REVIEW doi:10.

1038/nature17042

Antibacterial drug discovery

in the resistance era
Eric D. Brown1,2 & Gerard D. Wright1,2

The looming antibiotic-resistance crisis has penetrated the consciousness of clinicians, researchers, policymakers, poli-
ticians and the public at large. The evolution and widespread distribution of antibiotic-resistance elements in bacterial
pathogens has made diseases that were once easily treatable deadly again. Unfortunately, accompanying the rise in global
resistance is a failure in antibacterial drug discovery. Lessons from the history of antibiotic discovery and fresh under-
standing of antibiotic action and the cell biology of microorganisms have the potential to deliver twenty-first century
medicines that are able to control infection in the resistance era.

W
e are entering a period in infection control that has not been
experienced for more than three-quarters of a century. In
this era of ever-increasing resistance (Fig. 1), the manage-
ment of bacterial infection through the use of safe, cheap and plenti-
ful antibiotics can no longer be taken for granted. The serendipitous
discovery of penicillin in 1929 (ref. 1) enabled the effective control of
infections caused by Gram-positive pathogens such as Staphylococcus
and Streptococcus, and the isolation of streptomycin in 1943 (ref. 2)
facilitated the control of tuberculosis agent Mycobacterium tubercu-
losis — for the first time in history. These discoveries propelled the
golden era of antibiotics, in which natural scaffolds and alternative
versions of these pioneering drugs were uncovered by mining the
specialized metabolism of bacteria and fungi or by the chemical modi-
fication of existing scaffolds (Table 1). The results were revolutionary.
Released from the hegemony of infection, clinicians began to trans-
form medicine. Invasive surgeries became routine, immune-system-
shattering chemotherapy was introduced to fight the ‘war’ on cancer,
organ transplantation extended lives and the radical replacement of
deteriorating joints, diseased corneas and burnt skin improved quality
of life for millions of people. In the resistance era, these breakthroughs
— and the consequent improvements in our quality of life and life
expectancy — are at risk.
The microbial world has always had the molecular tools to drive
resistance. Surveys of microorganisms in the environment and in sam-
ples of ancient permafrost revealed that the antibiotic resistome —
the global collection of all microbial resistance genes — is genetically
diverse3,4, widespread across all environmental niches5–8 and pre-dates
the modern antibiotic era by millennia9. The use of large quantities of
antibiotics to control infection in human and animal diseases and in
agriculture has created unprecedented conditions for the mobiliza-
tion of resistance elements in bacterial populations and their capture
by previously antibiotic-sensitive pathogens. Over time, these condi-
tions have enabled the selection of evermore drug-resistant bacteria
through three main mechanisms: the sequential capture of a myriad
of resistance genes, mostly through mobilization and horizontal trans-
fer from environmental sources; the ‘freezing’ of polymorphisms in
antibiotic target genes through secondary mutations that neutralize
the effects on fitness that drug-resistance mutations often impart10;
and the upregulation of intrinsic mechanisms of resistance, such as
efflux11,12 or antibiotic-inactivating enzymes13. Multidrug resistance
1
Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario L8S 4L8, Canada. 2Department of Biochemistry and Biomedical Sciences, McMaster
University, Hamilton, Ontario L8S 4L8, Canada.
in pathogens is typical in the resistance era14 and is eroding our ability
to control infections with antibiotics. Pathogenic strains of bacteria
that are resistant to most, or all, available antibiotics are now isolated
routinely. We have entered the post-antibiotic age.
Mechanisms and origins of antibiotics
Antibiotics perturb important biochemical processes, which results in
the inhibition of cell growth and division and, in the case of bacteri-
cidal agents, cell death15. The first antimicrobial agents were synthetic
molecules that were discovered by screening libraries of chemicals, in
particular dyes. This screening was superseded by the realization that
bacteria and fungi in the environment produce metabolites that could
treat bacterial infections in humans with remarkable efficacy and mini-
mal toxic side effects. The strategy adopted by Selman Waksman, a co-
discoverer of streptomycin, involved screening soil-dwelling bacteria,
and in particular the spore-forming Actinomycetes, for the production
of metabolites that block the growth of pathogens — a process that was
later termed the Waksman platform16. The simplicity and effectiveness
of the platform ushered in the golden era of antibiotic discovery, the
period in which most of the microbial natural scaffolds that serve as our
antibiotic arsenal were discovered (Fig. 1). The platform also imposed
measures of success that subsequent drug-discovery efforts would need
to use, in particular use of the inhibition of cell growth in vitro, which
is assessed on rich media, as the main way to determine the minimal
inhibitory concentration (MIC) of a compound.
By the mid-1960s, new and effective antibiotic scaffolds were
becoming harder to identify using the Waksman platform. Because
these specialized metabolites are the product of microbial evolution
within a specific environment and were not designed as drugs, most
had considerable pharmacological or toxicological drawbacks. And
resistance to these early antibiotics, the result of the horizontal transfer
of resistance genes between bacteria or chromosomal mutation, was
also becoming a problem. These issues spawned the era of medici-
nal chemistry, the next period of innovation in antibiotic discovery
(Fig. 1). The development of antibiotics in this period was dominated
by cycles of innovation that focused largely on creating synthetic ver-
sions of the natural scaffolds of the golden era of discovery. These
derivatives led to outstanding improvements in the application of
antibiotics, which included lower doses, an expanded antimicrobial
spectrum against various pathogens, and the avoidance of resistance.

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These newer drugs substantially changed the practice of medicine
and ushered in the ‘miracles’ of modern medicine that we now take
for granted.
Most existing antibiotics are therefore derived from natural products
and tend to target the bacterial cell wall, DNA or ribosomes. With a few
exceptions, these compounds exert pleiotropic and complex effects on
the bacterial cell and often have more than one molecular target. The
β-lactam antibiotics, such as penicillin, covalently modify a number of
target enzymes known as penicillin-binding proteins (PBPs). Collec-
tively, these enzymes are responsible for the synthesis and remodelling
of the bacterial cell wall for growth and division. Antibiotics that inhibit
protein synthesis target the ribosome, which is encoded by multiple
copies of ribosomal DNA, and those that block DNA synthesis act on
several topoisomerase enzymes. Importantly, this effect on multiple
cellular targets limits the frequency of spontaneous resistance that can
arise from mutation in the target gene. Inhibition of the molecular
targets of antibiotics commonly results in complex downstream effects
that exceed those of simple enzyme inhibition. Mounting evidence sug-
gests that β-lactam antibiotics disorganize the activity of the bacterial
cell-wall synthesis machinery in a manner far more complicated than
simple inhibition17. The ribosome is a macromolecular complex with
many enzymatic functions, regulatory sites and components. When it
is targeted by aminoglycosides, for example, the resulting synthesis of
aberrant proteins leads to pleiotropic and toxic effects on the bacterial
cell18,19. Systems-biology approaches suggest that reactive oxygen spe-
cies have been overlooked as contributors to cell death20. Although this
hypothesis remains controversial, there is an increasing appreciation
that bacterial cell death is complex and probably requires the involve-
ment of several cellular pathways. Many natural-product antibiotics
are the product of selection for these complex traits over millions of
years of evolution. Therefore, it is perhaps unsurprising that modern
methods of drug discovery have yet to deliver compounds with efficacy
comparable to that of the first generation of natural antibiotics and
their semisynthetic derivatives.
Modern antibacterial drug discovery
Successes in the golden era were followed by a considerable hiatus
in the identification of new scaffolds that began in the 1960s and
lasted until the early 1990s, when a renaissance in discovery efforts
was precipitated by a fresh wave of resistance. This coincided with
the emergence of innovative drug-discovery approaches in all thera-
peutic areas. These new methods were underpinned by remarkable
advances in technology, such as breakthroughs in the manipulation
of recombinant DNA to produce desired proteins at high yields and in
high-throughput synthesis to create large chemical libraries. Improve-
ments in facile protein-structure determination enabled rational
drug design, and robotic liquid handling facilitated high-throughput
screens of biochemical assays. Furthermore, a computing revolution
made it possible to handle much larger data sets. When coupled with
emergent genomic technologies, these advances enabled a ‘genes-to-
drugs’ model that has been applied with success to many therapeutic
areas21 and become the convention. Yet more than two decades into
the practice of modern antibacterial drug discovery, no new medicines
have been discovered through this approach.
Antibacterial drug discovery has been especially influenced by
genomic data, technology and innovation. The first free-living organ-
ism to be sequenced was the pathogen Haemophilus influenzae22 and,
since then, thousands of bacterial genomes have been sequenced.
High-throughput techniques that can create precise deletions at the
genome scale have been used to explore methodically the dispen-
sability of each gene in both the model Gram-positive bacterium
Bacillus subtilis23 and in the model Gram-negative bacterium Escheri-
chia coli24. Pathogens such as Staphylococcus aureus and Pseudomonas
aeruginosa have also been the subject of systematic approaches to
mutagenesis25,26, as is the fastidious pathogen M. tuberculosis27. The
goal of these large-scale surveys has been to identify new targets for a
© 2016 Macmillan Publishers Limited. All rights reserved
REVIEW INSIGHT
1925
Golden era
• Natural products
• Whole-cell screens
1950
• High success
Medicinal chemistry era
• Synthetic tweaking
1975 • Whole-cell screens
• Broad spectrum
• High success
Resistance era
• Modern discovery
2000
• Target-based
• Broad spectrum
• Low success
Narrow-spectrum era
2025 • Unconventional discovery
• In vivo essential targets
• Combinatorial approaches
• Diagnostic development
• Predicted success
Figure 1 | Models of antibiotic drug discovery and development.  Organized
with respect to the prevalent models of each era, this timeline highlights the
history and future of antibiotic drug discovery. The golden era was initiated by
the discovery of sulfonamide and penicillin and is typified by the methods of
Selman Waksman and his contemporaries, who used whole-cell screening of
natural product extracts to find new antibiotic scaffolds. In subsequent years,
termed the medicinal-chemistry era, these scaffolds were modified chemically.
Emphasis in the resistance era has fallen on target-based drug discovery to find
broad-spectrum agents — a model that has failed to provide new antibiotics. In
the future, a focus on innovative methods and unconventional targets should
help to create narrow-spectrum agents and associated diagnostics.
generation of antibiotics that are insusceptible to existing mechanisms
of resistance28.
In the face of increasing resistance, the failure to yield new anti-
biotics has been the cause of much concern. Unsuccessful antibac-
terial programmes have been documented by industry experts29–31
and common themes are evident. Large-scale gene dispensability
studies generated numerous targets — 160 in one account29 — and
many were tested in in vitro screening campaigns. However, these
high-throughput biochemical screens of large collections of synthetic
chemicals often were unable to find promising compounds with the
necessary physical and chemical properties. When encouraging lead
compounds were found, these failed, largely because of difficulties in
developing antibacterial drugs with a suitable product profile — for
example, efficacy against a broad spectrum of pathogens or against
difficult-to-treat Gram-negative organisms. Gram-negative bacteria
have proved especially daunting owing to a poor understanding of
drug penetration and efflux systems that are pervasive in this group
of pathogens. There were also concerns about the rapid evolution of
resistance that is often evident in these single-target approaches. Aside
from its emphasis on target-based efforts, modern antibacterial drug
discovery has also employed large chemical screens of whole cells for
growth inhibition. However, such screens produce huge numbers of
active compounds that are difficult to follow up because of a lack of
tools for prioritization. Indeed, there has been a resurgence in cell-
based approaches that includes innovative strategies for focusing on
promising leads that target different aspects of bacterial survival32.
Ultimately, these approaches will focus on specific targets to facilitate
the lead-optimization strategies that are inherent to modern drug dis-
covery. Therefore, it will become increasingly important to under-
stand the failures of target-based approaches.
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The essential-gene paradox
With its focus on the optimization of lead compounds that target indi-
vidual proteins using state-of-the-art tools of chemistry, biochemistry and
pharmacology, modern industrial drug discovery is an inherently reduc-
tionist endeavour. Thus, an important premise is that the target is valid,
and the weight of evidence to support a campaign against any given target
can vary considerably. For example, although systematic surveys of gene
dispensability in bacteria have illuminated a large number of potential
targets, a substantial fraction of these genes encode proteins of unknown
function. A clear understanding of function is commonly inherent in
the process of target validation and — from a practical standpoint — it
is difficult to build a chemical screening assay for a protein of unknown
function. We are coming to understand that dispensability phenotypes
are contextual even for well-characterized protein targets, and that growth
conditions and genomic context can greatly influence the dispensability
of the corresponding gene33.
In vivo essential genes
Approximately 7% of the E. coli genome, some 303 genes, has been
shown to be essential for growth in rich media, which represents typi-
cal Waksman-screen conditions24. Nevertheless, systematic studies of
stress conditions such as nutrient deprivation and chemical perturba-
tion have demonstrated that a further 258 genes are conditionally essen-
tial34,35. These conditionally essential genes largely encode enzymes that
are important for the utilization of carbon sources or the biosynthesis
of the outer membrane as well as in the synthesis of amino acids, vita-
mins and nucleobases. Yet these processes have long been ignored by
antibacterial drug discovery. Indeed, comprehensive efforts to define
essential sets of bacterial genes have been performed only in vitro and,
most extensively, using model microorganisms. Thus, the set of genes
that is required for the viability of pathogens in the context of infection
is less well understood. These in vivo essential genes have been assessed
in various pathogens using random transposon mutagenesis approaches
that, coincidentally, were invented while the first bacterial genomes were
being sequenced36. The methodology has revealed candidate roles for
hundreds of genes in aspects of virulence or colonization, including the
bacterial stress response, nutrient biosynthesis, type III secretion and
attachment functions that are mediated by pili, fimbriae or surface car-
bohydrates37. Typically, these approaches assess large pools of mutants
and have been enabled by the introduction of massively parallel DNA
sequencing38. An intriguing application of this technology was an investi-
gation of the genomic requirements for growth of P. aeruginosa in sputum
Table 1 | Unique chemical classes of antibiotics discovered in the golden era
Chemical class Target
Sulfonamides* Folate synthesis
β-Lactams† Cell-wall synthesis
Aminoglycosides† Protein synthesis
Tetracyclines† Protein synthesis
Chloramphenicols† Protein synthesis
Macrolides† Protein synthesis
Glycopeptides† Cell-wall synthesis
Oxazolidinones* Protein synthesis
Ansamycins† RNA synthesis
Quinolones† DNA synthesis
Streptogramins† Protein synthesis
*Synthetic chemical †Natural product..
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© 2016 Macmillan Publishers Limited. All rights reserved
from a person with cystic fibrosis39. Vitamin biosynthesis was found to be
an important requirement for bacterial viability — noteworthy because
searches for antibacterial chemicals have overwhelmingly emphasized
rich media conditions.
In vivo essential genes represent a set of emerging targets that remain
untested in modern antibacterial drug discovery. However, they hold
great promise for expanding the pool of targets for new drugs. Inhibi-
tors of this class of targets often fail to show the standard phenotype of
cell-growth inhibition, which is used to calculate the MIC. The MIC is
an important driver of compound optimization in preclinical antibi-
otic development and a gold standard for efficacy that is understood
by regulators, discoverers and practitioners alike. In the absence of a
MIC, an assay that is amenable to high-throughput screening and down-
stream drug discovery needs to be built. One innovative solution involves
screening in a non-conventional growth media. This approach was used
to target the glyoxylate shunt of P. aeruginosa because of its involve-
ment in pulmonary infection40. The screening workflow prioritized
compounds that were active in nutrient-limited media that contained
acetate as the only source of carbon but inactive when glucose was the
only source, and led to the discovery of eight lead compounds. Another
study built on a platform to discover small molecules that block growth
solely under nutrient-limited conditions, and used it to target the bio-
synthesis of amino acids, nucleobases and vitamins41. After prioritizing
antibacterial compounds that were active only in the absence of nutri-
ent supplements, the approach used systematic supplementation with
individual and pools of metabolites to illuminate mechanisms of action.
Lead compounds that target glycine, folate and biotin synthesis in E. coli
were uncovered as a result. Research that targets the quorum-sensing
virulence pathway in the pathogen P. aeruginosa has generated promising
compounds that are active in mouse models of infection without per-
turbing growth in vitro42. Through an innovative screening platform, the
quorum-sensing system and expression of the toxic gene product SacB
were linked such that ‘hits’ in the screen led to cell viability. Another route
to the discovery of in vivo targets involves screening in host models of
disease. For example, a high-content microscopic screen of macrophages
that were infected with M. tuberculosis identified a series of lead com-
pounds that targeted the cytochrome bc1 complex in the bacterium43.
The essential-gene paradox reveals a fascinating interplay between
risk and the target set of genes that has consequences for industrial drug
discovery (Fig. 2). For instance, a pathogen might have a set of genes that
is essential for growth on rich media and includes conventional targets
that are already under investigation. A greater innovation risk would be to
Mode of action Examples
Bacteriostatic Sulfanilamide
Bacteriocidal Penicillins
Cephalosporins
Carbapenems
Bacteriocidal Spectinomycin
Kanamycin
Neomycin
Bacteriostatic Tetracycline
Doxycycline
Bacteriostatic Chloramphenicol
Bacteriostatic Erythromycin
Clarithromycin
Bacteriocidal Vancomycin
Teicoplanin
Bacteriostatic Linezolid
Bacteriocidal Rifamycin
Bacteriocidal Ciprofloxacin
Bacteriocidal Pristinamycin

place the focus on the discovery of targets that are required for growth on
nutrient-limited media. Such targets are in vivo essential genes but would
also have in vitro phenotypes that facilitate conventional approaches to
antibiotic drug development. And an even greater innovation risk would
be to focus on genes that are uniquely essential for infection. Studies so
far suggest that infection genes could represent the largest set of targets.
However, the lack of in vitro phenotypes, such as growth inhibition, means
that it can be a challenge to enable established methods of drug discovery
for such targets.
Genetic-interaction networks
As modern drug discovery has come of age, we have witnessed a trans-
formation in our understanding of the cell that is changing our view
of the druggable genome. Studies of gene and protein interactions in
yeast44,45 and bacteria46,47 have revealed a network of abundant func-
tional interactions. The classic maps of biochemical metabolism and
cell signalling are being supplanted by a network model of the cell that
is characterized by a highly connected web of proteins and genes that
exhibits remarkable complexity and redundancy. In the model micro-
organism S. cerevisiae, in which genome-scale experiments to create a
matrix of pairwise deletions for all 5,000 dispensable genes are nearly
complete, around 200,000 pairs of genes were revealed to be synthetic
lethal in vitro44. These double-deletion strains suffered a substantial
deficiency in fitness compared with the corresponding single-deletion
strains, for which the network revealed about 40 interactions per gene.
This is in contrast with the approximately 1,000 essential genes that
have been mapped for this yeast. Early studies of synthetic lethality in
the model bacterium E. coli revealed a similar density of gene interac-
tions47 and predicted a dramatically expanded body of targets for drugs
or combinations of drugs that inhibit interacting gene products. Indeed,
these realities make a compelling case for combinatorial approaches to
new antibiotics.
The contextual nature of gene essentiality is perhaps best demon-
strated by synthetic-viable gene interactions. Here, the double mutant
is considerably more fit than expected with respect to the fitness of the
corresponding single mutants. An example is the interaction of toxin
and antitoxin genes that have a role in bacterial stress responses48. The
gene that encodes the antitoxin has an essential phenotype, but becomes
dispensable in a genetic background that lacks the toxin gene. Another,
more remarkable, example of synthetic viability can be found in the wall
teichoic acid (WTA) biosynthetic pathway of Gram-positive bacteria.
Although early steps in the pathway are dispensable, genes that encode
late-step enzymes have an essential phenotype49. Curiously, the deletion
of an early gene renders any of the late genes dispensable. The essential
phenotype of the late genes is thought to arise from the accumulation
of WTA biosynthetic intermediates that are linked to undecaprenyl
moieties and the sequestration of undecaprenyl phosphate from its
role as a lipid carrier in the truly essential process of peptidoglycan-
wall assembly50. The unique dispensability patterns of WTA genes have
provided the basis for sophisticated platforms for the discovery of new
lead compounds that target early and late steps in the WTA pathway in
S. aureus 51–53 as well as the related pathway of undecaprenyl-phosphate
synthesis54. Primary and secondary screens for antagonism that are ena-
bled by synthetic-viable phenotypes facilitate the rapid elimination of
nuisance compounds that often affect whole-cell screening campaigns.
By taking advantage of such knowledge, innovative screens for new anti-
biotics can be developed.
Chemical genomics
Further examples of the complexity of bacterial viability are provided
by studies that map enhancers or suppressors of antibiotic action on a
genome scale55–57. Such efforts have revealed that antibiotics have dense
and complicated networks of chemical and genetic interactions that seem
to be designed to resist perturbation (Fig. 3). These networks offer a rich
vista of new targets for antibiotic adjuvants, which are nonantibiotic
compounds that enhance the activity of antibiotics or help to overcome
© 2016 Macmillan Publishers Limited. All rights reserved
REVIEW INSIGHT
In vivo infection
2 In vitro growth on
nutrient-limited media
In vitro growth on
nutrient-rich media
3
1
innovation risk
Relative
1 2 3
Target set
Figure 2 | Target gene sets and innovation risks for a bacterial pathogen. 
A Venn diagram to show a relative profile of target genes for a hypothetical
bacterial pathogen. Three potential target sets can be imagined. Each has
a relative innovation risk within the context of modern industrial drug
discovery, which is indicated on the graph. By far the biggest set can be found
in the genes that are needed for infection in a relevant animal model, but
much validation remains to be done for these targets. The set that is required
for growth on nutrient-limited media is well charted. However, not all of
the genes in this set will be required for in vivo infection. Antibiotic drug
discovery under nutrient-rich conditions has been the convention for many
years, and the corresponding set of target genes is the smallest.
resistance. Therefore, emerging knowledge of bacterial cell systems is
challenging the modern genes-to-drugs approach through the idea that
target-validation measures must capture the implications for the network
as a whole. An important example of this is the growing appreciation for
the role of metabolic states and reactive oxygen species in the effective-
ness of existing antibiotics58,59. Indeed, the inherent complexity of cell
systems is a compelling argument for the use of whole-cell, phenotype-
based screens over biochemical, target-focused efforts in the discovery
of antibacterial compounds. Once a new potential compound has been
identified, chemical-genomic characterization can provide important
information and predictions about its mechanism and resistance profile.
Compounds that are discovered and characterized in this way therefore
provide a ready probe for target validation and a greater understanding
of the network that underpins the target.
Combinatorial discovery
The effectiveness of combinations of antibiotics has been demonstrated
in the clinic and is gaining favour as a design strategy. An effective
pairing is trimethoprim and sulfamethoxazole, which have a spectacu-
larly synergistic impact on folate metabolism. Another combination
combines amoxicillin, a β-lactam antibiotic, with clavulanic acid, an
inhibitor of β-lactamase drug resistance enzymes. Clinicians have
long combined antibiotics from different classes, such as β-lactams
with aminoglycosides, to achieve synergy, to cover a broad spectrum
of pathogens when the infectious agent is unknown or to suppress the
emergence of resistance. In fact, combinations are the mainstay of treat-
ment for stubborn infections such as tuberculosis. With the exception
of β-lactam–β-lactamase inhibitor pairs, existing antimicrobial drug
combinations are largely the product of clinical experimentation with
established antibiotics. However, a new model of discovery is emerging
that recognizes the network view of the cell and searches for efficacious
combinations of small molecules in a systematic manner.
Platforms that aim to discover active combinations have almost exclu-
sively searched for adjuvants. This is because the systematic examination
of combinations, even in relatively small chemical collections, leads to
prohibitively large screens. Typically, an existing antibiotic is tested for
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Genetic lethal interaction
Folate metabolism
Cell-wall biogenesis
DNA replication
Protein translation
RNA synthesis
growth inhibition at subinhibitory concentrations in combination with a
chemical collection. A screen for synthetic compounds that augment the
activity of the narrow-spectrum Gram-positive antibiotic novobiocin in
the Gram-negative bacterium E. coli identified four new compounds60.
They were found to affect cell shape and membrane permeability, which
provided an explanation for the observed synergies. Another combination
screen that used natural product antibiotics aimed to reverse carbapenem
resistance in metallo-β-lactamase-positive Gram-negative pathogens61.
It found that aspergillomarasmine A could block the notorious carbap-
enemase NDM-1 and was efficacious in a mouse model of infection by
NDM-1-containing Klebsiella pneumoniae.
Drugs that have already been approved have attracted considerable
interest as sources of bioactive chemical matter for combination stud-
ies62. This stems from a growing understanding that small molecules with
proven therapeutic activity are privileged chemicals that bind to biologi-
cal polymers and often have potential for alternative uses. Compounds
that are used in the clinic have a proven track record in patients and are
accompanied by a deep record of study that offers a substantial edge over
chemicals for which little is known. Several discovery campaigns that
used such libraries have yielded exciting combinations of drugs. For
example, the antidiarrhoeal drug loperamide was shown to destabilize
the membrane potential of bacterial cells, which facilitated the uptake of
the antibiotic minocycline in Gram-negative bacteria63. And the antihy-
pertensive drug ticlopidine was found to strengthen the action of β-lactam
antibiotics against methicillin-resistant S. aureus (MRSA) by inhibiting
the synthesis of WTA53.
Combinatorial approaches are consistent with an increased under-
standing that the most successful antibiotics seem to interact with
more than one target30. Furthermore, such approaches are aligned
with the network view of bacteria and their resistome, which resides
within the cell to buffer against perturbation. Nevertheless, they have
received relatively little attention from industry. Indeed, modern
target-based discovery approaches seek overwhelmingly to achieve
exquisite target selectivity to minimize toxicity that might arise from
drug promiscuity.
The adjuvant approach offers advantages such as the potential to
decrease the concentration of antibiotic that is needed to achieve a thera-
peutic effect, which should reduce the emergence of resistance and lessen
issues of off-target toxicity. Combinations can extend the effective lifespan
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Figure 3 | A chemical–genomic interaction
network.  The network map illustrates the dense
connectivity of enhancing interactions that
affect the activity of 39 antibiotics that target the
bacterium Escherichia coli. Grey balls represent
genes and coloured ones are for antibiotics of
various mechanistic classes. Interactions are
denoted by connecting lines. The map is derived
from data in ref. 35 and was prepared with the
BioLayout Express3D network visualization and
analysis tool83.
of legacy drugs, and because the delivery and utility of such drugs are
familiar to clinicians, barriers to entry into clinical practice might be
minimized. However, the combination approach also presents challenges.
These include the need to match the pharmacology and efficacy of each
component drug, which adds to the burden of drug discovery. Resistance
is also a concern — as it is for any antimicrobial therapy. The potential for
unexpected drug–drug interactions must also be examined thoroughly.
Nevertheless, multicomponent therapies, which are well established in
treatment of cancer and of HIV, tuberculosis and bacterial infections —
with combinations of β-lactam antibiotics and β-lactamase inhibitors,
in particular — offer an alternative to traditional antibiotic discovery in
the resistance era.
What chemical matter should be the focus?
The history of antibiotic discovery suggests that natural products, espe-
cially those produced by microorganisms, represent privileged chemical
matter for the discovery of antibiotics. Because they are the result of natu-
ral selection, these compounds have been proved to be highly effective
and are the source of most of our antibiotic medicines. Yet they have many
drawbacks. Natural-product antibiotics are often chemically complex
with challenging and intractable routes to synthesis in the laboratory that
make it difficult to prepare derivatives. The pharmacology of most first-
generation antibiotics is also not ideal, which reflects the origins of these
drugs as specialized microbial metabolites not medicines. Furthermore,
because these ancient metabolites are important components of microbial
chemical ecology, genetic elements that provide resistance against them
have been retained by and are often widely dispersed in communities of
microorganisms in the environment64,65. Finally, although screens that use
the Waksman platform can readily detect antimicrobial metabolites from
libraries of environmental microbes, most such metabolites have already
been identified and catalogued. More than 20,000 microbial natural
products have been described over the past several decades66. Waksman-
platform screens of typical libraries of natural products that are derived
from the actinomycetes group of bacteria, a favoured source of antibiot-
ics, tend to result in the identification of known compounds67. A further
complication is that the collection of libraries of natural-product produc-
ing microorganisms, the generation of compound extracts for screening
and the purification and characterization of active compounds is very
labour intensive and costly. Overall, this has resulted in the abandonment

of microbial natural products by most large pharmaceutical companies.
However, libraries of synthetic chemicals can exceed several million
compounds and, in principle, provide access to a vast spectrum of chemi-
cal space. By definition, they are chemically tractable, which enables their
scale up and relatively facile expansion by medicinal chemistry to explore
structure–activity relationships, improve efficacy and optimize pharma-
cology. Indeed, one of the most successful classes of antibiotics over the
past four decades has been the synthetic fluoroquinolones. Synthetic
libraries are the mainstay of most pharmaceutical companies and are the
source of many medicines that are crucial for human health, including
inhibitors of protein kinases, G-protein-coupled receptors and ion chan-
nels. Although these collections have been explored as sources of new
antibiotics using modern drug-discovery platforms for more than three
decades, they have yet to deliver new medicines — partly because they are
sampling such a narrow area of chemical space. Driven by metrics such as
the Lipinski rule of five for orally available drugs68, the synthetic libraries
of most pharmaceutical companies tend to be dominated by compounds
that are optimized for human biology. But most antibiotics in clinical use
do not conform to these parameters69 because they are natural products or
their derivatives. The Achilles’ heel of synthetic compounds as new anti-
biotic drugs seems to be their general inability to penetrate readily the cell
envelope of bacteria, which comprises inner and outer membranes, porins
and complex carbohydrate polymers, coupled with their susceptibility
to active efflux by membrane-associated pumps. Because they are prod-
ucts of evolution, antibiotic natural products have evolved to overcome
these challenges. The overall result is a stalemate in discovery: screens of
natural-product libraries identify bioactive but known compounds, and
screens of synthetic libraries identify potent ligands of biochemical targets
but with poor bioactivity.
A solution to this impasse is the development of synthetic libraries
that capture the chemical diversity and physicochemical properties of
natural products. These are being pursued for a number of therapeutic
classes70, including antibiotics71,72. Another solution is to capitalize on
BOX 1
Hurdles and solutions for antibiotic discovery
If not for the emergence of resistance to existing antibiotics, these
medicines might be thought of as ‘perfect’ therapeutics with an
extraordinary record of success in the clinic. Thus, the bar is set high
for the next generation of these medicines. Here are four hurdles and
solutions to the antibiotic-discovery problem.
●●Resistance has arisen partly because only a narrow selection
of chemical compounds are available, and they have a limited
range of mechanisms. Genomics-driven efforts to exploit new
potential targets, however, have been unsuccessful. New targets
and approaches to antibiotic drug discovery are fraught with risk.
Thus, there is a substantial need for fundamental research to
mitigate them. Academic research on unconventional targets and
discovery platforms has made much progress in past decades.
However, most resources in new antibiotic drug discovery have been
invested conservatively in conventional targets and approaches. With
growing potential for unconventional targets and new approaches
to antibiotic drug discovery, there is great opportunity for successful
efforts to industrialize these platforms.
●●The complex mechanisms of action of existing antibiotics could
be crucial to their success and elusive to modern drug-discovery
platforms. The solution is to understand these mechanisms better.
Biology is complex and our understanding of the action of antibiotics
is incomplete. Research that uses both traditional and systems
approaches will be required to expand our knowledge and, if this
hypothesis proves to be true, our approaches to drug discovery will
© 2016 Macmillan Publishers Limited. All rights reserved
REVIEW INSIGHT
the ability of synthetic compounds to inhibit essential bacterial targets
by developing delivery systems that solve the cell-envelope penetration
and efflux challenges. Conjugation to siderophores is an example of such
a ‘Trojan horse’ approach that has been explored but not yet proved to be
clinically efficacious73. A further solution is to combine these compounds
with molecules that enhance their transport or otherwise help to breach
the permeability barrier of the bacterial cell. In-depth study of the bacte-
rial cell envelope and the development of rules to guide the synthesis of
potential antibiotics should enhance the ability of such compounds to
penetrate cells and avoid efflux.
The crisis in antibiotic discovery is prompting a return to the inves-
tigation of natural products. Known chemical scaffolds that were aban-
doned in the past are being revisited with some success. Daptomycin,
for example, is now being used to treat serious infections caused by
Gram-positive pathogens. Eli Lilly and Company had discarded the
compound in the 1980s owing to toxic adverse effects that were iden-
tified during clinical trials. But in the late 1990s, Cubist Pharmaceu-
ticals licensed the compound and were able to overcome the adverse
effects by altering the formulation and dosage. The US Food and Drug
Administration (FDA) approved the drug in 2003 (ref. 74), and by
2014, annual sales of daptomycin had exceeded US$1 billion. Similarly,
the poorly absorbed antibiotic fidaxomicin, which was discovered in
the 1970s at Hoechst Marion Roussel, saw no clinical use until infec-
tions associated with the gut bacterium Clostridium difficile started to
rise around a decade ago75. Fidaxomicin’s shortcomings as a broad-
spectrum, orally available antibiotic were revisited as strengths in an
agent that targets C. difficile by taking advantage of high concentra-
tions of the drug in faeces, and fidaxomicin was approved for use by
the FDA in 2011.
Over the past 20 years, we have taken amazing strides in our under-
standing of the mechanisms and genomics of microbial natural-product
biosynthesis. Genome sequencing of microorganisms has revealed a
spectrum of potential for specialized metabolite production that greatly
need to adapt. In fact, promising new strategies are already being
used in the search for combinations of compounds that will target
cell networks in ways that are reminiscent of the complex action of
existing antibiotics.
●●Physical and chemical properties of existing antibiotics are
not compatible with conventional collections of compounds and
medicinal-chemistry approaches. There is great need for research
into the antibiotic permeability barriers of bacteria, particularly of
Gram-negative organisms, and the physical properties of chemicals
that can overcome these barriers. Those who have a role in drug
discovery must look critically at existing compound collections and
assess medicinal chemistry strategies within this context. Natural
sources of chemical compounds have been eschewed in past decades
in favour of synthetic chemicals that are better suited to conventional
models of drug discovery. It is time to revisit natural products as an
alternative to synthetic collections and to embrace new technologies
that will help to overcome problems of compound rediscovery.
●●The yardstick for early stage antibiotic discovery is the inhibition
of cell growth and the determination of the MIC. The ease of
identifying antibiotics through the Waksman platform, and its
success in the golden era, solidified a protocol that narrowly focused
success on this simple laboratory test. The realization that numerous
targets, such as virulence factors and aspects of metabolism, are not
captured by the traditional MIC approach requires innovation in the
laboratory and an acceptance that twenty-first century antibiotics
might not have the same properties as existing drugs.
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 INSIGHT REVIEW
exceeds the diversity of compounds that have been purified and char-
acterized over the past century. Automated prediction of the structure
and regulation of compounds from genome data is improving rapidly.
The manipulation of products with techniques from synthetic biology is
becoming more common, and offers an unprecedented opportunity for
tapping into the bioactive chemical space. Techniques such as metagen-
omics and use of specialized growth chambers are enabling exploration of
the microbial dark matter — microorganisms that have resisted easy cul-
tivation in the laboratory76 — as well as identification of new chemistry77
and fresh leads for antibiotic drugs78.
Of the thousands of natural-product antibiotics that have been identi-
fied over the past decades, only a select few have been seriously investi-
gated as drugs. The examples that we describe in this Review suggest that
it would be fruitful to revisit some of these old scaffolds and to search for
new ones using twenty-first century technology through the lens of the
clinical needs of the resistance era. Large-scale screens will be needed to
identify these candidate compounds. At present, no collection of micro-
bial natural products adequately reflects the chemical diversity that has
already been reported in the literature, let alone that which is yet to be
discovered. Indeed, many ‘old’ compounds are essentially lost to modern
drug discovery because there is no supply of them available. A multi-
national effort to develop a truly representative library of antimicrobial
natural products that are readily available to antibiotic researchers would
be transformative.
The future
Ultimately, clinical need and the marketplace will dictate the product pro-
file of new antibiotics. The clinical need for such drugs is growing, and
in some cases, such as multidrug-resistant Gram-negative pathogens, we
are already experiencing a profound crisis. In the resistance era, we must
accept and tackle this global challenge to continue to enjoy the benefits
of antimicrobial agents and the improvements in quality of life and life
expectancy that they bring. Although considerable, the regulatory chal-
lenges of developing new antibiotics are being slowly addressed to stream-
line approvals for drugs. For example, the FDA approved a combination
treatment that contains ceftazidime and avibactam on the basis of data
gathered in phase II clinical trials, therefore avoiding the need for phase
III data. And the economic hurdles associated with drugs that cure rather
than control diseases are being addressed by proposals such as the GAIN
(Generating Antibiotics Incentives Now) act in the United States, which
seeks to extend market exclusivity for new antibiotics79, public–private
partnerships such as the Innovative Medicines Initiative in Europe, and
by incentives such as financial rewards for companies that successfully
launch new antimicrobial drugs80.
The scientific challenges in identifying and developing new antibiotic
drugs remain substantial (Box 1). It is clear from the past two decades of
effort that antibiotics that are highly effective, safe and broad spectrum
are incredibly difficult to find. Consequently, we must ask whether our
current medical practices and ideas about infection are appropriate in
the resistance era. The availability and properties of the antibiotics of the
golden and medicinal-chemistry eras radically changed the practice of
infection control and treatment. For example, the efficacy of antibiotics
enabled empirical therapy and prophylaxis. And broad-spectrum anti-
biotics facilitated the practice of ‘best guess’ medicine and of overpre-
scription. But we no longer have the luxury of empirical therapy, and
infectious-disease medicine must adopt twenty-first century innova-
tions. Just as oncology has been transformed by fundamental research
and the realization that cancer encompasses many diseases that require
individualized therapies, so too must infection research. This requires a
much greater understanding of the specific microorganisms that cause
disease, their biology and their interactions with the host. As oncology
has done already, the treatment of infection should adopt the model of
personalized medicine, which would move the focus of the field from
broad-spectrum agents to narrow, disease-specific antibiotics. The result-
ing narrow-spectrum era (Fig. 1) would have considerable impact on the
next generation of antibiotics and generate fewer off-target effects on the
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© 2016 Macmillan Publishers Limited. All rights reserved
microbiome and decrease the pressure to evolve resistance. Such narrow-
spectrum agents can be identified by mining the catalogue of discarded
antibiotics — as evidenced by daptomycin — and through systematic
testing of combinations of compounds that selectively target genus- and
even species-specific pathways53,63. The switch to narrow-spectrum agents
should also expand the target base of antibiotics. Antibiotic discovery
is considered to be target poor, with the drugs limited to targeting cell-
wall synthesis, membrane integrity, translation, transcription and DNA
synthesis. This might simply reflect common targets that are available
to broad-spectrum agents. Narrow-spectrum agents offer a much larger
vista of targets that are specific to bacteria and pathogenesis. For example,
many of the processes that enable in vivo growth and virulence are targets
for such antibiotics.
Because of time pressures and a lack of sensitive and reliable diagnostic
agents, clinicians most often favour broad-spectrum agents. Advances
in the accuracy of point-of-care diagnostics will be necessary to move
forward the field of antibiotic discovery. And the business case for narrow-
spectrum agents must address the consequences of lower sales volumes81.
However, a spate of prizes for the development of point-of-care diagnos-
tics, such as the UK Longitude Prize that seeks to tackle global antibiotic
resistance, together with stunning advances in genome sequencing and
informatics tools that can identify pathogens and their resistance profiles82
are creating the conditions necessary to support the discovery of narrow-
spectrum antibiotics and their implementation through improved diag-
nostics. The narrow-spectrum era requires innovation, new economic
models and collaboration across disciplines. Despite these challenges,
the field of infectious-disease medicine can learn from other therapeutic
areas to move from broad-spectrum empirical therapy that is no longer
effective in the resistance era to the personalized and strategic approach
of the emerging narrow-spectrum era. ■
Received 25 August; accepted 16 November 2015.
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Acknowledgements The authors thank M. Farha for stimulating discussion with
regards to the manuscript and S. French for creating Fig. 3. This work was supported
by Discovery grants from the Natural Sciences and Engineering Research Council
of Canada to E.D.B. (RGPIN 04384-2014) and G.D.W. (RGPIN 237480), by salary
awards from the Canada Research Chairs Program to both E.D.B. and G.D.W., and by
grants from the Canadian Institutes of Health Research to E.D.B. (MOP-81330 and
MOP-15496) and G.D.W. (MT-13536).
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing interests. Readers are
welcome to comment on the online version of this paper at go.nature.com/srti9i.
Correspondence should be addressed to E.D.B. (ebrown@mcmaster.ca) or G.D.W.
(wrightge@mcmaster.ca).
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