Behind Enemy Lines
A close look at the inner workings of microbes in this era of escalating antibiotic
resistance is offering new strategies for designing drugs
BY K. C. NICOLAOU and CHRISTOPHER N. C. BODDY [Sci Amer]
In the celebrated movie Crouching Tiger, Hidden Dragon, two warriors face
each other in a closed courtyard whose walls are lined with a fantastic array
of martial-arts weaponry, including iron rods, knives, spears and swords.
The older, more experienced warrior grabs one instrument after another from the
arsenal and battles energetically and fluidly with them. But one after another, the
weapons prove useless. Each, in turn, is broken or thrown aside, the shards of an era
that can hold little contest against a young, triumphant, upstart warrior who has
learned not only the old ways but some that are new.
One of the foundations of the modern medical system is being similarly overcome.
Health care workers are increasingly finding that nearly every weapon in their
arsenal of more than 150 antibiotics is becoming useless. Bacteria that have
survived attack by antibiotics have learned from the enemy and have grown
stronger; some that have not had skirmishes themselves have learned from
others that have. The result is a rising number of antibiotic-resistant strains.
Infections - including tuberculosis, meningitis and pneumonia - that would once
have been easily treated with an antibiotic are no longer so readily thwarted.
More and more bacterial infections are proving deadly.
Bacteria are wily warriors, but even so, we have given them - and continue to give
them - exactly what they need for their stunning success. By misusing and overusing
antibiotics, we have encouraged super-races of bacteria to evolve. We don't finish a
course of antibiotics. Or we use them for viral and other inappropriate infections - in
fact, researchers estimate that one third to one half of all antibiotic prescriptions are
unnecessary. We put 70 percent of the antibiotics we produce in the U.S. each year
into our livestock. We add antibiotics to our dishwashing liquid and our hand soap.
In all these ways, we encourage the weak to die and the strong to become stronger
[see "The Challenge of Antibiotic Resistance," by Stuart B. Levy; Scientific
American, March 1998].
Yet even absent the massive societal and medical misuse of these medications, the
unavoidable destiny of any antibiotic is obsolescence. Bacteria - which grow quickly
through many cell divisions a day - will always learn something new; some of the
strongest will always survive and thrive. So we have had to become ever more wily
ourselves.
In the past 10 years, long-standing complacency about vanquishing infection has
been replaced by a dramatic increase in antibacterial research in academic,
government and industrial laboratories. Scientists the world over are finding
imaginative strategies to attack bacteria. Although they will have a limited life span,
new antibiotics are being developed using information gleaned from genome and
protein studies. This exciting research and drug development is no panacea, but if
combined with the responsible use of antibiotics, it can offer some hope. Indeed, in
April 2000 the Food and Drug Administration approved the first new kind of clinical
antibiotic in 35 years - linezolid - and several agents are already in the
pharmaceutical pipeline.
Dismantling the Wall
Almost all the antibiotics that have been developed so far have come from nature.
Scientists have identified them and improved on them, but they certainly did not
invent them. Since the beginning of life on this planet, organisms have fought over
limited resources. These battles resulted in the evolution of antibiotics. The ability to
produce such powerful compounds gives an organism - a fungus or plant or even
another species of bacteria - an advantage over bacteria susceptible to the antibiotic.
This selective pressure is the force driving the development of antibiotics in nature.
Our window onto this biological arms race first opened with the discovery of
penicillin in 1928. Alexander Fleming of St. Mary's Hospital Medical School at
London University noticed that the mold Penicillium notatum was able to kill
nearby Staphylococcus bacteria growing in agar in a petri dish. Thus was the field of
antibiotics born. By randomly testing compounds, such as other molds, to see if they
could kill bacteria or retard their growth, later researchers were able to identify a
whole suite of antibiotics.
One of the most successful of these has been vancomycin, first identified by Eli Lilly
and Company in 1956. Understanding how it works - a feat that has taken three
decades to accomplish - has allowed us insight into the mechanism behind a class of
antibiotics called the glycopeptides, one of the seven or so major kinds of antibiotics.
This insight is proving important because vancomycin has become the antibiotic of
last resort, the only remaining drug effective against the most deadly of all
hospital-acquired infections: methicillin-resistant Staphylococcus aureus. And yet
vancomycin's power - like that of the great, experienced warrior - is itself in
jeopardy.
Vancomycin works by targeting the bacterial cell wall, which surrounds the cell and its
membrane, imparting structure and support. Because human and other mammalian cells
lack such a wall (instead their cells are held up by an internal structure called a
cytoskeleton), vancomycin and related drugs are not dangerous to them. This bacterial
wall is composed mostly of peptidoglycan, a material that contains both peptides and
sugars (hence its name). As the cell assembles this material - a constant process,
because old peptidoglycan needs to be replaced as it breaks down -
sugar units are linked together by an enzyme called transglycosidase
to form a structural core. Every other sugar unit along this core has a short peptide
chain attached to it. Each peptide chain is composed of five amino acids, the last
three being an L-lysine and two D-alanines. An enzyme called transpeptidase then
hooks these peptide chains together, removing the final D-alanine and attaching the
penultimate D-alanine to an L-lysine from a different sugar chain. As a result, the
sugar chains are crocheted together through their peptide chains. All this linking and
cross-linking creates a thickly woven material essential for the cell's survival: without
it, the cell would burst from its own internal pressure.
Vancomycin meddles in the formation of this essential material. The antibiotic is
perfectly suited to bind to the peptide chains before they are linked to one another by
transpeptidase. The drug fastens onto the terminal D-alanines, preventing the enzyme
from doing its work. Without the thicket of cross-linking connections, peptidoglycan
becomes weak, like an ill-woven fabric. The cell wall rends, and cell death rapidly
occurs.
Resisting Resistance
Vancomycin's lovely fit at the end of the peptide chain is the key to its effectiveness
as an antibiotic. Unfortunately, its peptide connection is also the key to resistance on
the part of bacteria. In 1998 vancomycin-resistant S. aureus emerged in three
geographic locations. Physicians and hospital workers are increasingly worried that
these strains will become widespread, leaving them with no treatment for lethal staph
infections.
Understanding resistance offers the possibility of overcoming it, and so scientists
have focused on another bacterium that has been known to be resistant to the
powerful drug since the late 1980s: vancomycin-resistant enterococci (VRE).
In most enterococci bacteria, vancomycin does what it does best: it binds to the
terminal two D-alanines. At a molecular level, this binding entails five hydrogen bonds -
think of them as five fingers clasping a ball. But in VRE, the peptide chain is slightly
different. Its final D-alanine is altered by a simple substitution: an oxygen replaces a
pair of atoms consisting of a nitrogen bonded to a hydrogen. In molecular terms, this
one substitution means that vancomycin can bind to the peptide chain with only four
hydrogen bonds. The loss of that one bond makes all the difference. With only four
fingers grasping the ball, the drug cannot hold on as well, and enzymes pry it off,
allowing the peptide chains to link up and the peptidoglycan to become tightly woven
once again. One atomic substitution reduces the drug's activity by a factor of 1,000.
Researchers have turned to other members of the glycopeptide class of antibiotics to
see if some have a strategy that vancomycin could adopt against VRE. It turns out
that some members of the group have long, hydrophobic - that is, oily - chains
attached to them that have proved useful. These chains prefer to be surrounded by
other hydrophobic molecules, such as those that make up the cell membrane, which
is hidden behind the protective peptidoglycan shield. Researchers at Eli Lilly have
borrowed this idea and attached hydrophobic chains to vancomycin, creating an
analogue called LY333328. The drug connects to the cell membrane in high
concentrations, allowing it more purchase and, as a consequence, more power
against peptidoglycan. This analogue is effective against VRE and is now in clinical
trials.
Other glycopeptide antibiotics use a different strategy: dimerization. This process
occurs when two molecules bind to each other to form a single complex. By creating
couples, or dimers, of vancomycin, researchers can enhance the drug's strength. One
vancomycin binds to peptidoglycan, bringing the other half of the pair - the other
molecule of vancomycin - into proximity as well. The drug is more effective because
more of it is present. One of the aims of our laboratory is to alter vancomycin so it
pairs up more readily, and we have recently developed a number of dimeric
vancomycin molecules with exceptional activity against VRE.
Even so, the good news may be short-lived. A second mechanism by which VRE
foils vancomycin has recently been discovered. Rather than substituting an atom in
the final D-alanine, the bacterium adds an amino acid that is much larger than
D-alanine to the very end of the peptide chain. Like a muscular bouncer blocking a
doorway, the amino acid prevents vancomycin from reaching its destination.
One method by which the deadly S. aureus gains resistance is becoming clear as
well. The bacterium thickens the peptidoglycan layer but simultaneously reduces the
linking between the peptide fragments. So it makes no difference if vancomycin binds
to D-alanine: thickness has replaced interweaving as the source of the
peptidoglycan's strength. Vancomycin's meddling has no effect.
The Cutting Edge
As the story of vancomycin shows, tiny molecular alterations can make all the difference,
and bacteria find myriad strategies to outwit drugs. Obviously, the need for new, improved
or even revived antibiotics is enormous. Historically, the drug discovery process identified
candidates using whole-cell screening, in which molecules of interest were applied to living
bacterial cells. This approach has been very successful and underlies the discovery of many
drugs, including vancomycin. Its advantage lies in its simplicity and in the fact that every
possible drug target in the cell is screened. But screening such a large number of targets
also has a drawback. Various targets are shared by both bacteria and humans; compounds
that act against those are toxic to people. Furthermore, researchers gain no information
about the mechanism of action: chemists know that an agent worked, but they have no
information about how. Without this critical information it is virtually impossible to bring
a new drug all the way to the clinic.
Molecular-level assays provide a powerful alternative. This form of screen identifies
only those compounds that have a specified mechanism of action. For instance, one
such screen would look specifically for inhibitors of the transpeptidase enzyme.
Although these assays are difficult to design, they yield potential drugs with known
modes of action. The trouble is that only one enzyme is usually investigated at a time.
It would be a vast improvement in the drug discovery process if researchers could
review more than one target simultaneously, as they do in the whole-cell process, but
also retain the implicit knowledge of the way the drug works. Scientists have
accomplished this feat by figuring out how to assemble the many-enzyme pathway of
a certain bacterium in a test tube. Using this system, they can identify molecules that
either strongly disrupt one of the enzymes or subtly disrupt many of them.
Automation and miniaturization have also significantly improved the rate at which
compounds can be screened. Robotics in so-called high-throughput machines allow
scientists to review thousands of compounds per week. At the same time,
miniaturization has cut the cost of the process by using ever smaller amounts of
reagents. In the new ultrahigh-throughput screening systems, hundreds of thousands
of compounds can be looked at cost-effectively in a single day. Accordingly,
chemists have to work hard to keep up with the demand for molecules. Their work is
made possible by new methods in combinatorial chemistry, which allows them to
design huge libraries of compounds quickly [see "Combinatorial Chemistry and New
Drugs," by Matthew J. Plunkett and Jonathan A. Ellman; Scientific American, April
1997]. In the future, some of these new molecules will most likely come from
bacteria themselves. By understanding the way these organisms produce antibiotics,
scientists can genetically engineer them to produce new related molecules.
The Genomic Advantage
The methodology of drug design and screening has benefited tremendously from
recent developments in genomics. Information about genes and the synthesis of their
proteins has allowed geneticists and chemists to go behind enemy lines and use inside
information against the organism itself. This microbial counterintelligence is taking
place on several fronts, from sabotaging centrally important genes to putting a
wrench in the production of a single protein and disrupting a bacterium's ability to
infect an organism or to develop resistance.
Studies have revealed that many of the known targets of antibiotics are essential
genes, genes that cause cell death if they are not functioning smoothly. New
genetic techniques are making the identification of these essential genes much faster.
For instance, researchers are systematically analyzing all 6,000 or so genes of the
yeast Saccharomyces cerevisiae for essential genes. Every gene can be experimentally
disrupted and its effect on yeast determined. This effort will ultimately catalogue all the
essential genes and will also provide insight into the action of other genes that could serve
as targets for new antibiotics.
The proteins encoded by essential genes are not the only molecular-level targets that
can lead to antibiotics. Genes that encode for virulence factors are also important.
Virulence factors circumvent the host's immune response, allowing bacteria to
colonize. In the past, it has been quite hard to identify these genes because they are
"turned on," or transcribed, by events in the host's tissue that are very difficult to
reproduce in the laboratory. Now a technique called in vivo expression technology
(IVET) can insert a unique sequence of DNA, a form of tag that deactivates a gene,
into each bacterial gene. Tagged bacteria are then used to infect an organism. The
bacteria are later recovered and the tags identified. The disappearance of any tags
means that the genes they were attached to were essential for the bacteria's survival -
so essential that the bacteria could not survive in the host without the use of those
genes.
Investigators have long hoped that by identifying and inhibiting these virulence
factors, they can allow the body's immune system to combat pathogenic bacteria
before they gain a foothold. And it seems that the hypothesis is bearing fruit. In a
recent study, an experimental molecule that inhibited a virulence factor of the
dangerous S. aureus permitted infected mice to resist and overcome infection.
In addition to identifying essential genes and virulence factors, researchers are
discovering which genes confer antibiotic resistance. Targeting them provides a
method to rejuvenate previously ineffective antibiotics. This is an approach used with
ß-lactam antibiotics such as penicillin. The most common mechanism of resistance to
ß-lactam antibiotics is the bacterial production of an enzyme called ß-lactamase,
which breaks one of the antibiotic's chemical bonds, changing its structure and
preventing it from inhibiting the enzyme transpeptidase. If ß-lactamase is silenced, the
antibiotics remain useful. A ß-lactamase inhibitor called clavulanic acid does just that
and is mixed with amoxicillin to create an antibiotic marketed as Augmentin.
In the near future, with improvements in the field of DNA transcriptional profiling, it
will become routine to identify resistance determinants, such as ß-lactamase, and
virulence factors. Such profiling allows scientists to identify all the genes that are in
use under different growth conditions in the cell. Virulence genes can be determined
by identifying bacterial genes whose expression increases on infecting a host. Genes
that code for antibiotic resistance can be determined by comparing expression levels
in bacteria treated with the antibiotic and those not treated. Though still in its infancy,
this technique monitored tiny changes in the number of transcription events. With
DNA transcriptional profiling, researchers should also be able to determine whether
certain drugs have entirely new mechanisms of action or cellular targets that could
open up new fields of antibiotic research.
Killing the Messenger
Another interesting line of genomic research entails interfering with bacterial RNA.
Most RNA is ribosomal RNA (rRNA), which forms a major structural component
of ribosomes, the cellular factories where proteins are assembled. Ribosomal RNA
is vulnerable because it has various places where drugs can attach and because it
lacks the ability to repair itself. In 1987 scientists determined that antibiotics in the
aminoglycoside group - which includes streptomycin - bind to rRNA, causing the
ribosome to misread the genetic code for protein assembly. Many of these antibiotics,
however, are toxic and have only limited usefulness. Recently scientists at the Scripps
Research Institute in La Jolla, Calif., have reported a new synthetic aminoglycoside
dimer that may have less toxicity.
Investigators can also interfere with messenger RNA (mRNA), which directs the
assembly of proteins and travels between the genetic code and the ribosome.
Messenger RNA is created by reading one strand of the DNA, using the same
nucleic acid, or base pair, interactions that hold the double helix together. The
mRNA molecule then carries its message to the ribosome, where a protein is
assembled through the process of translation. Because each mRNA codes for a
specific protein and is distinct from other mRNAs, researchers have the opportunity
to create interactions between small organic molecules - that is, not proteins - and
specific mRNAs. Parke-Davis chemists have been able to use such an approach to
combat HIV infection. They identified molecules that bind to a part of an mRNA
sequence and prevent it from interacting with a required protein activator, thus
inhibiting the replication of HIV. This proof-of-principle experiment should help pave
the way for further studies of mRNA as a drug development target.
Scientific interest has been intense in another approach, called antisense therapy. By
generating sequences of nucleotides that bind perfectly with a specific mRNA
sequence, investigators can essentially straitjacket the mRNA. It cannot free itself
from the drug, which either destroys it or inhibits it from acting. Although the FDA
has recently approved the first antisense drug to treat human cytomegalovirus
infections, antisense for bacterial infections has not succeeded yet for several
reasons, including toxicity and the challenge of getting enough of the drug to the right
spot. Nevertheless, the approach holds promise.
As is clear, all these genomic insights are making it possible to identify and evaluate a
range of new biological targets against which chemists can direct their small, bulletlike
molecules. A number of antibiotics developed in the past century cannot be used,
because they harm us. But by comparing a potential target's genetic sequence with
the genes found in humans, researchers can identify genes that are unique to bacteria
and can focus on those. Similarly, by comparing a target's genetic sequence to those
of other bacteria, they are able to evaluate the selectivity of a drug that would be
generated from it. A target sequence that appears in all bacteria would very likely
generate an antibiotic active against many different bacteria: a broad-spectrum
antibiotic. In contrast, a target sequence that appears in only a few bacterial genomes
would generate a narrow-spectrum antibiotic.
If physicians can identify early on which strain is causing an infection, they can hone
their prescription to a narrow-spectrum antibiotic. Because this drug would affect
only a subset of the bacterial population, selective pressure for the development of
resistance would be reduced. Advances in the high-speed replication of DNA and
transcriptional profiling may soon make identification of bacterial strains a routine
medical procedure.
Although the picture looks brighter than it has for several decades, it is crucial that
we recognize that the biological arms race is an ancient one. For every creative
counterattack we make, bacteria will respond in kind - changing perhaps one atom in
one amino acid. There will always be young warriors to challenge the old ones. The
hope is that we exercise restraint and that we use our ever expanding arsenal of
weapons responsibly, not relegating them so quickly to obsolescence.
Photographs by Eric O'Connell
Further Information:
The Coming Plague: Newly Emerging Diseases in a World out of Balance.
Laurie Garrett. Penguin USA, 1995.
The Chemistry, Biology, and Medicine of the Glycopeptide Antibiotics. K. C.
Nicolaou, Christopher N. C. Boddy, Stefan Bräse and Nicolas Winssinger in
Angewandte Chemie International Edition, Vol. 38, No. 15, pages 2096 – 2152;
August 2, 1999.
Genome Prospecting. Barbara R. Jasny and Pamela J. Hines in Science, Vol. 286,
pages 443 – 491; October 15, 1999.
The Authors
K. C. NICOLAOU and CHRISTOPHER N. C. BODDY have worked together at
the Scripps Research Institute in La Jolla, Calif., where Nicolaou is chairman of the
department of chemistry and Boddy recently received his Ph.D. Nicolaou holds the
Darlene Shiley Chair in Chemistry, the Aline W. and L. S. Skaggs Professorship in
Chemical Biology and a professorship at the University of California, San Diego. His
work in chemistry, biology and medicine has been described in more than 500
publications and 50 patents. Boddy’s research has focused on the synthesis of
vancomycin. He will soon be moving to Stanford University, where as a postdoctoral
fellow he will continue work on antibiotics and anticancer agents. The authors are
indebted to Nicolas Winssinger and Joshua Gruber for valuable discussions and
assistance in preparing this article.
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