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.

Mode-of-action profiling reveals glutamine synthetase as a collateral metabolic vulnerability of M. tuberculosis to bedaquiline.

Combination chemotherapy can increase treatment efficacy and suppress drug resistance. Knowledge of how to engineer rational, mechanism-based drug combinations, however, remains lacking. Although studies of drug activity have historically focused on the primary drug-target interaction, growing evidence has emphasized the importance of the subsequent consequences of this interaction. Bedaquiline (BDQ) is the first new drug for tuberculosis (TB) approved in more than 40 y, and a species-selective inhibitor of the Mycobacterium tuberculosis (Mtb) ATP synthase. Curiously, BDQ-mediated killing of Mtb lags significantly behind its inhibition of ATP synthase, indicating a mode of action more complex than the isolated reduction of ATP pools. Here, we report that BDQ-mediated inhibition of Mtb's ATP synthase triggers a complex metabolic response indicative of a specific hierarchy of ATP-dependent reactions. We identify glutamine synthetase (GS) as an enzyme whose activity is most responsive to changes in ATP levels. Chemical supplementation with exogenous glutamine failed to affect BDQ's antimycobacterial activity. However, further inhibition of Mtb's GS synergized with and accelerated the onset of BDQ-mediated killing, identifying Mtb's glutamine synthetase as a collateral, rather than directly antimycobacterial, metabolic vulnerability of BDQ. These findings reveal a previously unappreciated physiologic specificity of ATP and a facet of mode-of-action biology we term collateral vulnerability, knowledge of which has the potential to inform the development of rational, mechanism-based drug combinations.

Linking High-Throughput Screens to Identify MoAs and Novel Inhibitors of Mycobacterium tuberculosis Dihydrofolate Reductase.

Though phenotypic and target-based high-throughput screening approaches have been employed to discover new antibiotics, the identification of promising therapeutic candidates remains challenging. Each approach provides different information, and understanding their results can provide hypotheses for a mechanism of action (MoA) and reveal actionable chemical matter. Here, we describe a framework for identifying efficacy targets of bioactive compounds. High throughput biophysical profiling against a broad range of targets coupled with machine learning was employed to identify chemical features with predicted efficacy targets for a given phenotypic screen. We validate the approach on data from a set of 55 000 compounds in 24 historical internal antibacterial phenotypic screens and 636 bacterial targets screened in high-throughput biophysical binding assays. Models were built to reveal the relationships between phenotype, target, and chemotype, which recapitulated mechanisms for known antibacterials. We also prospectively identified novel inhibitors of dihydrofolate reductase with nanomolar antibacterial efficacy against Mycobacterium tuberculosis. Molecular modeling provided structural insight into target-ligand interactions underlying selective killing activity toward mycobacteria over human cells.

The within-host population dynamics of Mycobacterium tuberculosis vary with treatment efficacy.

BACKGROUND: Combination therapy is one of the most effective tools for limiting the emergence of drug resistance in pathogens. Despite the widespread adoption of combination therapy across diseases, drug resistance rates continue to rise, leading to failing treatment regimens. The mechanisms underlying treatment failure are well studied, but the processes governing successful combination therapy are poorly understood. We address this question by studying the population dynamics of Mycobacterium tuberculosis within tuberculosis patients undergoing treatment with different combinations of antibiotics. RESULTS: By combining very deep whole genome sequencing (~1000-fold genome-wide coverage) with sequential sputum sampling, we were able to detect transient genetic diversity driven by the apparently continuous turnover of minor alleles, which could serve as the source of drug-resistant bacteria. However, we report that treatment efficacy has a clear impact on the population dynamics: sufficient drug pressure bears a clear signature of purifying selection leading to apparent genetic stability. In contrast, M. tuberculosis populations subject to less drug pressure show markedly different dynamics, including cases of acquisition of additional drug resistance. CONCLUSIONS: Our findings show that for a pathogen like M. tuberculosis, which is well adapted to the human host, purifying selection constrains the evolutionary trajectory to resistance in effectively treated individuals. Nonetheless, we also report a continuous turnover of minor variants, which could give rise to the emergence of drug resistance in cases of drug pressure weakening. Monitoring bacterial population dynamics could therefore provide an informative metric for assessing the efficacy of novel drug combinations.

Some Nigerian anti-tuberculosis ethnomedicines: a preliminary efficacy assessment.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Nigerian herbalists possess indigenous ethnomedicinal recipes for the management of tuberculosis and related ailments. A collaborative preliminary modern scientific evaluation of the efficacy of some Nigerian ethnomedicines used by traditional medicine practitioners (TMPs) in the management of tuberculosis and related ailments has been carried out. MATERIALS AND METHODS: Ethnomedicinal recipes (ETMs) were collected from TMPs from locations in various ecological zones of Nigeria under a collaborative understanding. The aqueous methanolic extracts of the ETMs were screened against Mycobacterium bovis, BCG and Mycobacterium tuberculosis strain H37Rv using the broth microdilution method. RESULTS: Extracts of ETMs screened against BCG showed 69% activity against the organism. The activities varied from weak, ≤2500 µg/mL to highly active, 33 µg/mL 64% of the extracts were active against Mycobacterium tuberculosis The activities of the extracts against Mycobacterium tuberculosis varied from weak, ≤2500 µg/mL to highly active, 128 µg/mL. There was 77% agreement in results obtained using BCG or Mycobacterium tuberculosis as test organisms. CONCLUSION: The results show clear evidence for the efficacy of the majority of indigenous Nigerian herbal recipes in the ethnomedicinal management of tuberculosis and related ailments. BCG may be effectively used, to a great extent, as the organism for screening for potential anti-Mycobacterium tuberculosis agents. A set of prioritization criteria for the selection of plants for initial further studies for the purpose of antituberculosis drug discovery research is proposed.

Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis.

Identification of new drug targets is vital for the advancement of drug discovery against Mycobacterium tuberculosis, especially given the increase of resistance worldwide to first- and second-line drugs. Because traditional target-based screening has largely proven unsuccessful for antibiotic discovery, we have developed a scalable platform for target identification in M. tuberculosis that is based on whole-cell screening, coupled with whole-genome sequencing of resistant mutants and recombineering to confirm. The method yields targets paired with whole-cell active compounds, which can serve as novel scaffolds for drug development, molecular tools for validation, and/or as ligands for co-crystallization. It may also reveal other information about mechanisms of action, such as activation or efflux. Using this method, we identified resistance-linked genes for eight compounds with anti-tubercular activity. Four of the genes have previously been shown to be essential: AspS, aspartyl-tRNA synthetase, Pks13, a polyketide synthase involved in mycolic acid biosynthesis, MmpL3, a membrane transporter, and EccB3, a component of the ESX-3 type VII secretion system. AspS and Pks13 represent novel targets in protein translation and cell-wall biosynthesis. Both MmpL3 and EccB3 are involved in membrane transport. Pks13, AspS, and EccB3 represent novel candidates not targeted by existing TB drugs, and the availability of whole-cell active inhibitors greatly increases their potential for drug discovery.

Structure-activity relationships of antitubercular nitroimidazoles. 3. Exploration of the linker and lipophilic tail of ((s)-2-nitro-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazin-6-yl)-(4-trifluoromethoxybenzyl)amine (6-amino PA-824).

The (S)-2-nitro-6-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine named PA-824 (1) has demonstrated antitubercular activity in vitro and in animal models and is currently in clinical trials. We synthesized derivatives at three positions of the 4-(trifluoromethoxy)benzylamino tail, and these were tested for whole-cell activity against both replicating and nonreplicating Mycobacterium tuberculosis (Mtb). In addition, we determined their kinetic parameters as substrates of the deazaflavin-dependent nitroreductase (Ddn) from Mtb that reductively activates these pro-drugs. These studies yielded multiple compounds with 40 nM aerobic whole cell activity and 1.6 µM anaerobic whole cell activity: 10-fold improvements over both characteristics from the parent molecule. Some of these compounds exhibited enhanced solubility with acceptable stability to microsomal and in vivo metabolism. Analysis of the conformational preferences of these analogues using quantum chemistry suggests a preference for a pseudoequatorial orientation of the linker and lipophilic tail.

Prospects for clinical introduction of nitroimidazole antibiotics for the treatment of tuberculosis.

Nitroaromatic antibiotics have a long and controversial history in human and veterinary medicine. This controversy lies behind the presumption of many pharmaceutical companies that nitroaromatic compounds should be filtered from the list of drug-like compounds but stands at odds with the remarkably safe clinical record of use of such compounds. In this review, we will describe the whole-cell structure-activity relationships that have been reported for antimycobacterial nitroimidazoles as well as the available in vivo data supporting efficacy with a particular emphasis on nitroimidazo[2,1-b]oxazines such as PA-824. We will also explore the unique potential of such compounds to shorten the course of tuberculosis therapy by exerting a bactericidal effect on non-replicating bacilli. We will consider the mode of action of such compounds in sensitive organisms and discuss the mechanisms by which resistance may emerge. Finally, we will review the pharmacokinetics, toxicology and laboratory and animal studies linking nitroimidazoles with carcinogenicity and mutagenicity and assess the prospects for the clinical introduction of nitroimidazoles for the treatment of tuberculosis.

Prospects for new antitubercular drugs.

The inexorable rise in cases of tuberculosis worldwide, fuelled by the HIV epidemic, highlights the need for new drugs and particularly those that can shorten the duration of treatment. Clinical trials of existing broad-spectrum agents such as the fluoroquinolone moxifloxacin are proceeding, on the basis of efficacy in models of infection and preliminary clinical data. These may provide a stopgap, but the real breakthrough will come when novel agents with potent sterilising activity are discovered. Few such novel pre-clinical drug candidates exist and therefore considerable effort is being exerted to employ new tools to identify drug targets essential for survival of Mycobacterium tuberculosis.

Unique mechanism of action of the thiourea drug isoxyl on Mycobacterium tuberculosis.

The thiourea isoxyl (thiocarlide; 4,4'-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 microg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Delta9-stearoyl desaturase as the drug target. Development of a cell-free assay for Delta9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Delta9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Delta9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Delta9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development.

Combinatorial lead optimization of [1,2]-diamines based on ethambutol as potential antituberculosis preclinical candidates.

Despite relatively modest potency, ethambutol (EMB, (S,S)-[N,N-di-2-amino-1-butanol]ethylenediamine) is a mainstay of contemporary chemotherapy for the treatment of tuberculosis. We have developed a solid-phase synthesis of 1,2-diamine analogues of EMB using a novel acylation-reduction sequence that is compatible with high-throughput 96-well format chemistry. Using this procedure, we have synthesized 63 238 diamine analogues in pools of 10 that are suitable for testing. MIC and a target-based reporter assay were used to direct deconvolution of 2796 individual compounds from these mixtures, and the 69 most potent molecules were resynthesized in milligram quantities for hit confirmation. Purification of these individual active diamine analogues allowed the identification of 26 compounds with activity equal to or greater than EMB. Amines which occurred most frequently in active compounds included many with large hydrophobic moieties, suggesting that optimization was perhaps selecting for the isoprenoid binding site of the arabinosyltransferase target of EMB. N-Geranyl-N'-(2-adamantyl)ethane-1,2-diamine (109), the most active of these diamines, displayed a 14-35-fold improvement in activity in vitro against Mycobacterium tuberculosis, as compared to EMB.