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

Abstract

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.

Publication DOI: https://doi.org/10.1096/fba.2020-00022
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences
Additional Information: ©2020 The Authors. FASEB BioAdvances published by The Federation of American Societies for Experimental Biology This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Publication ISSN: 2573-9832
Full Text Link:
Related URLs: https://faseb.o ... /fba.2020-00022 (Publisher URL)
PURE Output Type: Article
Published Date: 2020-10
Published Online Date: 2020-08-20
Accepted Date: 2020-07-31
Authors: Burke, Christopher
Jankute, Monika
Moynihan, Patrick
Gonzalez del Rio, Ruben
Li, Xiaojun
Esquivias, Jorge
Lelièvre, Joël
Cox, Jonathan A. G. (ORCID Profile 0000-0001-5208-4056)
Sacchettini, James
Besra, Gurdyal S.

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