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

Christopher Burke, Monika Jankute, Patrick Moynihan, Ruben Gonzalez del Rio, Xiaojun Li, Jorge Esquivias, Joël Lelièvre, Jonathan A. G. Cox, James Sacchettini, Gurdyal S. Besra

Research output: Contribution to journalArticle

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.
Original languageEnglish
Pages (from-to)600-612
Number of pages13
JournalFASEB BioAdvances
Volume2
Issue number10
Early online date20 Aug 2020
DOIs
Publication statusPublished - Oct 2020

Bibliographical note

©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.

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    Burke, C., Jankute, M., Moynihan, P., Gonzalez del Rio, R., Li, X., Esquivias, J., Lelièvre, J., Cox, J. A. G., Sacchettini, J., & Besra, G. S. (2020). Development of a novel secondary phenotypic screen to identify hits within the mycobacterial protein synthesis pipeline. FASEB BioAdvances, 2(10), 600-612. https://doi.org/10.1096/fba.2020-00022