminator codons, 90
Tetracyclines
active uptake by bacterial cells, 131
antimicrobial actions, 96
binding to ribosomes, 97
chelating activity, 97
efflux of, 131
inhibition of aminoacyl tRNA-ribosome
interaction, 96-97
mechanisms of resistance to, 167-169.
171-171
ribosormd site of action, 97
selectivity of antibacterial action, 96
structure-activity relationships, 96-98
structures, 97, 99
Tetra hydrofolate
biosynthesis, 67
double blockage of synthesis, 69
importance in nucleotide synthesis,
69-70
181
Index
Thienamycin
antibacterial action, 36
resistance to P-lactamase, 36
structure, 35
Thiol groups possible involvement in an-timicrobial action of arsenicals, 118
Thymidine kinase
activation of acyclovir, 72
activation of azidothymidine, 73–74
Thymidylate synthase, inhibition by 5-fluo-rodeoxyuridine, 70
TicarciUin, 34, 35
Tigicycline
action against tetracycline-resistant bac-teria, 168
structure, 99
Tolnaftate
clinical use, 63
inhibition of squalene epoxidation, 63
structure, 64
Topoisomerases
functions of, 7.5-76
DNA gyrase, 76
inhibitors, 76-79
supercoiling of DNA, 75-76
types of, 76
Toxophore, 5
Transduction
generalized, 146
possible contribution to spread of drug-resistance, 146
specialized, 146
Transfer RN.4
binding to ribosomes, 89
conversion to aminoacyl transfer (t)
RNA, 87
translocation in protein biosynthesis, 89
Transformation
contribution to the spread of drug-resist-ance, 147
mechanism of, 146-147
Transglycolase
component of penicillin-binding pro-teins, 27
inhibition by vancomycin, 33
role in peptidoglycan biosynthesis, 24
Translational domain on ribosomes, 86
Translocation
inliibition by cycloheximide, 104
inhibition by fusidic acid, 103
in protein biosynthesis, 89
Transpeptidase
inhibition by P-lactams, 37-39
in peptidoglycan cross-linking, 27
Transposons
in drug-resistance, 140-142
insertion mechanism, 140, 141, 142
structure, 141
see also Conjugative transposons
Triclosan
iintiseptic, 50
inliibitor of fatty acid biosynthesis,
51-52
structure, 49
Tiidemorph, 64
Trimethoprim
resistance, 164-165
selective inhibition of bacterial dihydro-folate reductase, 69
structure, 68
Trypan red, antitrypanosomal action 4
Trypanosomes
kinetoplast DNA and intercalating mole-cules, 83
glycolysis in, 117, 118
polyamines synthesis in, 119
redox balance in, 118
Trypanosomiasis
current treatments, 117-119
forms of, 117
Trypanothione
role in redox control in trypanosomes,
118
interaction with melarsoprol, 117
Tuberculosis
resurgence of, 1
treatment with synthetic compounds,
39-41
treatment with antibiotics, 79, 92
Tyrocidin A
action on membranes, 52, 53
structure, 52
UDP-A’-acetylmuramic acid biosynthesis,
22
Undecaprenyl phosphate in peptidoglycan
biosynthesis, 24
Undecaprenyl pyrophosphatase, 24
Uptake of antimicrobial drugs, 129-133
Uridine nucleotides of A’-acetyl muramic
acid
accumulation caused by inhibitors of
peptidoglycan biosynthesis, 30
biosynthesis, 22
Valinomycin
mechanism of potassium complex for-mation, 54-56
mobile carrier of ions, 54-56
structure, 55
Vancomycin
antibacterial activity, 32
binding to D-alanyl-D-alanine group,
32-33
dimerization, 33
inhibition of transglycolase, 33
resistance, 71-72
stracture, 32
Variable surface glycoprotein in try-panosomes, 119
Vidarabine see Arabinosyl adenine
Virus infections, chemotherapy of, 70-75,
109-113
Voriconazole
antifungal spectrum, 62
structure, 63
Waksman, Selman, 8
West Nile fever, 2
Zanaraavir
action against influenza, 111-112
inhibitor of viral neuraminidsase, 112
structure, 112
182

One comment on “1 THE DEVELOPMENT OF ANTIMICROBIAL AGENTS, PAST, PRESENT AND FUTURE 1

  1. Apple now has Rhapsody as an app, which is a great start, but it is currently hampered by the inability to store locally on your iPod, and has a dismal 64kbps bit rate. If this changes, then it will somewhat negate this advantage for the Zune, but the 10 songs per month will still be a big plus in Zune Pass’ favor.

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