Common Biological Properties of <i>Mycobacterium tuberculosis</i> MmpL3 Inhibitors

MmpL3 is a promising new target for antitubercular drugs, but the microbiological properties of MmpL3 inhibitors are not fully understood. We compared the activity and mode of action of 11 structurally diverse compound series that target MmpL3. We confirmed the activity was via MmpL3 using strains with differential expression of MmpL3. MmpL3 inhibitors had potent activity against replicating <i>M. tuberculosis</i>, with increased activity against intramacrophage bacilli and were rapidly bactericidal. MmpL3 inhibition induced cell wall stress concomitantly with a boost in the ATP levels in <i>M. tuberculosis</i>. Mutation in MmpL3 conferred resistance to all series at different levels. The molecules did not negatively impact membrane potential, pH homeostasis, or induce reactive oxygen species and were inactive against starved bacilli. Our study revealed common features related to the chemical inhibition of MmpL3, enabling the identification of off-target effects and highlighting the potential of such compounds as future drug candidates.