Abstract
BackgroundWhole‐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.MethodsUsing 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.ResultsThe 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.ConclusionFollowing 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.
Highlights
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains one of the most successful bacterial pathogens
The protein synthesis pipeline, containing RNA polymerase (RNAP), approximately 22 tRNA synthases,[3] and the ribosome represents one of the most crucial aspects of biological life. Both RNAP and the ribosome have been successfully inhibited in M. tuberculosis by rifampicin (RIF)[4,5,6,7,8] and streptomycin,[9,10,11,12,13,14] respectively, drugs that see extensive use in the clinic for treating TB.[15]
Phenotypic screening of large compound libraries is the current gold standard for the discovery of novel anti-tubercular agents with promising pharmacokinetic properties.[32,33,34]. Large screens, such as these excel at finding novel inhibitors but fall short when it comes to target identification. This results in a reliance on whole genome sequencing (WGS) to identify the drug targets from spontaneous drug-resistant mutants (DRMs), which can be time consuming to generate
Summary
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains one of the most successful bacterial pathogens. The protein synthesis pipeline, containing RNA polymerase (RNAP), approximately 22 tRNA synthases,[3] and the ribosome represents one of the most crucial aspects of biological life Both RNAP and the ribosome have been successfully inhibited in M. tuberculosis by rifampicin (RIF)[4,5,6,7,8] and streptomycin,[9,10,11,12,13,14] respectively, drugs that see extensive use in the clinic for treating TB.[15] New scaffolds that target these complexes at different sites are an attractive proposition for further drug development and clinical use. This screen in addition to other, recently developed,[28,29] whole-cell phenotypic screening methods is focused on reducing the time required for mode of action studies into known effective targets
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