Microbiological evidence for the trisubstituted benzimidazoles targeting MmpL3 in <i>Mycobacterium tuberculosis</i>

New anti-tuberculosis (TB) drugs with novel modes of action are in great demand due to the complex treatment regimens as well as the rising number of multidrug-resistant TB cases. We recently re-evaluated a few 2,5,6-trisubstituted benzimidazole derivatives (SBZ) previously demonstrated to have potent antitubercular activity. These compounds displayed favorable MICs and significantly reduced bacterial counts in an acute mouse infection model. Although this antitubercular lead series was initially reported to inhibit mycobacterial cell division, our findings suggest that its primary activity likely involves other cellular targets. By using bacterial cytological profiling, we observed that SBZ-treated <i>Mycobacterium tuberculosis</i> cells exhibit cell wall-damaging phenotypes resembling those caused by known cell wall biosynthesis inhibitors, such as AU1235 and SQ109, that mostly target the membrane protein large 3 (MmpL3). Whole-cell assays further supported the findings by showing activation of the <i>iniBAC</i> operon and accumulation of intracellular ATP. The antitubercular activity of SBZs was tested against engineered mycobacterial strains that have the transcriptionally regulated <i>mmpL3</i> gene expression, confirming that SBZs engage the MmpL3 target in the cell. Strains with mutations in <i>mmpL3</i> exhibited either low- or high-level resistance to the SBZs. A molecule docking model is proposed, based on a high-resolution crystal structure of MmpL3, which could be useful in reconciling the inhibition mechanism and suggesting a further development of MmpL3 inhibitor starting with the SBZ scaffold.