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.
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