Ann. N.Y. Acad. Sci.

ISSN 0077-8923

A N N A L S O F T H E N E W Y O R K A C A D E M Y O F SC I E N C E S
Issue: Antimicrobial Therapeutics Reviews

Challenges of antibacterial discovery revisited

Michael N. Gwynn, Alison Portnoy, Stephen F. Rittenhouse, and David J. Payne
Antibacterial Discovery Performance Unit, Infectious Diseases Center of Excellence for Drug Discovery, GlaxoSmithKline, Collegeville, Pennsylvania Address for correspondence: David J. Payne, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426. David.J.Payne@gsk.com

The discovery of novel antibiotic classes has not kept pace with the growing threat of bacterial resistance. Antibiotic candidates that act at new targets or via distinct mechanisms have the greatest potential to overcome resistance; however, novel approaches are also associated with higher attrition and longer timelines. This uncertainty has contributed to the withdrawal from antibiotic programs by many pharmaceutical companies. Genomic approaches have not yielded satisfactory results, in part due to nascent knowledge about unprecedented molecular targets, the challenge of achieving antibacterial activity by lead optimization of enzyme inhibitors, and the limitations of compound screening libraries for antibacterial discovery. Enhanced diversity of compound screening banks, entry into new chemical space, and new screening technologies are currently being exploited to improve hit rates for antibacterial discovery. Antibacterial compound lead optimization faces hurdles associated with the high plasma exposures required for efcacy. Lead optimization would be enhanced by the identication of new antibiotic classes with improved tractability and by expanding the predictability of in vitro safety assays. Implementing multiple screening and target identication strategies is recommended for improving the likelihood of discovering new antibacterial compounds that address unmet needs. Keywords: antibacterial discovery; novel antibiotic; gene essentiality; HTS; DNA-encoded library; boron chemistry

Introduction The effectiveness of all antibiotic agents in current use is compromised by the prevalence of drug resistance.1,2 Increasingly, multiple drugresistant strains of Gram-positive and Gramnegative pathogens (so called superbugs) are encountered that are resistant to even last-line agents (e.g., vancomycin).2,3 The main classes of antibiotic drugs most commonly used today were discovered, largely by empirical screening, over 50 years ago and exploit a limited range of bacterial physiology: cell wall biosynthesis ( -lactams, glycopeptides), cell membranes (daptomycin, colistin), type II topoisomerases (uoroquinolones), ribosomes (macrolides, aminoglycosides, tetracyclines, the oxazolidinone linezolid, streptogramins), transcription (rifampicin), and folate biosynthesis (sulfonamides and trimethoprim).4,5 These classes of agents represent a relatively small set of chemical scaffolds that have been modied over the past few
doi: 10.1111/j.1749-6632.2010.05828.x

decades by synthetic tailoring into new-generation agents1 that generally offer incremental improvements over existing therapies. Novel-class antibiotic agents have greater potential to address the deciencies of existing classes of antibiotics and are vitally important to address the ever-increasing problem of bacterial resistance.6 Unfortunately, development and marketing approval of new antibiotics have not kept pace with the growing public health threat of drug-resistant bacteria2,3,7 and examination of the current industry pipeline shows a marked innovation gap.7 There has been a decline in new antibiotic approvals over the past 20 years7 and existing antibiotics are losing effectiveness more rapidly than they can be replaced. This is due in part to the extensive cost and timelines associated with drug development (8 15 years8,9 and $800M10 ). The availability of lowcost generic agents, coupled with the potential that use of novel agents may be reserved exclusively for the treatment of infections due to highly resistant

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pathogens, can reduce the nancial returns associated with antibiotic development. The numbers of approved or late-phase development novel-class antibacterials are even lower,7 with only two systemic antibiotics based on novel chemical scaffolds achieving marketing approval in the past 40 years: the protein synthesis inhibitor linezolid (an oxazolidinone) and the lipopeptide daptomycin.1,7 Considering the signicant unmet medical need for new antibiotics to address bacterial resistance, why is the pharmaceutical pipeline so poorly equipped to handle this important and growing deciency? The answer lies in the difculties associated with discovering novel antibacterials and the reality that the most innovative approaches (e.g., new targets or new chemical space) have longer development times when factoring in target validation and lead generation. With greater burden of unknowns, new targets or new chemical space also carry the highest risk of failure and attrition. Yet, novel agents offer the greatest potential against bacterial resistance, so we must continue to apply what we have learned over the past decade and use the latest technological tools to support the development of transformational medicines. This review addresses the discovery hurdles associated with small molecule antibacterial agents. Alternative therapeutic approaches, such as exogenous antibodies, vaccines, and small molecule immune modulators, are associated with unique difculties and opportunities that are not fully covered in this paper.

(e.g., faropenem), or have an alternative safety prole;11,a (ii) Agents that act at an established target but contain modications to an existing chemical scaffold that modify spectrum or overcome resistance (e.g., anti-methicillin-resistant Staphylococcus aureus (MRSA) uoroquinolones, ketolides, and cephalosporins);12 (iii) Agents that act at a known target but exploit a new mechanism of action, binding site, or new chemical space (e.g., novel topoisomerase inhibitors1316 and pleuromutilins17 ); and (iv) Novel chemical classes that act at a completely new target (e.g., peptide deformylase inhibitors, leucyl tRNA synthetase inhibitors).18 Compounds that act at a previously unexploited target, or via new mechanisms at existing targets, have the greatest potential to address bacterial drug resistance because no precedence for clinical resistance due to target-mediated or drug-inactivation mechanisms exists. There is greater risk, however, when developing novel mechanism or novel target agents. The earlier in the drug development cycle where novel approaches are attempted, the greater the risk of failure and attrition due to the unknown properties waiting downstream. For example, pursuing a novel target leaves room for surprises regarding the essentiality of the target across the bacterial spectrum. Even when essentiality is established, can adequate bacterial cell penetration be achieved for antibacterial activity by an enzyme inhibitor? Can a suitable molecule be optimized to include physicochemical properties that warrant candidate selection for clinical development? What unknown toxicology, clinical safety, or tolerability considerations might be uncovered by working in a new chemical space?

A drug called novel Every antibiotic development candidate is considered novel by those who make them, but the multiple approaches to developing new compounds do not carry equal potential to overcome preexisting resistance mechanisms, nor are they associated with equal development risk. As represented below, there is a spectrum of innovation that ranges from developments within established classes, to completely new bacterial pharmacophores and molecular targets. Spectrum of innovation for new antibacterials:
(i) Agents that act at an established target and within preexisting chemical space but have new physicochemical properties compared to existing agents of the same class such that they can be delivered via a new route of administration

Higher risk affects investment Many factors have contributed to the reduced industry investment in antibacterial discovery, including

Faropenem, an orally administered penem antibacterial marketed in Japan, received a nonapprovable letter by the U.S. Food and Drug Administration (FDA), requesting additional studies for all indications, including superiority studies for acute bacterial sinusitis and acute exacerbation of chronic bronchitis.

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cost, timelines, attrition, and the potential for a better return on investment for other disease areas that require longer term treatment. These factors can be unpalatable to investors and contributes to pharmaceutical companies increasingly abandoning the search for truly novel agents. The already low rate of development candidates launched as new medicines (8% in 20002002)19 largely reects compounds within existing classes, and may underestimate the contest faced by novel class therapeutics. The technical difculties associated with novel antibacterial discovery to produce differentiated medicines factor strongly into the costbenet calculations. Improving the probability of success is paramount to securing renewed investment in antibacterial discovery. The technical challenges to identifying new antibacterial leads that address novel genomic targets have been previously described8 based on the experience of evaluating over 300 genes for essentiality and conducting greater than 70 high throughput screens over a period of 7 years starting in 1995. Productivity in generating new leads was very low, and this represents only part of the overall drug discovery process with all of the attendant attrition at each stage. In this paper, the technical challenges in validating new bacterial targets and in achieving successful screening will be reviewed along with strategies that increase opportunities for discovering new class inhibitors for novel targets. Challenges with validating targets At the outset of the bacterial genomic era in the mid 1990s, new target discovery was the major focus for antibacterials and was anticipated to address rate limiting steps in antibacterial discovery. While genomics may yet transform antibacterial discovery, the learning curve and translational challenges have proven greater than initially appreciated. The current challenges for antibacterial lead generation include validating the potential targets discovered over the past decade, understanding the risks associated with unprecedented targets, and identifying inhibitor leads that have the potential to be developed into drugs.

genomes of relevant pathogens are compared to reveal genes that are highly conserved among bacteria but are absent or poorly conserved in the human genome. While certain pathogen-specic genes may be of interest, for example Mycobacterium tuberculosis, Helicobacter pylori, Pseudomonas aeruginosa, and Staphylococcus aureus, genes that are well conserved across the diversity of pathogens that can be encountered in therapeutic indications, such as respiratory infection, wound infection, and hospital acquired infections, offer greater commercial attraction to balance the cost of discovery and drug development. Based on genomic analyses, it has been estimated that less than 300 bacterial genes have appropriate conservation across species and in vitro essentiality to represent potential broad-spectrum targets.20 As of January 2010, genomes for over 100 bacterial pathogen species and hundreds of individual strains have been reported.21 Additional genomes may help rene understandings of genetic conservation and spectrum but the number of good molecular targets will likely diminish as more is learned about individual target exploitability. Although genomic databases dene all potential new bacterial targets based on their genes, other important structures like bacterial cytoplasmic and outer membranes (targeted by Daptomycin and colistin, respectively) and peptidoglycan (bound by vancomycin-class antibacterials), represent targets that are not dened by discrete gene products. Nongenomic starting points (such as empirical whole cell screening described later) may be needed to nd agents with mechanisms not dened by reductionist approaches. In addition, the full functionality of many highly integrated genes products may be best screened for inhibitors in the context of functional pathways, such as cell division.22

Target prospecting Previously unexploited targets are initially prospected by bioinformatic analysis in which

Target essentiality Essentiality for in vitro growth is the usual prerequisite for a gene product to be regarded as a potential antibacterial target for inhibitors. However, essentiality cannot be extrapolated across bacterial species and sometimes not even across most strains of the same species without additional supporting data. While target heterogeneity remains a consideration for any drug discovery effort, the extent of microbial diversity means that bacterial targets exhibit far greater phylogenetic range than

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human or other mammalian targets, hindering the identication of novel targets that are valid across bacterial groups. As the premise for a new antibacterial is its effectiveness against the majority of strains in a disease indication, a failure due to microbial diversity in the target would result in an unacceptable level of built in drug resistance. This is illustrated by the following examples where bioinformatic and in vitro genetic assessment of essentiality and predicted spectrum were confounded by phylogenetic variation or other factors. The protein synthesis gene product methionyl tRNA synthetase (MetRS) is one example of a target believed to be broad spectrum based on high sequence homology between S. pneumoniae, S. aureus, and Escherichia coli. Potent inhibitors were identied and optimized for antibacterial activity against strains of S. aureus and Enterococcus sp.; however, approximately 30% of S. pneumoniae clinical isolates were found to be resistant due to the presence of an additional nonhomologous MetRS gene product insensitive to the inhibitor class.23 The gap in S. pneumoniae coverage was unacceptable for treatment against respiratory infections, though potential for this class remains for treatment of MRSA infections. Literature supporting essentiality of the E. coli fatty acid biosynthesis gene product FabI (enoylACP-reductase) across all bacterial pathogens led to the exploration of FabI for use as a broad-agent target. FabI was later found to be absent from some bacteria whose function was encoded by nonhomologous genes. Despite the identication of leads with potent activity against S. aureus, the absence of activity against S. pneumoniae meant this target could never achieve broad-spectrum or respiratoryspectrum activity.24 Another lesson learned about pursuing genomic targets is the case of duplicate genes in some species. UDP-N -acetylglucosamine-enolpyruvyl transferase (MurA) is the clinically validated antibiotic target of fosfomycin. Du et al.25 reported that, unlike E. coli, several Gram-positive species actually have two forms of essential MurA gene products. An effective inhibitor must therefore inhibit both Mur enzymes in order to have antibacterial activity, representing an additional challenge for lead optimization.25 The examples above show that unexpected challenges, such as nonhomologous and duplicate genes,

can undermine essentiality even for targets that have been validated across multiple species and strains, and therefore need to be anticipated. Such surprises were particularly hazardous at the outset of the genomic era. Today, our greater maturity of knowledge helps reduce risk through essentiality testing in multiple species and the use of deep sequencing and bioinformatics analysis across multiple strains and species. Highly cost-effective Next Generation Sequencing technologies will broaden our knowledge of microbial target heterogeneity and the frequency in clinical settings. For example, Harris et al.26 describes the application of Next Generation DNA sequencing technology to compare the near complete genomes of 63 strains of S. aureus. Brinster et al.27 provides another salutary lesson in target validation using the fatty-acid synthesis (FASII) pathway by demonstrating that exogenous fatty acids, provided in both in vitro and in vivo systems, allow microbial pathogens to fully bypass inhibition of a pathway that was otherwise widely viewed as essential. Though the conclusions remain controversial,28,29 this work reminds us that targets broadly validated using optimized in vitro conditions might not always remain essential when applied to in vivo systems.

Tractability of bacterial targets Not all bacterial targets have equal potential to be exploited for use by antibacterial agents, even if they were all equally essential and conserved. Most successful drug targets are associated with deep active site clefts30 that offer potential for strong inhibitor binding. Other targets, such as those associated with proteinprotein interactions, are less tractable and are thus far devoid of examples of successful exploitation for antibacterials.22 The bacterial cell division septation process, for example, is a complex interplay of essential proteins involving multiple proteinprotein interactions22 and is an attractive target in terms of bacterial specicity and the potential to be accessed by extra-cytoplasmic inhibitors for some of the components. While complex protein interactions have not been successfully targeted in the past, examples are emerging of smallmolecule inhibitors of proteinprotein interactions, and the scientic community is beginning to understand which interactions are more amenable to antagonism.31

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Discovery of successful antibacterial targets and inhibition mechanisms have often been preceded by the identication of tool compounds that were found by nontarget-based approaches, such as whole cell screening. These tool compounds increase condence in nding new inhibitors for the target with antibacterial activity that can be exploited in lead optimization programs. Exploitation of the bacterial cell division protein FtsZ was in part encouraged by knowledge of effective inhibitors of the homologous human cytoskeleton protein, tubulin, such as taxol. A new small molecule inhibitor of the GTPase activity of FtsZ had potent antistaphylococcal activity and efcacy in animal models of infection.32 Where no previously known inhibitors of targets exist, extensive screening campaigns have frequently failed to nd exploitable inhibitors.8 Why some targets appear to be less amenable for nding inhibitors is not always obvious, and might be a function of the diversity of the compounds screened or the physiology of the targets.33 Ribosome and gyrase targets, for example, have multiple classes of published leads and marketed drugs, whereas other essential gene products have no known inhibitors despite a long history of antibacterial research. Pursuing novel chemical classes of inhibitors for tractable, pharmacologically validated targets by new mechanisms or binding modes that overcome preexisting resistance mechanisms, such as DNA replication, ribosome function, cellwall biosynthesis, and -lactamase inhibition, is a rational strategy that reduces the risks associated with unprecedented targets and exploits their established druggability.8,15,34 In addition to activity at the bacterial target, antibiotic compounds face the added requirement of penetrating across bacterial cell membranes, or for intracellular pathogens, penetrating across both mammalian and bacterial cell membranes. The requirement to cross challenging biological barriers, while not unique to antibacterials (e.g., central nervous system active agents and some anticancer drugs must cross the bloodbrain barrier), does place signicant demands on the discovery and lead optimization of antibiotics and may contribute to the lower success rates of antibacterials. Simultaneous to the challenge of passive diffusion across membrane(s), Gram-negative bacteria in particular express powerful multidrug

efux systems that can reduce antibacterial activity by orders of magnitude.35 Targets outside the bacterial cytoplasmic membrane obviate penetration barriers to the inhibitor reaching the target, but the choice of essential, well-conserved broadspectrum targets outside the membrane is greatly reduced. -lactam antibiotics exemplify successful targeting outside the cytoplasmic membrane and achieve a superb spectrum of activity, but this example has not been matched. An example of a potent new-target, narrow-spectrum (P. aeruginosa) agent that addresses an exposed outer membrane target involved in its assembly is the synthetic protein epitope mimetic (PEM) POL7080, which is currently in preclinical development and is described later.36

Spontaneous resistance to new antibacterials Target selection for antibiotics also has a complex temporal aspect: selective pressure generated by clinically used classes of antibacterials will alter bacterial population genetics. Proliferative diseases, including bacterial infection, are different from other therapeutic areas in that emergence of resistance diminishes effectiveness of therapies with time. Single-enzyme targets are particularly susceptible to spontaneous resistance.37 Examples of agents that rapidly became compromised as single-agent therapies due to resistance include: fosfomycin, novobiocin, fusidic acid, and rifampicin. Agents with nonsingle-enzyme targets fare better against the development of resistance. These include -lactams (which act at multiple penicillin binding proteins), uoroquinolones (which target the homologous topoisomerases DNA gyrase and topoisomerase IV enzymes), vancomycin-class antibacterials (which target the peptidoglycan substrate rather than the processing enzymes), and a wide range of ribosomally targeted antibacterials (which experience low target-mediated spontaneous resistance because rRNA is encoded by multiple genes). These are notably also the targets for most clinically used antibacterials that have stood the test of time. Most antibacterial classes in wide clinical use benet from acting at molecular targets associated with intrinsically reduced levels of spontaneous resistance. Novel, single-enzyme bacterial targets can still be considered for development, but the risk of high spontaneous resistance causing expensive failure during early clinical use is

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arguably higher for single-enzyme targets, and also presents an additional risk associated with unprecedented targets. Careful preclinical assessment of spontaneous resistance and virulence of laboratory mutants is important when evaluating new drug leads in order to understand the potential for rapid reduction in therapeutic effectiveness due to emergent resistance. Screening strategies to address target challenges Following target identication and validation, screening for molecules with inhibitory activity at the selected target is needed; however, the output of high throughput screening (HTS) campaigns against novel bacterial targets has been inadequate to support the antibacterial pipeline.8 This was attributed in part to the screening of available chemical libraries that were limited in relevant diversity for antibacterial discovery. Marketed antibacterials do not generally follow Lipinskis rule of ve, and are on average less lipophilic and slightly larger than many other drug classes.38 Fortunately, advances in small molecule libraries and the creation of innovative screening technologies could potentially transform the identication of suitable molecules for new targets.39

HTS libraries and enhanced diversity Pharmaceutical industry small molecule screening collections are generally composed of around one million compounds, derived from a number of sources. HTS in the new genomic era was unsuccessful in nding hits for many targets (particularly in the antibacterial arena), thus driving the need for compound screening collections with more biologically relevant and diverse compounds.39 Several sources of compounds have been used for expanding and enhancing screening collections. Compounds from previous and current lead optimization efforts across diverse therapeutic areas are useful within screening collections as they have demonstrated relevance to biological systems and tend to have desirable physicochemical properties, such as solubility, stability, and lack of unwanted reactivity. However, while such molecules can be drug-like, they can also represent a limited range of scaffolds and they tend to have higher molecular weight than smaller, lead-like molecules with greater potential as inhibitors.40

Specialized vendors are now available who can add diversity for screening collections by lling in perceived diversity gaps present in preexisting collections.41 Further enrichment of synthetic compound screening libraries is based on use of Diversity-Oriented Synthesis (DOS), which applies combinatorial synthesis of diverse small molecules within biologically relevant and underexploited chemical space,42 and Galloway et al.43 describe an example applied to antibacterial lead discovery. Biology-Oriented Synthesis (BIOS) centers on the generation of small compound libraries based on scaffolds of proven biological relevance, natural products, and drugs.44 The aims of DOS and BIOS are to generate a relevant diversity of scaffolds and functionalities,42 while avoiding the downsides of traditional natural product screening, such as longer discovery process and low synthetic tractability. In addition to biologically inspired diversity, there is also renewed interest in natural products themselves as a source of screening leads. Thirtyfour percent of all small molecule New Chemical Entity (NCE) approvals between 1981 and 2006 were either natural products or semisynthetic derivatives.45 Natural products have an even greater relevance in the history of antibiotic discovery.4648 Despite that success, pharmaceutical research into natural products declined during the past two decades. At the start of the HTS era, many companies moved from natural-product extract libraries toward more convenient synthetic compound libraries. The reemerging interest in natural products for lead discovery is partly driven by the realization that competing technologies, such as combinatorial chemistry, have failed to deliver new drug leads in signicant numbers.41 Analysis of chemical space associated with marketed drugs, natural products, and molecules made from combinatorial synthesis demonstrated that the structures of drugs in use today more closely resemble those of natural products.49 In addition, recent analytical technological advances, such as spectroscopy and HTS, have been crucial breakthroughs in separation of natural products and in determining structures of screening hits.4651 A recent study looking at hit rates from HTS campaigns with a collection at Novartis suggests natural products are a more diverse class than traditional synthetic and combinatorial molecules, and that natural products have the highest hit rate.52

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The enduring value of enhancing the diversity of HTS screening collections to industry drug pipelines remains undetermined given the lengthy research and development time scale, but investment in these approaches by both industry and academia suggests promise for the future.

and selectivity data, before functional biochemical assays are developed. This rapid and inexpensive platform for the discovery of small molecule drug leads has the potential to revolutionize the discovery of small-molecule modulators of biological targets including bacterial targets.

Specialized chemical libraries The potential for specialized chemical libraries to yield promising novel small molecule leads and therapeutics is illustrated by the boron chemistry platform of Anacor Pharmaceuticals (Palo Alto, CA). Boron as boric acid is ubiquitous in the natural environment and known to be safe in many everyday items and is a trace mineral believed to contribute to growth, brain function, and inammatory response.53 Boron-containing molecules are increasingly being explored for their chemotherapeutic potential. The properties and electrophilicity of boron and boronic acids confer to boron-based compounds the ability to undergo novel interactions with biological targets.54 In December 2009, the commencement of a Phase I clinical study was announced for AN3365, a novel boron-based, small molecule drug candidate in development for the treatment of hospital infections caused by Gramnegative bacteria, including E. coli, K. pneumoniae, and Enterobacter spp. AN3365 targets the bacterial enzyme leucyl tRNA synthetase, a previously unexploited target.55 The potential to act at this bacterial target was presaged by the activity of this class of inhibitor against a fungal homolog, and illustrates an exciting new mechanism of action.56,57 DNA-encoded small molecule libraries Clark et al.58 report a technology for the synthesis and screening of an 800-million-member DNA-encoded library, in which small molecules are covalently attached to an encoding, doublestranded oligonucleotide. The library was interrogated by afnity selection and underwent deconvolution through DNA barcode sequencing of the library, resulting in the discovery of inhibitors for two human enzymes: Aurora A kinase and p38 MAP kinase. This process increases library screening throughput by several orders of magnitude over previous methods with only modest instrumentation and protein requirements. By using high throughput sequencing techniques, the selection output can be deconvoluted to yield families of ligands with emerging trends of structureactivity relationships

Fragment-based drug discovery One complementary approach to HTS includes fragment screening, which has become a mainstream discovery strategy in the pharmaceutical industry over the last few years. Fragment-based drug discovery (FBDD) screens molecules less than 300 Da (compared to standard HTS of molecules from 450 to 500 Da). The hit rates are higher than with traditional HTS, but the afnities are typically relatively low ( MmM range). The higher hit rates are achieved because smaller, less complex molecules have lower chances of a mismatch that would otherwise negate binding.40 Chemists can later enhance afnity by increasing structural complexity with the benet of structure-guided design. FBDD is typically conducted in conjunction with high-resolution structural techniques. X-ray crystallography is most widely used for protein structure determination and advances have solved technical constraints to enable crystallographic techniques to be used on a higher throughput basis and serve as part of fragment screening strategies. The highresolution structural data can then be used as a starting point for lead optimization and also forms part of an iterative process in the creation of larger, more potent compounds.30 FBDD approach has been successful across a range of targets, either when the target is not amenable to HTS or as a parallel strategy to increase probability of success. FBDD-related discovery approaches have been credited with at least 13 examples of molecules entering Phase I and Phase II clinical trials; however, none of these examples thus far are antibacterials.59 A recent application to antibacterial lead discovery is reported in an elegant study by Pzer, which demonstrated the use of FBDD and virtual screening to identify antibacterially active leads targeting biotin carboxylase, a component of the multienzyme complex acetyl-CoA carboxylase, which catalyzes the rst committed step in fatty acid biosynthesis. This further validates biotin carboxylase as a tractable antibacterial target and illustrates the applicability of FBDD in antibacterial

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discovery.60 FBDD was used with virtual screeningin which shape and electrostatic properties were screened in silicoto identify weak-binding but exploitable fragments as ATPcompetitive inhibitors. Lead optimization then resulted in up to a 3,000-fold improvement in afnity and achievement of antibacterial activity while maintaining desirable physicochemical properties.

Whole cell screening: pros and cons Most currently used antibacterial classes were discovered by whole cell screening and are composed of relatively few structural families. Molecular target screening became fashionable, but the lack of tractable leads, together with the known difculty of converting compounds lacking whole cell activity into ones with activity, has led to renewed consideration of nontarget-based, whole cell antibacterial screens. More holistic approaches, such as viable cell screening and biochemical pathway screens, do not limit the concept of target to a dened gene product, and also allow biological complexity to be screened with the potential to identify inhibitors with unpredicted mechanisms. This is well illustrated by the investigation of an unexploited class of natural product antibacterial from the patent literature that was found to dysregulate bacterial proteolytic machinery.61 Whole cell screening evaluates all targets essential for in vitro growth in a physiological context. Although potentially less sensitive than molecular screens, whole cell screening selects for antibacterial activity as a starting property. The availability of improved methods for ruling out nuisance compounds62 and for dening molecular targets of new antibacterial leads contributes to the renewed efciency of empirical whole cell screening, affording it a back to the future quality. Mechanism-based whole cell screens can combine a targeted approach with a biologically integrated context. This is elegantly illustrated by the discovery of a broad-spectrum FtsZ inhibitor in a mechanistic whole cell screen63 and the use of a whole cell antisense screening strategy to identify a novel natural product inhibitor of the fatty acid biosynthesis condensing enzymes, FabF/B.64 Once a screening hit has been identied, dening the molecular target is a key step before entering into lead optimization. Knowing the molecular target enables bioinformatic analysis of the

potential spectrum and of selectivity (with respect to human homologs), and supports lead optimization with target Structure Activity Relationship (SAR) and cocrystallography. Macromolecular synthesis (RNA, protein, DNA, peptidoglycan, and fatty acids) encompasses more than 75% of the bacterial essential genome. Inhibition of these pathways can be detected using radiolabeled precursor uptake and can be screened in high throughput microtiter format. Genetic approaches can be applied to rapid denition of mode of action including resistant mutant analysis, gene overexpression and under-expression (e.g., antisense), the use of conditional lethal mutants to identify compounds with synergistic effects, and gene-expression assays (such as DNA microarrays) that identify compounds that elicit specic stress responses.65 Sequencing technology now makes it practical to locate a single point mutation in the genome of a mutant selected by a novel antibacterial, in order to identify the molecular target.66 A recent application of genomic methods to rapidly analyze a mechanism of action was described by Pathania et al.,67 who assembled an ordered, high-expression clone set of all the essential genes from E. coli and used it to systematically screen for suppressors of antibacterial action. This method led to the discovery of MAC13243, a new antibacterial with a unique mechanism that inhibits the function of the LolA protein (a key component of the lipoprotein targeting pathway in bacteria). Miller et al.68 used whole cell screening to identify a series of antibacterial pyridopyrimidines derived from a repurposed protein kinase inhibitor compound collection. The leads were found to target the ATP-binding site of the fatty acid biosynthesis target, biotin carboxylase, and were effective in vitro and in vivo against Gram-negative pathogens including Haemophilus inuenzae. While the enzyme has homology to the human counterpart, the inhibitors were selective for bacterial biotin carboxylase as the binding mode was distinct from the protein kinasebinding mode of mammalian enzyme inhibitors. This illustrates the creative combination of whole cell screening with the exploitation of an enhanced compound collection based on inhibitors of a structurally related human target. This resulted in a mechanistically and structurally new class of selective antibacterial leadsderived from a protein kinase inhibitor pharmacophore.68

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Focus on spectrum Antibacterial spectrum is a major consideration when selecting a target for lead optimization. Many targets have the potential to be broad in spectrum but are limited by the requirement to penetrate the outer membrane of Gram-negative bacteria. In addition, multidrug efux systems present in many bacteria transport a wide range of chemically diverse molecules out of the bacterial cell.69,70 Medicinal chemists are therefore challenged with the task of identifying molecules whose physicochemical properties allow it to penetrate the cell and evade efux, but retain potent activity at the molecular target. The design of small molecules to inhibit bacterial efux has also been explored as a means of broadening the antibacterial spectrum and overcoming bacterial resistance. While a clinical candidate has yet to emerge, this approach has been validated across a range of in vitro and in vivo assays.71 One approach to circumvent the challenges of discovering and optimizing broad-spectrum agents is to focus on agents that target a specic pathogen. In addition to offering fewer drug discovery barriers, narrow-spectrum agents have the potential for reduced disruption of normal intestinal ora.72 Clostridium difcile is an obvious candidate as it accounts for 2030% of antibiotic-associated diarrhea and nearly all cases of antibiotic-associated colitis.7375 Episodes of recurrent C. difcile infection are difcult to treat and require repeated and prolonged treatment. Several strategies have been taken to develop narrow-spectrum agents against C. difcile associated diarrhea (CDAD). Fidoximicin (Opt80) is a novel, bactericidal, macrocyclic compound that inhibits RNA polymerase of specic bacteria, such as C. difcile.76,77 Because of its narrow spectrum, doximicin is believed to prevent relapse in CDAD since alterations of the normal ora of the gastrointestinal tract are a prerequisite for C. difcile proliferation.78 Furthermore, doximicin is primarily retained in the gut, with low levels in serum following oral administration, making it an attractive agent to treat a gut-specic pathogen.79 Monoclonal antibodies (Mabs) have also been added to the armamentarium of potential agents for narrow-spectrum antibacterial therapy. One of the most advanced agents is the C. difcile Mab combination MDX-066 and MDX-1388, which targets and neutralizes the two main C. difcile tox-

ins. The results reported so far are promising; in a Phase II clinical trial involving 200 patients with C. difcile infection receiving standard-of-care antibiotics, the addition of intravenous MDX-066MDX1388 treatment reduced the rate of recurrent infection by 70%.80 As reviewed by Bebbington and Yarranton,81 there are a number of antibodies to bacterial targets primarily in S. aureus, Bacillus anthracis, and P. aeruginosa that are currently in clinical studies. As Mabs are highly directed against a specic bacterial target without a human homolog, the safety prole using this approach is predicted to be very good. While there are currently no FDAapproved Mab products for treating bacterial infections, this promising approach warrants further investigation. PEM technology is a new approach in drug discovery in use for the development of narrowspectrum therapies, that may represent a novel class of therapeutics. As described by Obrecht et al.,82 PEM molecules can mimic the beta-hairpin and alpha-helical secondary structure elements often observed in proteinprotein interactions. In rapid iterative cycles that include design, synthesis, purication, and biological proling, optimized PEM molecules can be obtained with greatly improved Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties compared to the starting natural products. The PEM approach thus efciently generates fully synthetic compounds with drug-like properties, which display natural productlike structural complexity. A new structure-activity trail has provided access to derivatives with a much higher potency and selectivity against P. aeruginosa. One molecule, POL7080, showed potent in vitro activity against a broad panel of Pseudomonas strains (including multidrug-resistant clinical isolates from cystic brosis patients) with an MIC90 = 0.25 g/mL. In addition, POL7080, which is believed to perturb outer-membrane biogenesis, showed potent activity in an in vivo septicemia mouse model against P. aeruginosa PAO1 (EC50 = 0.3 mg/kg) and was 10 times more potent than gentamicin tested in the same model. These data demonstrate the potential utility of this new class of compound.83 Lead optimization Identication of a lead molecule is considered a milestone achievement although most leads require substantial chemistry (optimization) in order

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to achieve the potency, selectivity, spectrum, and pharmacokinetic properties necessary for an antibiotic. Traditional approaches to synthetic antibiotic drug design focused on incremental changes to lead molecules that altered the molecular structure to improve or reduce a particular feature, such as potency, spectrum, absorption, distribution, metabolism, or toxicity. With the discovery of three-dimensional crystal structures of essential antibacterial targets, X-ray crystallography now enables improved binding predictability associated with proposed modications to the molecule. A review of structure-based drug design using the bacterial ribosome crystal structure describes the signicant improvements in the potency and spectrum obtained for the oxazolidinones class of antibacterials.84 Antibiotic lead optimization has some advantages over other therapeutic areas in that validated in vitro assays and in vivo models mitigate a great deal of efcacy risk against the bacterial target at the time of drug candidate selection. On the other hand, the high plasma exposures needed for antibiotic efcacy create additional hurdles in optimizing candidate leads due to the difculties associated with achieving high systemic exposures, such as increased toxicity associated with off-target effects. High plasma exposures are required for antibiotics as they must cross multiple biological barriers in order to access their target, which generally resides within the bacterial cell. In comparison, targets for many other disease areas are represented by receptors or ligands that are outside of the cell. This is exemplied in Table 1, which illustrates the dose levels and therapeutic exposures needed for amoxicillin/clavulanate compared to rosiglitazone (treatment of type 2 diabetes) and sumatriptan (treatment of migraine headaches). With the need for relatively high plasma exposure for antibacterials comes the burden of both engineering acceptable
Table 1. Plasma concentrations associated with therapeutic use of selected marketed drugs

Drug (therapeutic dose) Amoxicillin/clavulanate (875/125 mg) Rosiglitazone (2 mg) Sumatriptan (25 mg)

Plasma Cmax ( g/mL) 11.6 (amoxicillin)85 0.15686,87 0.02888,89

ADME properties and increased potential for tolerability and toxicological effects. The maximum concentration (Cmax) of unbound drug associated with efcacious exposure for antibiotics is often 1,000-fold higher than for other therapeutic areas (antibiotic Cmax range is generally 110 uM). Even where the primary pharmacokinetic parameter for efcacy is not Cmax, antibiotics must still achieve plasma levels comparable to bacterial MIC levels in order to achieve efcacious exposures. Since antibacterial activity requires presence of unbound drug, it is not uncommon to nd antibacterials that are >50% unbound to plasma proteins (whereas 1% of unbound drug may be adequate for other therapeutic areas). Therefore, antibiotics can be associated with up to 50,000-fold greater free Cmax levels than other therapeutics. High free Cmax concentrations contribute to the potential drug-induced arrhythmia and electrocardiogram QT prolongation safety hurdles facing many antibiotic leads, because acute functional cardiovascular toxicity is generally related to Cmax. Over the past decade our understanding of certain drug related toxicities, in particular those related to cardiovascular anomalies, have substantially improved. Drug-induced QT prolongation, which currently serves as a biomarker for increased risk for torsade de pointes, has been the reason why several antibiotics have been rejected by regulatory agencies or withdrawn from the market.90 While such an outcome is warranted, it sacrices large investments, especially when this occurs after a full clinical program has been conducted. To better manage this risk, assays such as those for hERG ion channel effects, as well as other in silico, ex vivo, and in vitro models can be implemented during the lead optimization stage to deselect compounds that have potential cardiac ion channel risk.91,92 Lead optimization to reconcile all the required attributes for a drug candidate remains a highly challenging, resource-intensive, and time-consuming phase of drug discovery. Drug leads from established classes have to demonstrate improvements that adequately differentiate them from marketed drugs enough to warrant development investment. Leads from drug classes that exploit novel antibacterial targets have greater risk for encountering unanticipated class liabilities. Despite the widespread acceptance of the physical properties associated with drug-like molecules, analysis of current

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medicinal chemistry efforts, as reected in patent applications from AstraZeneca, GlaxoSmithKline, Merck and Co., and Pzer, indicates that recent compounds synthesized by pharmaceutical companies have been larger and more lipophilic than marketed oral drugs. Higher lipophilicity is recognized as a correlate to reduced pharmacological specicity and potentially increased risk of future attrition due to safety ndings during the drug optimization and development process.93,94 Improved choices and diversity of starting points for antibacterial lead optimization programs and greater chemical tractability of the classes selected would help this situation. However, lead optimization failure is also driven by incomplete target validation as described in an earlier section, the learning curve around toxicity prediction, and the need to avoid a narrow drive for molecular potency that can result in a level of lipophilicity that is more prone to off-target activity. The ability to predict clinical effectiveness and spectrum using in vitro and in vivo models is a distinct advantage in antibacterial lead optimization efforts over other therapeutics. Animal models of bacterial infection have substantially improved the ability to predict clinical efcacy of discovery stage antibiotics. Additionally, these models can be used to study the efcacy of a compound against strains that may be encountered infrequently in clinical studies.95,96 These models offer insight into the utility of a compound across a range of pathogens, infection sites, and resistance proles. Over the past two decades our understanding of the pharmacokinetic and pharmacodynamic (PK/PD) relationship of conventional antibiotics has become well established. Understanding the PK/PD properties that drive efcacy allows selection of a clinically relevant dose and dose regimen to optimize efcacy and mitigate against the selection of bacterial resistance.97,98 This is exemplied by a number of approved antibiotics including doripenem and linezolid for which the doses selected for use in Phase II/ III studies were based on animal derived PK/PD combined with human Phase I pharmacokinetic data.99,100 Animal models generally show excellent correlation with clinical outcome; however, these models do have some limitations, such as between animal and human physiology. For example, the lipoglycopeptide antibiotic daptomycin failed to meet the noninferiority criteria in severe communityacquired pneumonia trials.101 The antibiotic was

subsequently discovered to be inactivated by human lung surfactant, a phenomenon not clearly observed in animals.102 The activity of daptomycin is strictly dependent on the presence of physiological levels of free calcium. Daptomycin has a novel mechanism of action that results from insertion into and disruption of the functional integrity of the Gram-positive bacterial membrane. This produces a rapid loss of membrane potential, cessation of macromolecular synthesis, and cell death. The calcium dependence of daptomycinsurfactant interactions, the absence of surfactant inhibition of other classes of antibiotic, and the demonstration of direct insertion of daptomycin into surfactant aggregates in vitro all support a model whereby daptomycin in the lungs is unable to efciently distinguish between the vast surfactant layer and the relatively small surface area of the bacteria. Insertion into the surfactant layer is predicted to be an essentially irreversible process resulting in sequestration and inactivation of the antibiotic. This effect102 represents the rst example of a pulmonary-specic inactivation of an antibiotic. Other potential limitations include differences in drug concentrations at specic sites of infection, protein binding, and immune response. Local tissue concentrations at the site of infection may differ between animals and humans. Decades of experience gained across these models, however, have minimized this risk by showing good correlation using blood levels with clinical outcome.95 Differences in protein binding can also be mitigated by using the free drug concentrations to compare blood concentration levels between species.96 While recognizing the potential limitations of the animal models, there remains a high level of condence that these models provide insight to potential human efcacy. Strategies to address the innovation gap There is now high-level political, medical, and public concern about the perfect storm of increasing antibiotic resistance and the thin industry pipeline. For example, in 2009 the U.S. and European Community Presidencies have established a Transatlantic Task Force to address antimicrobial resistance,103 and the Infectious Diseases Society of America (IDSA) has announced a call to action for a global commitment to develop 10 novel antibacterial drugs by 2020 (10 20).104 Recognition of the challenge is also evident in publicprivate partnerships,

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including government agencies and charities (e.g., Wellcome Trust, Bill and Melinda Gates Foundation) to address the public health threat from conventional and weaponizable pathogens, a sign of the importance and difculty of the challenge. However, while incentives for antibacterial drug development and optimization of the regulatory environment are key, the principal barrier is technological. The technical challenges we described in 2007 remain.8 The ability of new antibiotics to overcome bacterial resistance is enhanced when more novel approaches are pursued; however, greater novelty introduces more technical and nancial risks. Furthermore, efforts at the earliest stages of drug discovery, which employ the newest technologies, can require 1215 years of effort before a licensed antibacterial drug becomes available on the market. Development of new-class antibacterial agents is imperative, but nancial and scientic considerations also drive a nearer term strategy to augment the antibacterial armamentarium by continued exploitation of proven antibacterial classes. This in some ways reduces the technical challenge, but on the other hand only offers more incremental therapy improvements. Challenges with identifying tractable new targets, coupled with the poor performance of HTS to generate leads for new antibacterial targets, has driven the search for screening methodologies that use greater compound diversity and novel assay designs, as well as innovative approaches to nding new targets. Promising discovery strategies include improvements in the diversity of compound collections for screening, such as prefractionated natural product libraries, improved synthetic diversity libraries, DNA-encoded small molecule libraries, new chemical platforms such as boron chemistry, and fragment-based screening. Back to the future use of whole cell antibacterial screening with reverse genomics to dene molecular targets is an attractive strategy, and also some industry successes have been reported in exploiting new narrow-spectrum and broad-spectrum molecular targets. Recognizing that not all targets have equal potential to yield drugs, pursuing novel inhibitors of validated targets, such as ribosomes and topoisomerases, can overcome existing resistance mechanisms, while reducing early stage discovery risk.

Acknowledgments We thank James R. Brown, Michael H. Hann, Jianzhong Huang, Andrew R. Leach, and Richard Jarvest for valuable input, and Ricardo Macarron and David Drewry for valuable input and generous access to prepublication information. Conicts of interest The authors are employees of GSK. References
1. Fischbach, M.A. & C.T. Walsh. 2009. Antibiotics for emerging pathogens. Science 325: 10891093. 2. Infectious Diseases Society of America. 2004. Bad bugs, no drugs: as antibiotic R&D stagnates, a public health crisis brews, http://www.idsociety.org/ (accessed October 23, 2010). 3. Talbot, G.H., J. Bradley Jr., J.E. Edwards, et al. 2006. Bad bugs need drugs: an update on the development pipeline from the antimicrobial availability task force of the Infectious Diseases Society of America. Clin. Infect. Dis. 42: 657668. 4. Fernandes, P. 2006. Antibacterial discovery and developmentthe failure of success? Nat. Biotechnol. 24: 14971503. 5. Lange, R.P., H.H. Locher, C.P. Wyss, et al. 2007. The targets of currently used antibacterial agents: lessons for drug discovery. Curr. Pharm. Design 13: 31403154. 6. Royal Society Policy Document. 2008. Innovative mechanisms for tackling antibacterial resistance, http:// royalsociety.org/Innovative-mechanisms-for-tacklingantibacterial-resistance/ (accessed October 23, 2010). 7. Spellberg, B., J.H. Powers, E.P. Brass, et al. 2004. Trends in antimicrobial drug development: implications for the future. Clin. Infect. Dis. 38: 12791286. 8. Payne, D.J., M.N. Gwynn, D.J. Holmes, et al. 2007. Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nat. Rev. Drug Discov. 6: 2940. 9. IDSA. 2008. Challenges in the pathway to antibiotic approvals. http://www.idsociety.org/WorkArea/ DownloadAsset.aspx?id=12134 (accessed October 23, 2010). 10. DiMasi, J.A., R.W. Hansen & H.G. Grabowski. 2003. The price of innovation: new estimates of drug development costs. J. Health Econ. 22: 151185. 11. Schurek, K.N., R. Wiebe, J.A. Karlowsky, et al. 2007. Faropenem: review of a new oral penem. Expert Rev. AntiInfect. 5: 185198. 12. Theuretzbacher, U. 2009. Future antibiotics scenarios: is the tide starting to turn? Int. J. Antimicrob. Agents 34: 15 20. 13. Mitscher, L.A. 2005. Bacterial topoisomerase inhibitors: quinolone and pyridone antibacterial agents. Chem. Rev. 105: 559592. 14. Black, M.T., T. Stachyra, D. Platel, et al. 2008. Mechanism of action of the antibiotic NXL101, a novel

16

Ann. N.Y. Acad. Sci. 1213 (2010) 519 c 2010 New York Academy of Sciences.

Gwynn et al.

Challenges of antibacterial discovery

15.

16.

17.

18.

19.

20.

21. 22.

23.

24.

25.

26.

27.

28.

29. 30.

31.

nonuoroquinolone inhibitor of bacterial type II topoisomerases. Antimicrob. Agents Chemother. 52: 3339 3349. Bax, B.D., P.F. Chan, D.S. Eggleston, et al. 2010. Type IIA topoisomerase inhibition by a new class of antibacterial agents. Nature 466: 935940. Miller A.A., G.L. Bundy, J.E. Mott, et al. 2008. Discovery and characterization of QPT-1, the progenitor of a new class of bacterial topoisomerase inhibitors. Antimicrob. Agents Chemother. 8: 28062812. Novak R. & D.M. Shlaes. 2010. The pleuromutilin antibiotics: a new class for human use. Curr. Opin. Invest. Drugs 11: 182191. Ochsner, U.A., X. Sun, T. Jarvis, et al. 2007. AminoacyltRNA synthetases: essential and still promising targets for new anti-infective agents. Expert Opin. Inv. Drug. 16: 573 593. Gilbert, J., P. Henske & A. Singh. 2003. Rebuilding big pharmas business model. Business Med. Rep. http://www. bain.com/bainweb/PDFs/cms/Marketing/rebuilding big pharma.pdf (accessed October 23, 2010). Hughes, D. 2003. Exploiting genomics, genetics and chemistry to combat antibiotic resistance. Nat. Rev. Genet. 4: 432441. NCBI Genome Project. http://www.ncbi.nih.gov/genomes/ lproks.cgi (accessed October 23, 2010). Lock, R.L., & E.J. Harry. 2008. Cell-division inhibitors: new insights for future antibiotics. Nat. Rev. Drug Discov. 7: 324338. Gentry, D.R., K.A. Ingraham, M.J. Stanhope, et al. 2003. Variable sensitivity to bacterial methionyl-tRNA synthetase inhibitors reveals subpopulations of Streptococcus pneumoniae with two distinct methionyl-tRNA synthetase genes. Antimicrob. Agents Chemother. 47: 17841789. Payne, D.J., W.H. Miller, V. Berry, et al. 2002. Discovery of a novel and potent class of FabI-directed antibacterial agents. Antimicrob. Agents.Chemother. 46: 3118 3124. Du, W., J.R. Brown, D.R. Sylvester, et al. 2000. Two active forms of UDP-N-Acetylglucosamine enolpyruvyl transferase in Gram-positive bacteria. J. Bacteriol. 182: 4146 4152. Harris, S.R., E.J. Feil, M.T.G. Holden, et al. 2010. Evolution of MRSA during hospital transmission and intercontinental spread. Science 327: 469474. Brinster, S., G. Lamberet, B. Staels, et al. 2009. Type II fatty acid synthesis is not a suitable antibiotic target for Gram-positive pathogens. Nature 458: 8386. Balemans, W., N. Lounis, R. Gilissen, et al. 2010. Essentiality of FASII pathway for Staphylococcus aureus [arising from Nature, 2009, 458: 8386]. Nature 463: E3. Brinster, S., G. Lamberet, B. Staels, et al. 2010. Replying to W. Balemans et al. 2009, Nature 463: E3. Nature 463: E4. Blundell, T.L., H. Jhoti & C. Abell. 2002. High-throughput crystallography for lead discovery in drug design. Nat. Rev. Drug Discov. 1: 4554. Arkin, M.R. & J.A. Wells. 2004. Small-molecule inhibitors of protein-protein interactions: progressing towards the dream. Nat. Rev. Drug Discov. 3: 301317.

32. Haydon, D.J., N.R. Stokes, R. Ure, et al. 2008. An inhibitor of FtsZ with potent and selective anti-staphylococcal activity. Science 321: 16731675. 33. Silver, L.L. 2008. Does the cell wall of bacteria remain a viable source of targets for novel antibiotics. Biochem. Pharmacol. 71: 9961005. 34. Stachyra, T., P. Levasseur, M.-C. P chereau, et al. 2009. e In vitro activity of the -lactamase inhibitor NXL104 against KPC-2 carbapenemase and Enterobacteriaceae expressing KPC carbapenemases. J. Antimicrob. Chemoth. 64: 326329. 35. Nikaido, H. 1994. Prevention of drug access to bacterial targets: permeability barriers and active efux. Science 264: 382388. 36. Page, M.G.P. & J. Heim. 2009. Prospects for the next antiPseudomonas drug. Curr. Opin Pharmacol. 9: 558565. 37. Silver, L.L. & K.A. Bostian. 1993. Discovery and development of new antibiotics: the problem of antibiotic resistance. Antimicrob. Agents Chemother. 37: 377383. 38. Gualtieri, M., F. Baneres-Roquet, P. Villain-Guillot, et al. 2009. The antibiotics in the chemical space. Curr. Med. Chem. 16: 390393. 39. Drewry, D.H. & R. Macarron. 2010. Enhancements of screening collections to address areas of unmet medical need: an industry perspective. Curr. Opin. Chem. Biol. 14: 289298. 40. Hann, M.M., A.R. Leach & G. Harper. 2001. Molecular complexity and its impact on the probability of nding leads for drug discovery. J. Chem. Inf. Model. 41: 856 864. 41. Snowden, M.A. & D.V.S. Green. 2008. The impact of diversity-based, high-throughput screening on drug discovery: chance favours the prepared mind. Curr. Opin. Drug Dis. Dev. 11: 553558. 42. Tan, D.S. 2005. Diversity oriented synthesis: exploring the intersections between chemistry and biology. Nat. Chem. Biol. 1: 7484. 43. Galloway, W.R.J.D., A. Bender, M. Welch & D.R. Spring. 2009. The discovery of antibacterial agents using diversityoriented synthesis. Chem. Commun. 18: 24462462. 44. Jacoby, E. & A. Mozzarelli. 2009. Chemogenomic strategies to expand the bioactive chemical space. Curr. Med. Chem. 16: 43744381. 45. Newman, D.J. & G.M. Cragg. 2007. Natural products as sources of new drugs over the last 25 years. J. Nat. Products 70: 461477. 46. Butler, M.S. & A.D. Buss. 2006. Natural productsthe future scaffolds for novel antibiotics? Biochem. Pharmacol. 71: 919929. 47. Pelaez, F. 2006. The historical delivery of antibiotics from microbial natural productscan history repeat? Biochem. Pharmacol. 71: 981990. 48. Li, J.W.H. & J. C. Vederas. 2009. Drug discovery and natural products: end of an era or an endless frontier? Science 325: 161165. 49. Feher, M. & J.M. Schmidt. 2003. Property distributions: differences between drugs, natural products, and molecules from combinatorial chemistry. J. Chem. Inf. Model. 43: 218227.

Ann. N.Y. Acad. Sci. 1213 (2010) 519 c 2010 New York Academy of Sciences.

17

Challenges of antibacterial discovery

Gwynn et al.

50. Koehn, F.E. & G.T. Carter. 2005. The evolving role of natural products in drug discovery. Nat. Rev. Drug Discov. 4: 206220. 51. Baltz, R.H. 2008. Renaissance in antibacterial discovery from actinomycetes. Curr. Opin. Pharmacol. 8: 557563. 52. Chetan S.C.K., J.L. Jenkins, R.E.J. Beckwith, et al. 2009. Plate-based diversity selection based on empirical HTS data to enhance the number of hits and their chemical diversity. J. Biomol. Screen. 14: 690699. 53. Nielsen, F.H. 2009. Micronutrients in parenteral nutrition: boron, silicon, and uoride. Gastroenterology 137: S55S60. 54. Baker, S.J., C.Z. Ding, T. Akama, et al. 2009. Therapeutic potential of boron-containing compounds. Future Med. Chem. 1: 12751288. 55. Systemic Anti-Infectives Pipeline. Anacor Pharmaceuticals. 2009. http://www.anacor.com/ (accessed October 23, 2010). 56. Rock, F.L., W. Mao, A. Yaremchuk, et al. 2007. An antifungal agent inhibits an aminoacyl-tRNA synthetase by trapping tRNA in the editing site. Science 316: 17591761. 57. Seiradake, E., W. Mao, V. Hernandez, et al. 2009. Crystal structures of the human and fungal cytosolic leucyl-tRNA synthetase editing domains: a structural basis for the rational design of antifungal benzoxaboroles. J. Mol. Biol. 390: 196207. 58. Clark, M.A., R.A. Acharya, C.C. Arico-Muendel, et al. 2009. Design, synthesis and selection of DNA-encoded smallmolecule libraries. Nat. Chem. Biol. 5: 647654. 59. Chessari, G. & A.J. Woodhead. 2009. From fragment to clinical candidatea historical perspective. Drug Discov. Today 14: 668675. 60. Mochalkin, I., J.R. Miller, L. Narasimhan, et al. 2009. Discovery of antibacterial biotin carboxylase inhibitors by virtual screening and fragment-based approaches. ACS Chem. Biol. 4: 473483. 61. Brotz-Oesterhelt, H., D. Beyer, H.-P. Kroll, et al. 2005. Dysregulation of bacterial proteolytic machinery by a new class of antibiotics. Nat. Med. 11: 10821087. 62. Gentry, D.R., I. Wilding, J.M. Johnson, et al. 2010. A rapid microtiter plate assay for measuring the effect of compounds on Staphylococcus aureus membrane potential. J. Microbiol. Methods. 83: 254256. 63. Stokes, N.R., J. Sievers, S. Barker, et al. 2005. Novel inhibitors of bacterial cytokinesis identied by a cell-based antibiotic screening assay. J. Biol. Chem. 280: 3970939715. 64. Wang, J., S.M. Soisson, K. Young, et al. 2006. Platensimycin is a selective FabF inhibitor with potent antibiotic properties. Nature 441: 358361. 65. Miesel, L., J. Greene & T.A. Black. 2003. Genetic strategies for antibacterial drug discovery. Nat. Rev. Genet. 4: 442 456. 66. Andries, K., V. Peter, J. Guillemont, et al. 2005. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 307: 223227. 67. Pathania, R., S. Zlitni, C. Barker, et al. 2009. Chemical genomics in Escherichia coli identies an inhibitor of bacterial lipoprotein targeting. Nat. Chem. Biol. 5: 849856. 68. Miller, J.R., S. Dunhama, I. Mochalkin, et al. 2009. A class of selective antibacterials derived from a protein kinase

69. 70.

71.

72.

73. 74. 75.

76.

77.

78.

79.

80.

81.

82.

83.

84.

85.

inhibitor pharmacophore. Proc. Natl. Acad. Sci. USA 106: 17371742. Poole, K. 2007. Efux pumps as antimicrobial resistance mechanisms. Ann. Med. 39: 162176. Lomovskaya, O., H.I. Zgurskaya, M. Totrov & W.J. Watkins. 2007. Waltzing transporters and the dance macabre between humans and bacteria. Nat. Rev. Drug Discov. 6: 56 66. Lomovskaya, O. & K.A. Bostian. 2006. Practical applications and feasibility of efux pump inhibitors in the clinic a vision for applied use. Biochem. Pharmacol. 71: 910 918. Surawicz, C.M. 2005. Antibiotic-associated diarrhea and pseudomembranous colitis: are they less common with poorly absorbed antimicrobials? Chemotherapy 51(Suppl. 1): 8189. Kelly, C.P., C. Pothoulakis & J.T. LaMont. 1994. Clostridium difcile colitis. N. Engl. J. Med. 330: 257262. Bartlett, J.G. 1992. Antibiotic-associated diarrhea. Clin. Infect. Dis. 15: 573579. Bouza, E., A. Burillo & P. Munoz. 2006. Antimicrobial therapy of Clostridium difcile-associated diarrhea. Med. Clin. North Am. 90: 11411163. Credito, K.L. & P.C. Appelbaum. 2004. Activity of OPT80, a novel macrocycle, compared with those of eight other agents against selected anaerobic species. Antimicrob. Agents Chemother. 48: 44304434. Finegold, S.M., D. Molitoris, M.-L. Vaisanenet, et al. 2004. In vitro activities of OPT-80 and comparator drugs against intestinal bacteria. Antimicrob. Agents Chemother. 48: 48984902. Larson, H.E. & S.P. Borriello. 1990. Quantitative study of antibiotic-induced susceptibility to Clostridium difcile enterocecitis in hamsters. Antimicrob. Agents Chemother. 34: 13481353. Shue, Y.K., P.S. Sears, S. Shangle, et al. 2008. Safety, tolerance, and pharmacokinetic studies of OPT-80 in healthy volunteers following single and multiple oral doses. Antimicrob. Agents Chemother. 52: 13911395. Lowry, I., D.C. Molrine, B.A. Leav, et al. 2010. Treatment with monoclonal antibodies against Clostridium difcile toxins. N. Engl. J. Med. 362: 197205. Bebbington, C. & G. Yarranton. 2008. Antibodies for the treatment of bacterial infections: current experience and future prospects. Curr. Opin. Biotech. 19: 613619. Obrecht, D., J.A. Robinson, F. Bernardini, et al. 2009. Recent progress in the discovery of macrocyclic compounds as potential anti-infective therapeutics. Curr. Med. Chem. 16: 4265. Srinivas, N., P. Jetter, B.J. Ueberbacher, et al. 2010. Peptidomimetic antibiotics target outer-membrane biogenesis in Pseudomonas aeruginosa. Science 327: 1010 1013. Wimberly, B.T. 2009. The use of ribosomal crystal structures in antibiotic drug design. Curr. Opin. Invest. Drugs 10: 750765. Augmentin label approved 12/04/2008. 2008. FDA.gov http://www.accessdata.fda.gov/drugsatfda docs/label/2008 /050564s050lbl.pdf (accessed October 23, 2010).

18

Ann. N.Y. Acad. Sci. 1213 (2010) 519 c 2010 New York Academy of Sciences.

Gwynn et al.

Challenges of antibacterial discovery

86. Avandia label approved 10/20/2008. 2008. FDA.gov http://www.accessdata.fda.gov/drugsatfda docs/label/2008 /021071s034lbl.pdf (accessed October 23, 2010). 87. Werner, A.L. & M.T. Travaglini. 2001. A review of rosiglitazone in type 2 diabetes mellitus. Pharmacotherapy 21: 10821099. 88. Imitrex label approved 10/13/2004. 2004. FDA.gov http://www.accessdata.fda.gov/drugsatfda docs/label/2004 /20626s004lbl.pdf (accessed October 23, 2010). 89. Perry, C.M. & M. Anthony. 1998. Sumatriptan. An updated review of its use in migraine. Drugs 55: 889922. 90. Lasser, K.E., P.D. Allen, S.J. Woolhandler, et al. 2002. Timing of new black box warnings and withdrawals for prescription medications. J. Am. Med. Assoc. 287: 2215 2220. 91. Raschi, E., L. Ceccarini, F. De Ponti & M. Recanatini. 2009. hERG-related drug toxicity and models for predicting hERG liability and QT prolongation. Expert Opin. Drug Metab. Toxicol. 5: 10051021. 92. Chaudhary, K.W. & B.S. Brown. 2010. Assessment of strategies utilized to minimize the potential for induction of acquired long QT syndrome and torsade de pointes. In Evaluation of Drug Candidates for Preclinical Development: Pharmacokinetics, Metabolism, Pharmaceutics, and Toxicology, Vol. 10. C. Han, B.D. Charles & B. Wang, Eds.: 253278. John Wiley & Sons, Inc. Hoboken. 93. Leeson, P.D. & A.M. Davis. 2004. Time-related differences in the physical property proles of oral drugs. J. Med. Chem. 47: 63386348. 94. Leeson, P.D. & B. Springthorpe. 2007. The inuence of drug-like concepts on decision-making in medician chemistry. Nat. Rev. Drug Discov. 6: 881890. 95. Czock, D., B. Hartman & F. Keller. 2009. Pharmacokinet-

96.

97.

98.

99.

100.

101.

102.

103.

104.

ics and pharmacodynamics of antimicrobial drugs. Expert Opin. Drug Metab. Toxicol. 5: 475487. Andes, D. & W.A. Craig. 2002. Animal model pharmacokinetics and pharmacodynamics: a critical review. Int. J. Antimicrob. Agents 19: 261288. Courvalin, P. 2008. Can pharmacokineticpharmacodynamic parameters provide dosing regimens that are less vulnerable to resistance? Clin. Microbiol. Infect. 14: 989 994. Drusano, G.L., S.L. Preston, C. Hardalo, et al. 2001. Use of preclinical data for selection of a phase II/III dose for evernimicin and identication of a preclinical MIC breakpoint. Antimicrob. Agents Chemother. 45: 1322. Bhavnani, S.M., J.P. Hammel, B.B. Cirincione, et al. 2005. Use of pharmacokinetic-pharmacodynamic target attainment analyses to support phase 2 and 3 dosing strategies for doripenem. Antimicrob. Agents Chemother. 49: 39443947. Andes, D., M.L. van Ogtrop, J. Peng, et al. 2002. In vivo pharmacodynamics of a new oxazolidinone (linezolid). Antimicrob. Agents Chemother. 46: 34833489. Pertel, P.E., P. Bernardo, C. Fogarty, et al. 2008. Effects of prior effective therapy on the efcacy of daptomycin and ceftriaxone for the treatment of community-acquired pneumonia. Clin. Infect. Dis. 46: 11421151. Silverman, J.A., L.I. Mortin, A.D.G. VanPraagh, et al. 2005. Inhibition of daptomycin by pulmonary surfactant: in vitro modeling and clinical impact. J. Infect. Dis. 191: 21492152. Guidos, R.J. 2009. New Transatlantic Task Force on Antimicrobial Resistance: A Path Forward http://www .cgdev.org/content/general/detail/1423276/ (accessed October 23, 2010). Infectious Diseases Society of America. Bad Bugs, No Drugs . . . 10 New Antibiotics by 2020, http://www. idsociety.org (accessed October 23, 2010).

Ann. N.Y. Acad. Sci. 1213 (2010) 519 c 2010 New York Academy of Sciences.

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