David J. Payne

Drugs for bad bugs: confronting the challenges of antibacterial discovery

The sequencing of the first complete bacterial genome in 1995 heralded a new era of hope for antibacterial drug discoverers, who now had the tools to search entire genomes for new antibacterial targets. Several companies, including GlaxoSmithKline, moved back into the antibacterials area and embraced a genomics-derived, target-based approach to screen for new classes of drugs with novel modes of action. Here, we share our experience of evaluating more than 300 genes and 70 high-throughput screening campaigns over a period of 7 years, and look at what we learned and how that has influenced GlaxoSmithKlines antibacterials strategy going forward.

Alternatives to antibiotics—a pipeline portfolio review

Antibiotics have saved countless lives and enabled the development of modern medicine over the past 70 years. However, it is clear that the success of antibiotics might only have been temporary and we now expect a long-term and perhaps never-ending challenge to find new therapies to combat antibiotic-resistant bacteria. A broader approach to address bacterial infection is needed. In this Review, we discuss alternatives to antibiotics, which we defined as non-compound approaches (products other than classic antibacterial agents) that target bacteria or any approaches that target the host. The most advanced approaches are antibodies, probiotics, and vaccines in phase 2 and phase 3 trials. This first wave of alternatives to antibiotics will probably best serve as adjunctive or preventive therapies, which suggests that conventional antibiotics are still needed. Funding of more than £1·5 billion is needed over 10 years to test and develop these alternatives to antibiotics. Investment needs to be partnered with translational expertise and targeted to support the validation of these approaches in phase 2 trials, which would be a catalyst for active engagement and investment by the pharmaceutical and biotechnology industry. Only a sustained, concerted, and coordinated international effort will provide the solutions needed for the future.

Discovery of a Novel and Potent Class of FabI-Directed Antibacterial Agents

ABSTRACT Bacterial enoyl-acyl carrier protein (ACP) reductase (FabI) catalyzes the final step in each elongation cycle of bacterial fatty acid biosynthesis and is an attractive target for the development of new antibacterial agents. High-throughput screening of the Staphylococcus aureus FabI enzyme identified a novel, weak inhibitor with no detectable antibacterial activity against S. aureus. Iterative medicinal chemistry and X-ray crystal structure-based design led to the identification of compound 4 [(E)-N-methyl-N-(2-methyl-1H-indol-3-ylmethyl)-3-(7-oxo-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)acrylamide], which is 350-fold more potent than the original lead compound obtained by high-throughput screening in the FabI inhibition assay. Compound 4 has exquisite antistaphylococci activity, achieving MICs at which 90% of isolates are inhibited more than 500 times lower than those of nine currently available antibiotics against a panel of multidrug-resistant strains of S. aureus and Staphylococcus epidermidis. Furthermore, compound 4 exhibits excellent in vivo efficacy in an S. aureus infection model in rats. Biochemical and genetic approaches have confirmed that the mode of antibacterial action of compound 4 and related compounds is via inhibition of FabI. Compound 4 also exhibits weak FabK inhibitory activity, which may explain its antibacterial activity against Streptococcus pneumoniae and Enterococcus faecalis, which depend on FabK and both FabK and FabI, respectively, for their enoyl-ACP reductase function. These results show that compound 4 is representative of a new, totally synthetic series of antibacterial agents that has the potential to provide novel alternatives for the treatment of S. aureus infections that are resistant to our present armory of antibiotics.

Desperately Seeking New Antibiotics

New approaches for discovering the next generation of antibiotics are needed to combat the rise in bacteria that are resistant to current drugs.

Bacterial fatty-acid biosynthesis: a genomics-driven target for antibacterial drug discovery

In this review we demonstrate how the interplay of genomics, bioinformatics and genomic technologies has enabled an in-depth analysis of the component enzymes of the bacterial fatty-acid biosynthesis pathway as a source of novel antibacterial targets. This evaluation has revealed that many of the enzymes are potentially selective, broad-spectrum antibacterial targets. We also illustrate the suitability of some of these targets for HTS. Furthermore, we discuss how the availability of a robust selectivity assay, mode-of-action assays and numerous crystal structures provide an excellent set of tools with which to initiate integrated programs of research to identify novel antibiotics targeted at these enzymes.

Challenges of antibacterial discovery revisited

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 efficacy. Lead optimization would be enhanced by the identification of new antibiotic classes with improved tractability and by expanding the predictability of in vitro safety assays. Implementing multiple screening and target identification strategies is recommended for improving the likelihood of discovering new antibacterial compounds that address unmet needs.

1,4-Disubstituted imidazoles are potential antibacterial agents functioning as inhibitors of enoyl acyl carrier protein reductase (FabI)

1,4-Disubstituted imidazole inhibitors of Staphylococcus aureus and Escherichia coli enoyl acyl carrier protein reductase (FabI) have been identified. Crystal structure data shows the inhibitor 1 bound in the enzyme active site of E. coli FabI.

Characterization of Streptococcus pneumoniae Enoyl- (Acyl Carrier Protein) Reductase (FabK)

The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to the Bacillus / Lactobacillus / Streptococcus group that includes Staphylococcus aureus and Bacillus subtilis. These two organisms also contain FabK-related genes, suggesting that they may also express a FabK-like enoyl-ACP reductase. However, the genes did not complement a fabI (Ts) mutant and the purified flavoproteins were unable to reduce enoyl-ACP in vitro and did not exhibit NAD(P)H oxidase activity, indicating they were not enoyl-ACP reductases. The restricted occurrence of the FabK enoyl-ACP reductase may be related to the role of substrate-independent NADH oxidation in oxygen-dependent anaerobic energy metabolism.

Two Active Forms of UDP-N-Acetylglucosamine Enolpyruvyl Transferase in Gram-Positive Bacteria

Gene sequences encoding the enzymes UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) from many bacterial sources were analyzed. It was shown that whereas gram-negative bacteria have only one murA gene, gram-positive bacteria have two distinct genes encoding these enzymes which have possibly arisen from gene duplication. The two murA genes of the gram-positive organism Streptococcus pneumoniae were studied further. Each of the murA genes was individually inactivated by allelic replacement. In each case, the organism was viable despite losing one of its murA genes. However, when attempts were made to construct a double-deletion strain, no mutants were obtained. This indicates that both genes encode active enzymes that can substitute for each other, but that the presence of a MurA function is essential to the organism. The two genes were further cloned and overexpressed, and the enzymes they encode were purified. Both enzymes catalyzed the transfer of enolpyruvate from phosphoenolpyruvate to UDP-N-acetylglucosamine, confirming they are both active UDP-N-acetylglucosamine enolpyruvyl transferases. The catalytic parameters of the two enzymes were similar, and they were both inhibited by the antibiotic fosfomycin.

Identification of a Series of Tricyclic Natural Products as Potent Broad-Spectrum Inhibitors of Metallo-β-Lactamases

ABSTRACT This work describes the discovery and characterization of a novel series of tricyclic natural product-derived metallo-β-lactamase inhibitors. Natural product screening of the Bacillus cereus II enzyme identified an extract from a strain of Chaetomium funicola with inhibitory activity against metallo-β-lactamases. SB236050, SB238569, and SB236049 were successfully extracted and purified from this extract. The most active of these compounds was SB238569, which possessed Ki values of 79, 17, and 3.4 μM for the Bacillus cereus II, Pseudomonas aeruginosa IMP-1, and Bacteroides fragilis CfiA metallo-β-lactamases, respectively, yet none of the compounds exhibited any inhibitory activity against the Stenotrophomonas maltophilia L-1 metallo-β-lactamase (50% inhibitory concentration > 1,000 μM). The lack of activity against angiotensin-converting enzyme and serine β-lactamases demonstrated the selective nature of these compounds. The crystal structure of SB236050 complexed in the active site of CfiA has been obtained to a resolution of 2.5 Å. SB236050 exhibits key polar interactions with Lys184, Asn193, and His162 and a stacking interaction with the indole ring of Trp49 in the flap, which is in the closed conformation over the active site groove. SB236050 and SB238569 also demonstrate good antibacterial synergy with meropenem. Eight micrograms of SB236050 per ml gave rise to an eightfold drop in the MIC of meropenem for two clinical isolates of B. fragilis producing CfiA, making these strains sensitive to meropenem (MIC ≤ 4 μg/ml). Consequently, this series of metallo-β-lactamase inhibitors exhibit the most promising antibacterial synergy activity so far observed against organisms producing metallo-β-lactamases.