Challenges of Antibacterial Discovery - PMC

Summary: The discovery of novel small-molecule antibacterial drugs has been stalled for many years. The purpose of this review is to underscore and illustrate those scientific problems unique to the discovery and optimization of novel antibacterial agents that have adversely affected the output of the effort. The major challenges fall into two areas: (i) proper target selection, particularly the necessity of pursuing molecular targets that are not prone to rapid resistance development, and (ii) improvement of chemical libraries to overcome limitations of diversity, especially that which is necessary to overcome barriers to bacterial entry and proclivity to be effluxed, especially in Gram-negative organisms. Failure to address these problems has led to a great deal of misdirected effort.

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The antibacterial product pipeline has not been empty during this time but has been filled with improved versions of previously registered classes. Many of these were true improvements, adding bacterial spectrum, safety, simpler dosing regimens, and most importantly, activity insusceptible to specific resistance mechanisms acting on the parent compound. A number of drugs with improved activity against resistant pathogens, such as oritavancin, iclaprim, and ceftobiprole, have reached the FDA but have met with regulatory problems, largely due to inadequacy of trials in proving noninferiority. Telavancin has been approved, and development of ceftaroline continues. Finding a derivative with an exploitable advantage over the original drug is not an easy path but is a process whose starting material has acceptable pharmacological properties and whose modification may be approached rationally. While the same caveats of meeting pharmacological and toxicological standards apply to this effort as to novel drug discovery, the leap is much greater for novel discovery, as it requires that the leads meet a tremendous number of criteria.

It is due in large part to this discovery void that Big Pharma has been withdrawing from research in the area, even though there has certainly been recognition of the continuing need for new antibacterials to combat the rise of resistant organisms.

Walsh has noted that “no major classes of antibiotics were introduced” between 1962 and 2000 and refers to the interim as an innovation gap ( 115 , 378 ). This understates the problem. The latest registered representatives of novel antibacterial classes, linezolid, daptomycin, and the topical agent retapamulin, were indeed introduced in 2000, 2003, and 2007, respectively, but these chemical classes (oxazolidinones, acid lipopeptides, and pleuromutilins) were first reported (or patented) in 1978 ( 124 ), 1987 ( 86 ), and 1952 ( 275 ), respectively. A timeline (Fig. ) of dates of discovery of distinct classes of antibacterials (as opposed to dates of introduction) illustrates that there have been no (as yet) successful discoveries of novel agents since 1987. There is a discovery void of unknown extent rather than a gap. While there are a small number of novel compounds in the early clinical phase that might portend the end of this hiatus, in most cases their eventual developmental success is unclear. Is the void due to a lack of innovation? While the simple definition of innovation is the act of introducing something new, the word implies creativity, intent, and experimentation. Almost all the discoveries shown in Fig. (with the exception of trimethoprim, monobactams, fosfomycin, and carbapenems) were serendipitous, made by screening fermentation products or chemicals for inhibition of bacterial growth (empirical screening). Not especially innovative, but it worked. In fact, since those last discoveries in the 1980s, there has been a great deal of creative, rational, technologically cutting-edge screening for and efforts at design of new antibacterials. But so far, little has reached serious development. The problem with the conduct of antibacterial discovery since the early 1980s is not a lack of innovation.

The challenges to antibacterial discovery have kept the output of novel antibacterial drug classes to extraordinarily low levels over the past 25 years, even though discovery programs have been in place at large and small pharmaceutical companies as well as academic laboratories over this period. This review focuses on the scientific challenges to the discovery of novel small-molecule antibacterials rather than on the commercial and regulatory considerations, which are well covered in a number of reviews ( 186 , 197 , 301 , 303 , 345 ). Rate-limiting steps to the discovery process are discussed, and some perspective on avenues to address those limitations is offered. An underlying thesis of this review is that the bleak picture of antibacterial discovery is due to an expenditure of effort and resources on non-rate-limiting steps of the process. While it is easy to find compounds that kill bacteria, it is hard to find novel antibacterial classes worthy of development. If new molecular entities with desirable properties and specificity had been discovered commonly throughout the past 25 years, it seems likely that large pharmaceutical companies (Big Pharma) would have viewed the area as productive and continued with antibacterial discovery. Even if unlimited money were poured into discovery and problematic regulatory guidelines were improved and stabilized, then it is probable that novel discovery would still be stymied because scientific obstacles remain to be overcome.

If success as a monotherapeutic is indeed due to multitargeting (or targets encoded by multiple genes) and single-targeted agents, prone as they are to single-step mutation to target-based resistance ( 347 , 388 ), are not optimal for monotherapy, then there are grave implications for antibacterial discovery. The impact of endogenous resistance (that occurring by antibiotic selection in the pathogen) on antibacterial drug discovery and development is covered below.

It remains a hypothesis that the low potential for target-based resistance is causally related to the success of multitarget agents in monotherapy. This is supported by the inverse, that most single targeted antibacterials in the clinic are indeed subject to single-step high-level resistance selection and are used as part of combination therapies, especially in therapy of M. tuberculosis or as topical agents (see Tables 3 and 4 of reference 337 ). Of course, all antibacterials with even moderate spectra have multiple homologous targets in that they must inhibit enzymes or bind to structures that are present and varied among the bacterial species of that spectrum.

The clinically used agents that target rRNA in their inhibition of protein synthesis provide another avenue of support for the hypothesis. These include macrolides, lincosamides, chloramphenicol, oxazolidinones, tetracyclines, aminoglycosides, and pleuromutilins (the last is not included in Table because it is not yet used systemically). They are useful in monotherapy against organisms that contain multiple copies of rRNA genes. Against the slow-growing mycobacteria, however, which contain only a single rRNA cistron ( 36 , 188 ), they are used in combination with other agents because single base changes in the rRNA gene lead to high-level resistance. With Helicobacter pylori, which contains 2 rRNA cistrons ( 55 ), clarithromycin resistance arises during therapy ( 2 , 376 ), and heterozygous strains display a resistant phenotype ( 376 ). Of course, anti-H. pylori therapy generally involves 2 or more antibacterial agents, although not strictly due to resistance development. For enterococci, it has been shown that MICs of linezolid-resistant isolates are highly correlated with the percentage of rRNA cistrons mutated ( 237 ). In a way similar to the rRNA case, the FQs which have dual targets in standard pathogens have only a single target (DNA gyrase) in both Mycobacterium tuberculosis and H. pylori ( 160 ) and do yield to single-step resistance ( 178 , 195 ).

Only two of the commonly used antibacterial classes actually target multiple different enzymes in a given species. The beta-lactams target the penicillin binding proteins (PBPs) ( 34 , 93 , 130 , 348 ), and the fluoroquinolones (FQs) target the catalytic subunit of DNA gyrase (GyrA) and topoisomerase IV (ParC) ( 65 , 112 , 189 ). The multitarget hypothesis was offered before the second target of the FQs, topoisomerase IV, was recognized ( 112 , 189 ). The FQs had appeared to be an exception to the rule ( 340 ), since resistance to the FQs was not extensive in the clinic by the early 1990s. This finding thus served to support the hypothesis.

The fact that successful systemic antibacterials have multiple molecular targets or targets encoded by multiple genes has been evident for the past 20 years ( 50 , 73 , 204 , 337 , 339 , 340 , 347 ). This is illustrated in Table , where the currently used systemic monotherapeutic agents and their targets are listed. These antibacterials are not subject to high-level target-based resistance by single genetic changes in the host. The hypothesis is that these agents are successful monotherapeutics and not subject to such resistance because they are multitargeted ( 339 , 340 ).

Many challenges to candidate selection and subsequent development of antibacterials, including pharmacological properties, pharmacokinetic/pharmacodynamic (PK/PD) analysis, and toxicities (both mechanism and chemistry-based), are common to all drug discovery. They have been approached, addressed, and overcome by more-standardized medicinal chemistry magic for many generations of successful antibacterials and other human health drugs and are addressed only minimally in this review.

In regard to target selection, the emphasis here on the importance of choosing targets by their low propensity for rapid resistance selection may seem a narrow view of the problem. There are a number of other important considerations involved in choosing specific targets for rational antibacterial discovery projects. These include (i) essentiality to the organism of the function, enzyme, or structure so that inhibition of enzyme action or blockage of the function leads to inhibition of bacterial growth or, better, death; (ii) conservation of structure of the target enzyme across bacterial species sufficient to provide a useful antibacterial spectrum; (iii) a lack of structural homology with the same or similar functions in the mammalian host in order to avoid mechanism-based toxicity; and (iv) in common with other areas of human drug discovery, “druggability” of the chosen target, in that there should exist sites on the target enzyme or structure that small drug-like molecules can bind to and, in so doing, exert a biological effect. These are important considerations and, in practice, generally lead to the selection of single enzymes as targets to pursue. However, one of the theses of this review is that single enzymes may not make good antibacterial targets due to their potential for rapid resistance development. This possibility has largely been neglected in the course of recent antibacterial discovery, to its detriment, and thus it is spotlighted here.

The purpose of this review is to underscore and illustrate some of those problems unique to the discovery and optimization of novel antibacterial agents that have adversely affected the output of the effort over the past 20 years. These are the rate-limiting steps of the antibacterial discovery process and can be divided into two main areas: (i) proper target selection, specifically the necessity of pursuing molecular targets that are not prone to rapid resistance development; and (ii) limitation of chemical diversity, especially that which is necessary to overcome barriers to bacterial entry and proclivity to be effluxed, especially in Gram-negative organisms.

The direction of novel antibacterial discovery research (as opposed to that of improving upon established classes) in the past 20 years has been to deploy an array of new technologies, based on genomics, bioinformatics, structural biology, and various high-throughput methods, in an effort to transform the giant leap of novel discovery into doable quantum steps. Indeed, the allure of the rational, engineering-oriented, stepwise application of techniques to make the discovery process a turnkey system is understandable. The concept has been to define broad-spectrum (or more species-specific) targets, screen for or design inhibitors, and then hope to address the subsequent obstacles of bacterial entry, non-mechanism-based toxicity, serum binding, pharmacokinetics, etc., in a piecemeal manner. But this approach has apparently not worked.

To summarize, after the Golden Age, antibacterial discovery became target oriented and largely abandoned natural product sources. Big Pharma evidently weighed the costs of maintaining the resources for natural product programs against the low probability of useful output and opted for the synthetic chemical route. Targets were pursued first as a means of dereplication in natural product screening and later to provide a rational basis for discovery and as a route to avoiding cross-resistance with other drugs, as discussed below.

Natural product screening (at least for novel antibacterials) waned with the low output of good leads, the advent of high-throughput liquid handling-based screening methods, for which crude microbial fermentation broths are a poor fit (since they require labor- and time-intensive culture isolation, fermentation, and extraction to produce a relatively limited number of samples), and the rise in the screening of chemical libraries, especially combinatorial chemicals. Antibacterial discovery largely became limited to screening these chemical libraries. This was not a fruitful source, as discussed below.

Much of early industrial antibacterial screening was carried out by cohesive groups that did natural product fermentation and both designed and ran the screens. The scientific direction and prioritization of resources were done within the group. But changes in the pharmaceutical industry led, in many cases, to a modularized system that is still more or less in effect. Drug discovery programs for different therapeutic areas (such as infectious diseases, cardiology, oncology, immunology, etc.) are generally organized such that biology and sometimes chemistry are committed to that area, but other functions (screening, animal testing, pharmacology, structural biology, etc.) may be shared. Since resources are always limiting, their allocation became a relatively high-level management decision (often at a remove from bench science), weighing the value to the company of a therapeutic area, the probability of success, the proximity to the “cutting edge” of current technology, and the ability of the scientists and their managers to push specific programs. For example, antibacterial discovery groups had to compete with other therapeutic areas for the opportunity (a so-called “slot”) to screen natural products. The awarding of natural product screening slots came to be based on the perceived attractiveness of the target and its amenability to downstream biochemical and physical analysis. Those antibacterial screens designed to find primarily novelty (over inhibitors of a specific target) were often given low priority. It could be argued that finding novelty is the goal of natural product screening for antibacterials and that concentration on a small number of preselected “desirable” targets (for which inhibitors might or might not be present) is an inefficient use of the natural product resource. Screening strategies for novelty among natural products are noted in a later section.

Importantly, in 1977, at a time when the output of novel antibiotic classes had decreased, the low-hanging fruit having been found, Cohen proposed rational chemotherapy of infectious organisms through a search for inhibitors of specific enzymes in the target organism ( 77 ). This, along with the growing ability to clone genes and manipulate bacterial strains to enhance whole-cell phenotypic screens for inhibitors of specific targets (and eventually allow the production of purified proteins which could be used for in vitro screening and assays), turned the whole of antibacterial discovery toward more target-directed screens.

In an effort to make dereplication easier, starting by the early 1960s ( 126 ), screening methods were modified in order to limit the hits to subsets of all possible antibiotic compounds. For example, many screens were developed over the years to detect inhibitors of the pathway of peptidoglycan (cell wall) synthesis ( 126 , 278 , 333 ). Each time a hit in such a screen was detected, it could be compared for biological and chemical similarity to the previously discovered cell wall synthesis inhibitors. Thus, pathway- or rudimentary target-based screening arose in part for dereplication purposes but also because certain pathways (cell wall and protein synthesis) appeared to be common targets for useful antibiotics. Furthermore, it was early recognized that cell wall inhibition was a very selective antibacterial target. The only clinically useful antibacterial classes discovered through directed screening thus far (monobactams, carbapenems, and fosfomycin) were discovered in these cell wall pathway screens.

The earliest history of antibacterial chemotherapeutic discovery was via screening dyestuffs and other chemicals for selective antibacterial activity, yielding salvarsan and the sulfa drugs. When the folate pathway inhibited by the sulfas was better understood, more directed screening of pyrimidine derivatives and analogs for inhibition of the bacterial folate pathway produced trimethoprim, an inhibitor of dihydrofolate reductase ( 57 , 158 , 375 ). But the so-called “Golden Age” of antibacterial discovery involved screening of natural products, especially fermentation broths and extracts of microorganisms, simply for the ability to inhibit growth of bacterial organisms of interest (pathogens or surrogates), without regard to their mechanism of action. This has been termed empirical screening ( 71 , 342 ). Selectivity was generally tested in secondary assays of toxicity in animals. This worked admirably for a number of years, as the most common antibiotics (natural product-derived antibacterials) were discovered and rediscovered rapidly. The prevalence of production of “common” antibiotics among standard Actinomycetes has been estimated by Baltz ( 22 ). To efficiently discard such previously described compounds, methods of so-called “dereplication” were quickly developed to identify them ( 1 , 104 , 352 ).

The fosfomycin case raises the possibility that endogenous resistance to single-enzyme targets may be avoided if drug levels at the infection site can be kept high without toxicity and/or the mutants are unfit or of low virulence. Clearly, fosfomycin has been successful (although for a limited indication), but is the fosfomycin/MurA scenario more broadly applicable? Should single-enzyme targets be avoided altogether, or has in vitro analysis of resistance frequencies had an unnecessary chilling effect on discovery programs within industry? Has the awareness of the potential for resistance to single-target agents led to the early demise of programs that would otherwise have proceeded—to optimization or even to the clinic? This is a chastening thought.

Although most single-enzyme-targeted agents are used in combination or topically and thus avoid rapid endogenous resistance development, there are a few exceptions, such as fosfomycin ( 272 ), which has been used successfully (outside the United States) against urinary tract infections (UTIs). Why is there a lack of clinically relevant endogenous fosfomycin resistance? Fosfomycin targets UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), the first committed step of pept