Biological Profiling Enables Rapid Mechanistic Classification of Phenotypic Screening Hits and Identification of KatG Activation-Dependent Pyridine Carboxamide Prodrugs With Activity Against .

Compounds with novel modes of action are urgently needed to develop effective combination therapies for the treatment of tuberculosis. In this study, a series of compounds was evaluated for activity against replicating and Vero cell line toxicity. Fourteen of the compounds with activities in the low micrometer range and a favorable selectivity index were classified using reporter strains of which showed that six interfered with cell wall metabolism and one disrupted DNA metabolism. Counter-screening against strains carrying mutations in promiscuous drug targets argued against DprE1 and MmpL3 as hits of any of the cell wall actives and eliminated the cytochrome complex as a target of any of the compounds. Instead, whole-genome sequencing of spontaneous resistant mutants and/or counter-screening against common isoniazid-resistant mutants of revealed that four of the six cell wall-active compounds, all pyridine carboxamide analogues, were metabolized by KatG to form InhA inhibitors. Resistance to two of these compounds was associated with mutations in that did not confer cross-resistance to isoniazid. Of the remaining seven compounds, low-level resistance to one was associated with an inactivating mutation in Rv0678, the regulator of the MmpS5-MmpL5 system, which has been implicated in non-specific efflux of multiple chemotypes. Another mapped to the mycothiol-dependent reductase, Rv2466c, suggesting a prodrug mechanism of action in that case. The inability to isolate spontaneous resistant mutants to the seven remaining compounds suggests that they act mechanisms which have yet to be elucidated.