Contribution of direct InhA inhibitors to novel drug regimens in a mouse model of tuberculosis.

Isoniazid is an important first-line medicine to treat tuberculosis (TB). Isoniazid resistance increases the risk of poor treatment outcomes and development of multidrug resistance, and is driven primarily by mutations involving katG, encoding the prodrug-activating enzyme, rather than its validated target, InhA. The chemical tractability of InhA has fostered efforts to discover direct inhibitors of InhA (DIIs). In this study, we bridge the gap in understanding the potential contribution of DIIs to novel combination regimens and demonstrate a clear distinction of DIIs, like GSK693 and the newly described GSK138, from isoniazid, based on activity against clinical isolates and contribution to novel drug regimens. The results suggest that DIIs, specifically GSK138 and GSK693, could be promising partners in novel drug regimens, including those used against isoniazid-resistant TB, potentially enhancing their efficacy and/or preventing the selection of resistant mutants and supporting the continued exploration of InhA as a promising target for TB drug development.

Activity of Oral Tebipenem-Avibactam in a Mouse Model of Mycobacterium abscessus Lung Infection.

The combination of the ß-lactam tebipenem and the ß-lactamase inhibitor avibactam shows potent bactericidal activity against Mycobacterium abscessus in vitro. Here, we report that the combination of the respective oral prodrugs tebipenem-pivoxil and avibactam ARX-1796 showed efficacy in a mouse model of M. abscessus lung infection. The results suggest that tebipenem-avibactam presents an attractive oral drug candidate pair for the treatment of M. abscessus pulmonary disease and could inform the design of clinical trials.

Development of an Intracellular Screen for New Compounds Able To Inhibit Mycobacterium tuberculosis Growth in Human Macrophages.

Here we describe the development and validation of an intracellular high-throughput screening assay for finding new antituberculosis compounds active in human macrophages. The assay consists of a luciferase-based primary identification assay, followed by a green fluorescent protein-based secondary profiling assay. Standard tuberculosis drugs and 158 previously recognized active antimycobacterial compounds were used to evaluate assay robustness. Data show that the assay developed is a short and valuable tool for the discovery of new antimycobacterial compounds.

Mycobacterium tuberculosis gyrase inhibitors as a new class of antitubercular drugs.

One way to speed up the TB drug discovery process is to search for antitubercular activity among compound series that already possess some of the key properties needed in anti-infective drug discovery, such as whole-cell activity and oral absorption. Here, we present MGIs, a new series of Mycobacterium tuberculosis gyrase inhibitors, which stem from the long-term efforts GSK has dedicated to the discovery and development of novel bacterial topoisomerase inhibitors (NBTIs). The compounds identified were found to be devoid of fluoroquinolone (FQ) cross-resistance and seem to operate through a mechanism similar to that of the previously described NBTI GSK antibacterial drug candidate. The remarkable in vitro and in vivo antitubercular profiles showed by the hits has prompted us to further advance the MGI project to full lead optimization.

Development of a novel secondary phenotypic screen to identify hits within the mycobacterial protein synthesis pipeline.

BACKGROUND: Whole-cell phenotypic screening is the driving force behind modern anti-tubercular drug discovery efforts. Focus has shifted from screening for bactericidal scaffolds to screens incorporating target deconvolution. Target-based screening aims to direct drug discovery toward known effective targets and avoid investing resources into unproductive lines of enquiry. The protein synthesis pipeline, including RNA polymerase and the ribosome, is a clinically proven target in Mycobacterium tuberculosis. Screening for new hits of this effective target pathway is an invaluable tool in the drug discovery arsenal. METHODS: Using M. tuberculosis H37Rv augmented with anhydrotetracycline-inducible expression of mCherry, a phenotypic screen was developed for the identification of protein synthesis inhibitors in a medium throughput screening format. RESULTS: The assay was validated using known inhibitors of protein synthesis to show a dose-dependent reduction in mCherry fluorescence. This was expanded to a proprietary screen of hypothetical protein synthesis hits and modified to include quantitative viability measurement of cells using resazurin. CONCLUSION: Following the success of the proprietary screen, a larger scale screen of the GlaxoSmithKline anti-tubercular library containing 2799 compounds was conducted. Combined single shot and dose-response screening yielded 18 hits, 0.64% of all screened compounds.

Triple oral beta-lactam containing therapy for Buruli ulcer treatment shortening.

The potential use of clinically approved beta-lactams for Buruli ulcer (BU) treatment was investigated with representative classes analyzed in vitro for activity against Mycobacterium ulcerans. Beta-lactams tested were effective alone and displayed a strong synergistic profile in combination with antibiotics currently used to treat BU, i.e. rifampicin and clarithromycin; this activity was further potentiated in the presence of the beta-lactamase inhibitor clavulanate. In addition, quadruple combinations of rifampicin, clarithromycin, clavulanate and beta-lactams resulted in multiplicative reductions in their minimal inhibitory concentration (MIC) values. The MIC of amoxicillin against a panel of clinical isolates decreased more than 200-fold within this quadruple combination. Amoxicillin/clavulanate formulations are readily available with clinical pedigree, low toxicity, and orally and pediatric available; thus, supporting its potential inclusion as a new anti-BU drug in current combination therapies.

Functional screening of selective mitochondrial inhibitors of Plasmodium.

Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors.

Repurposing clinically approved cephalosporins for tuberculosis therapy.

While modern cephalosporins developed for broad spectrum antibacterial activities have never been pursued for tuberculosis (TB) therapy, we identified first generation cephalosporins having clinically relevant inhibitory concentrations, both alone and in synergistic drug combinations. Common chemical patterns required for activity against Mycobacterium tuberculosis were identified using structure-activity relationships (SAR) studies. Numerous cephalosporins were synergistic with rifampicin, the cornerstone drug for TB therapy, and ethambutol, a first-line anti-TB drug. Synergy was observed even under intracellular growth conditions where beta-lactams typically have limited activities. Cephalosporins and rifampicin were 4- to 64-fold more active in combination than either drug alone; however, limited synergy was observed with rifapentine or rifabutin. Clavulanate was a key synergistic partner in triple combinations. Cephalosporins (and other beta-lactams) together with clavulanate rescued the activity of rifampicin against a rifampicin resistant strain. Synergy was not due exclusively to increased rifampicin accumulation within the mycobacterial cells. Cephalosporins were also synergistic with new anti-TB drugs such as bedaquiline and delamanid. Studies will be needed to validate their in vivo activities. However, the fact that cephalosporins are orally bioavailable with good safety profiles, together with their anti-mycobacterial activities reported here, suggest that they could be repurposed within new combinatorial TB therapies.

Antimicrobial susceptibility testing for Mycobacterium sp.

The concept of antimicrobial susceptibility testing is an essential part of clinical microbiology. Antimicrobial testing has played a central role in the identification of new antibiotics and defining their clinical uses. Here we describe different approaches to determine the activity of compounds in medium- or high-throughput format.

Release of 50 new, drug-like compounds and their computational target predictions for open source anti-tubercular drug discovery.

As a follow up to the antimycobacterial screening exercise and the release of GSK´s first Tres Cantos Antimycobacterial Set (TCAMS-TB), this paper presents the results of a second antitubercular screening effort of two hundred and fifty thousand compounds recently added to the GSK collection. The compounds were further prioritized based on not only antitubercular potency but also on physicochemical characteristics. The 50 most attractive compounds were then progressed for evaluation in three different predictive computational biology algorithms based on structural similarity or GSK historical biological assay data in order to determine their possible mechanisms of action. This effort has resulted in the identification of novel compounds and their hypothesized targets that will hopefully fuel future TB drug discovery and target validation programs alike.

A new molecular approach for cidal vs static antimalarial determination by quantifying mRNA levels.

In order to maximise compliance, the future antimalarial treatment should ideally require just a single-dose administration. This, in turn, demands new fast-acting effective drugs. Currently, methods to measure the in vitro killing rate of antimalarials are based on parasite growth. We have developed and validated a method to determine and classify antimalarial agents based on their cidal or static activity following quantitative Real Time PCR (RT-PCR) analysis. The method described here is a fast, reliable and user-friendly technique with a medium throughput. Metabolic activity of the parasite is followed by measuring mRNA expression levels of several genes during 5 parasite life cycles. mRNA from the parasite culture is then retrotranscribed to cDNA and quantified by RT-PCR. This new method provides a rapid and reproducible way to accurately measure the antimalarial activity of new compounds in vitro against Plasmodium falciparum.