Genetic andCheminformatic Characterization of Mycobacterium tuberculosis Inhibitors Discoveredin the Molecular Libraries Small Molecule Repository

High-throughput screening (HTS) of small molecules is a starting point for many drug development pipelines, including tuberculosis. These screens often result in multiple hits whose mechanisms of action remain unknown. From our initial HTS of the Molecular Libraries Small Molecule Repository (MLSMR), we cherry-picked 935 compounds that inhibited the growth of Mycobacterium tuberculosis and set out to provide an early assessment of their antimycobacterial properties and mechanism of action. To characterize the MLSMR Mtb growth inhibitors, a combination of cheminformatics and targeted mutant screening against mutants in <i>katG</i>, <i>hadAB</i>, and a mixed pool of <i>mmpL3</i> mutants was used to characterize the hits. As a validation of this approach, we identified 101 isoniazid analogs that predictably lose all their antimycobacterial activities against the <i>katG</i> mutant. Interestingly, eight isoniazid analogs retain part of their activity against the mutant, suggesting an alternative KatG-independent mechanism. This approach also identified new compounds belonging to already known scaffolds that target HadAB or MmpL3. Additionally, we explored the nitro-containing compounds in our data set and discovered nitrofuranyl benzothiazoles that show enhanced activity against the <i>mmpL3</i> and <i>katG</i> mutants, a phenomenon known as collateral sensitivity. Overall, this study will serve as an important resource for further follow-up studies of antitubercular small molecules in the MLSMR library and provide a well-characterized training set for artificial intelligence-driven antimycobacterial drug discovery.