Analysis of the Clinical Pipeline of Treatments for Drug-Resistant Bacterial Infections: Despite Progress, More Action Is Needed.

There is an urgent global need for new strategies and drugs to control and treat multidrug-resistant bacterial infections. In 2017, the World Health Organization (WHO) released a list of 12 antibiotic-resistant priority pathogens and began to critically analyze the antibacterial clinical pipeline. This review analyzes "traditional" and "nontraditional" antibacterial agents and modulators in clinical development current on 30 June 2021 with activity against the WHO priority pathogens mycobacteria and Clostridioides difficile. Since 2017, 12 new antibacterial drugs have been approved globally, but only vaborbactam belongs to a new antibacterial class. Also innovative is the cephalosporin derivative cefiderocol, which incorporates an iron-chelating siderophore that facilitates Gram-negative bacteria cell entry. Overall, there were 76 antibacterial agents in clinical development (45 traditional and 31 nontraditional), with 28 in phase 1, 32 in phase 2, 12 in phase 3, and 4 under regulatory evaluation. Forty-one out of 76 (54%) targeted WHO priority pathogens, 16 (21%) were against mycobacteria, 15 (20%) were against C. difficile, and 4 (5%) were nontraditional agents with broad-spectrum effects. Nineteen of the 76 antibacterial agents have new pharmacophores, and 4 of these have new modes of actions not previously exploited by marketed antibacterial drugs. Despite there being 76 antibacterial clinical candidates, this analysis indicated that there were still relatively few clinically differentiated antibacterial agents in late-stage clinical development, especially against critical-priority pathogens. We believe that future antibacterial research and development (R&D) should focus on the development of innovative and clinically differentiated candidates that have clear and feasible progression pathways to the market.

Characterization of a Carbapenem-Hydrolyzing Enzyme, PoxB, in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen often associated with severe and life-threatening infections that are highly impervious to treatment. This microbe readily exhibits intrinsic and acquired resistance to varied antimicrobial drugs. Resistance to penicillin-like compounds is commonplace and provided by the chromosomal AmpC ß-lactamase. A second, chromosomally encoded ß-lactamase, PoxB, has previously been reported in P. aeruginosa. In the present work, the contribution of this class D enzyme was investigated using a series of clean in-frame ampC, poxB, and oprD deletions, as well as complementation by expression under the control of an inducible promoter. While poxB deletions failed to alter ß-lactam sensitivities, expression of poxB in ampC-deficient backgrounds decreased susceptibility to both meropenem and doripenem but had no effect on imipenem, penicillin, and cephalosporin MICs. However, when expressed in an ampCpoxB-deficient background, that additionally lacked the outer membrane porin-encoding gene oprD, PoxB significantly increased the imipenem as well as the meropenem and doripenem MICs. Like other class D carbapenem-hydrolyzing ß-lactamases, PoxB was only poorly inhibited by class A enzyme inhibitors, but a novel non-ß-lactam compound, avibactam, was a slightly better inhibitor of PoxB activity. In vitro susceptibility testing with a clinical concentration of avibactam, however, failed to reduce PoxB activity against the carbapenems. In addition, poxB was found to be cotranscribed with an upstream open reading frame, poxA, which itself was shown to encode a 32-kDa protein of yet unknown function.

A Gestalt approach to Gram-negative entry.

A major obstacle confronting the discovery and development of new antibacterial agents to combat resistant Gram-negative (GN) organisms is the lack of a rational process for endowing compounds with properties that allow (or promote) entry into the bacterial cytoplasm. The major permeability difference between GN and Gram-positive (GP) bacteria is the GN outer membrane (OM) which is a permeability barrier itself and potentiates efflux pumps that expel compounds. Based on the fact that OM-permeable and efflux-deleted GNs are sensitive to many anti-GP drugs, recent efforts to approach the GN entry problem have focused on ways of avoiding efflux and transiting or compromising the OM, with the tacit assumption that this could allow entry of compounds into the GN cytoplasm. But bypassing the OM and efflux obstacles does not take into account the additional requirement of penetrating the cytoplasmic membrane (CM) whose sieving properties appear to be orthogonal to that of the OM. That is, tailoring compounds to transit the OM may well compromise their ability to enter the cytoplasm. Thus, a Gestalt approach to understanding the chemical requirements for GN entry seems a useful adjunct. This might consist of characterizing compounds which reach the cytoplasm, grouping (or binning) by routes of entry and formulating chemical 'rules' for those bins. This will require acquisition of data on large numbers of compounds, using non-activity-dependent methods of measuring accumulation in the cytoplasm.

Challenges of antibacterial discovery.

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.

Platensimycin is a selective FabF inhibitor with potent antibiotic properties.

Bacterial infection remains a serious threat to human lives because of emerging resistance to existing antibiotics. Although the scientific community has avidly pursued the discovery of new antibiotics that interact with new targets, these efforts have met with limited success since the early 1960s. Here we report the discovery of platensimycin, a previously unknown class of antibiotics produced by Streptomyces platensis. Platensimycin demonstrates strong, broad-spectrum Gram-positive antibacterial activity by selectively inhibiting cellular lipid biosynthesis. We show that this anti-bacterial effect is exerted through the selective targeting of beta-ketoacyl-(acyl-carrier-protein (ACP)) synthase I/II (FabF/B) in the synthetic pathway of fatty acids. Direct binding assays show that platensimycin interacts specifically with the acyl-enzyme intermediate of the target protein, and X-ray crystallographic studies reveal that a specific conformational change that occurs on acylation must take place before the inhibitor can bind. Treatment with platensimycin eradicates Staphylococcus aureus infection in mice. Because of its unique mode of action, platensimycin shows no cross-resistance to other key antibiotic-resistant strains tested, including methicillin-resistant S. aureus, vancomycin-intermediate S. aureus and vancomycin-resistant enterococci. Platensimycin is the most potent inhibitor reported for the FabF/B condensing enzymes, and is the only inhibitor of these targets that shows broad-spectrum activity, in vivo efficacy and no observed toxicity.

Multi-targeting by monotherapeutic antibacterials.

Antibacterial discovery research has been driven, medically, commercially and intellectually, by the need for new therapeutics that are not subject to the resistance mechanisms that have evolved to combat previous generations of antibacterial agents. This need has often been equated with the identification and exploitation of novel targets. But efforts towards discovery and development of inhibitors of novel targets have proved frustrating. It might be that the 'good old targets' are qualitatively different from the crop of all possible novel targets. What has been learned from existing targets that can be applied to the quest for new antibacterials?

Setting Our Sights on Infectious Diseases.

In May 2019, the Wellcome Centre for Anti-Infectives Research (WCAIR) at the University of Dundee, UK, held an international conference with the aim of discussing some key questions around discovering new medicines for infectious diseases and a particular focus on diseases affecting Low and Middle Income Countries. There is an urgent need for new drugs to treat most infectious diseases. We were keen to see if there were lessons that we could learn across different disease areas and between the preclinical and clinical phases with the aim of exploring how we can improve and speed up the drug discovery, translational, and clinical development processes. We started with an introductory session on the current situation and then worked backward from clinical development to combination therapy, pharmacokinetic/pharmacodynamic (PK/PD) studies, drug discovery pathways, and new starting points and targets. This Viewpoint aims to capture some of the learnings.

Perspective on Antibacterial Lead Identification Challenges and the Role of Hypothesis-Driven Strategies.

For the past three decades, the pharmaceutical industry has undertaken many diverse approaches to discover novel antibiotics, with limited success. We have witnessed and personally experienced many mistakes, hurdles, and dead ends that have derailed projects and discouraged scientists and business leaders. Of the many factors that affect the outcomes of screening campaigns, a lack of understanding of the properties that drive efflux and permeability requirements across species has been a major barrier for advancing hits to leads. Hits that possess bacterial spectrum have seldom also possessed druglike properties required for developability and safety. Persistence in solving these two key barriers is necessary for the reinvestment into discovering antibacterial agents. This perspective narrates our experience in antibacterial discovery-our lessons learned about antibacterial challenges as well as best practices for screening strategies. One of the tenets that guides us is that drug discovery is a hypothesis-driven science. Application of this principle, at all steps in the antibacterial discovery process, should improve decision making and possibly the odds of what has become, in recent decades, an increasingly challenging endeavor with dwindling success rates.

Analysis of the clinical antibacterial and antituberculosis pipeline.

This analysis of the global clinical antibacterial pipeline was done in support of the Global Action Plan on Antimicrobial Resistance. The study analysed to what extent antibacterial and antimycobacterial drugs for systemic human use as well as oral non-systemic antibacterial drugs for Clostridium difficile infections were active against pathogens included in the WHO priority pathogen list and their innovativeness measured by their absence of cross-resistance (new class, target, mode of action). As of July 1, 2018, 30 new chemical entity (NCE) antibacterial drugs, ten biologics, ten NCEs against Mycobacterium tuberculosis, and four NCEs against C difficile were identified. Of the 30 NCEs, 11 are expected to have some activity against at least one critical priority pathogen expressing carbapenem resistance. The clinical pipeline is dominated by derivatives of established classes and most development candidates display limited innovation. New antibacterial drugs without pre-existing cross-resistance are under-represented and are urgently needed, especially for geographical regions with high resistance rates among Gram-negative bacteria and M tuberculosis.

Fosfomycin: Mechanism and Resistance.

Fosfomycin, a natural product antibiotic, has been in use for >20 years in Spain, Germany, France, Japan, Brazil, and South Africa for urinary tract infections (UTIs) and other indications and was registered in the United States for the oral treatment of uncomplicated UTIs because of Enterococcus faecalis and Escherichia coli in 1996. It has a broad spectrum, is bactericidal, has very low toxicity, and acts as a time-dependent inhibitor of the MurA enzyme, which catalyzes the first committed step of peptidoglycan synthesis. Whereas resistance to fosfomycin arises rapidly in vitro through loss of active transport mechanisms, resistance is rarely seen during therapy of UTIs, seemingly because of the low fitness of the resistant organisms. Recently, interest has grown in the use of fosfomycin against multidrug-resistant (MDR) pathogens in other indications, prompting the advent of development in the United States of a parenteral formulation for use, initially, in complicated UTIs. Whereas resistance has not been problematic in the uncomplicated UTI setting, it remains to be seen whether resistance remains at bay with expansion to other indications.

What is an "ideal" antibiotic? Discovery challenges and path forward.

An ideal antibiotic is an antibacterial agent that kills or inhibits the growth of all harmful bacteria in a host, regardless of site of infection without affecting beneficial gut microbes (gut flora) or causing undue toxicity to the host. Sadly, no such antibiotics exist. What exist are many effective Gram-positive antibacterial agents as well as broad-spectrum agents that provide treatment of certain Gram-negative bacteria but not holistic treatment of all bacteria. However effectiveness of all antibacterial agents is being rapidly eroded due to resistance. This viewpoint provides an overview of today's antibiotics, challenges and potential path forward of discovery and development of new (ideal) antibiotics.

Does the cell wall of bacteria remain a viable source of targets for novel antibiotics?

Whether the bacterial cell wall remains a viable source of novel antibacterials is addressed here by reviewing screen and design strategies for discovery of antibacterials with a focus on their output. Inhibitors for which antibacterial activity has been shown to be due to specific inhibition of a reaction (antibacterially validated inhibitors) are known for 8 of the 14 conserved essential steps of the pathway. Antibacterially validated enzyme inhibitors exist for six of these steps. The possible obstacles to finding validated inhibitors of the remaining enzymes are discussed and some strategies are suggested.

Novel inhibitors of bacterial cell wall synthesis.

Over the past forty years, efforts to discover antibacterials have yielded a wide variety of chemical structures, almost exclusively natural products, which inhibit many steps in cell wall synthesis. Although screening for new cell wall inhibitors has been continuous during that period, there have been few reports of new drugs. With the advent of genomics, high resolution X-ray crystallography and the recognition of the need for new antibiotics to combat resistant organisms, there has been a resurgence in interest in this validated target area.

Appropriate Targets for Antibacterial Drugs.

Successful small-molecule antibacterial agents must meet a variety of criteria. Foremost is the need for selectivity and safety: It is easy to kill bacteria with chemicals, but difficult to do it without harming the patient. Other requirements are possession of a useful antibacterial spectrum, no cross-resistance with existing therapeutics, low propensity for rapid resistance selection, and pharmacological properties that allow effective systemic dosing. Choosing molecular targets for new antibiotics does seem a good basis for achieving these criteria, but this could be misleading. Although the presence of the target is necessary to insure the desired spectrum, it is not sufficient, as the permeability and efflux properties of various species, especially Gram-negatives, are critical determinants of antibacterial activity. Further, although essentiality (at least in vitro), lack of close human homologs, lack of target-based cross-resistance, and presence in important pathogens can be predicted based on the target, the choice of a single enzyme as a target may increase the likelihood of rapid resistance selection. In fact, it is likely that the low output of antibacterial target-based discovery is because of difficulty of endowing lead enzyme inhibitors with whole-cell activity and to the propensity for such inhibitors (if they can gain entry) to select rapidly for resistance. These potential problems must be reckoned with for success of novel target-based discovery.

A retrospective on the failures and successes of antibacterial drug discovery.

Antibacterial drug discovery has been notably unproductive in the past 20 years. A shift in the key players from large pharmaceutical companies to small biotechnology companies has been accompanied by high hopes for improvement in this field, but has, as yet, yielded little. This retrospective provides a brief review of antibacterial research and highlights the underlying obstacles to the discovery of novel antibacterial agents to offer an overview of the current state of the art.

Natural products as a source of drug leads to overcome drug resistance.

Novel antibacterials may be found that can enhance the pipeline of therapeutics capable of overcoming antibiotic resistance by a return to exploration of natural products. Such novel products may be derived from both standard and previously uncultivable sources, and enriched by expression of previously unseen antibiotics predicted by genome mining of productive bacterial genera. Hypersensitive whole cell phenotypic screens can be used to detect novel secondary metabolites from both standard and newly uncovered sources.

70th Anniversary Collection for the Microbiology Society: Journal of Medical Microbiology.

In the last 70 years, we have seen a radical change in our perception and understanding of the microbial world. During this period, we learned from Woese and Fox there exists a third kingdom called 'Archea' based on the phylogenetic studies of the 16S rRNA that revolutionized microbiology (Woese & Fox, 1977; Woese et al., 1978). Furthermore, we were forced to reckon with the fact that Koch and Pasteur's way of growing cells in test-tubes or flasks planktonically does not necessarily translate to the real-life scenario of bacterial lifestyle, where they prefer to live and function as a closely knit microbial community called biofilm. Thanks are due to Costerton, who led the crusade on the concept of biofilms and expanded its scope of inquiry, which forced scientists and clinicians worldwide to rethink how we evaluate and apply the data. Then progressively, disbelief turned into belief, and now it is universally accepted that the micro-organisms hobnob with the members of their community to communicate and coordinate their behaviour, especially in regard to growth patterns and virulence traits via signalling molecules. Just when we thought that we were losing the battle against bacteria, antimicrobials were discovered. We then witnessed the rise and fall of antibiotics and the development of antibiotic resistance. Due to space and choice limitation, we will focus on the three areas that caused this major paradigm shift (i) antimicrobial resistance (AMR), (ii) biofilm and (iii) quorum sensing (QS), and how the Journal of Medical Microbiology played a major role in advancing the shift.

Novel pyrazolo[3,4-d]pyrimidine-based inhibitors of Staphlococcus aureus DNA polymerase III: design, synthesis, and biological evaluation.

6-Anilinopyrazolo[3,4-d]pyrimidin-4-ones are novel dGTP analogues that inhibit the replication-specific enzyme DNA polymerase III (DNA pol III) of Staphlococcus aureus and other Gram-positive (Gr+) bacteria. To enhance the potential of these inhibitors as antimicrobial agents, a structure-activity relationship was developed involving substitutions at the 2, 4, and pyrazolo NH positions. All of the new inhibitors were tested for their ability to inhibit S. aureus DNA pol III and the growth of several other Gr+ bacteria in culture. 2-Anilino groups with small hydrophobic groups in the meta or para position enhanced both antipolymerase and antimicrobial activity. 2-Benzyl-substituted inhibitors were substantially less active. Substitution in the 4-position by oxygen gave the optimal activity, whereas substitution at the pyrazolo NH was not tolerated. These pyrazolo[3,4-d]pyrimidine derivatives represent a novel class of antimicrobials with promising activities against Gr+ bacteria.