Thienopyrimidine amide analogs target MmpL3 in Mycobacterium tuberculosis

The identification of novel agents with mechanisms of action distinct from those currently utilized in tuberculosis treatment remains a significant challenge. The mycobacterial protein MmpL3 has emerged as a promising drug target due to its essential role in the synthesis of the cell wall of Mycobacterium tuberculosis. We previously identified novel thienopyrimidine amides(TPAs) with good anti-tubercular activity. We profiled a subset of TPAs, determining activity against intracellular bacteria and bactericidal activity against replicating bacteria. We ran assays to determine the mode of action by measuring cell wall stress, ATP production, and bacterial cytological profiling. We determined activity against a strain of M. tuberculosis with mutations in MmpL3. We isolated and sequenced resistant mutants. We tested five analogs against a strain of M. tuberculosis with mutations in MmpL3 and determined that they lost potency. Analogs induced P_iniBAC, a reporter for cell wall stress, and led to an ATP boost characteristic of cell wall inhibitors. Bacterial cytological profiling of a representative compound revealed a morphological profile consistent with other MmpL3 inhibitors. Together, our data support MmpL3 as the most probable drug target for the TPA analogs and add to the growing list of scaffolds that can inhibit this vulnerable transporter.