Heterobenzamides exhibit bacteriostatic activity against intracellular Mycobacterium tuberculosis by targeting aerobic respiration

We previously identified a series of heterobenzamides (HBAs) with potent growth inhibitory activity against Mycobacterium tuberculosis in axenic culture. We also provided evidence that these target QcrB, a component of the terminal cytochrome oxidase in the electron transport chain. We expanded our studies to look at the full microbiological profile: key molecules from the series were tested for activity under different conditions and against additional strains. HBA analogs were active against intracellular bacteria where they exhibited bacteriostatic activity. A strain of M. tuberculosis with a mutation in QcrB (T313I) was resistant to HBAs in both axenic culture and inside macrophages. HBAs retained potency against lineages and mono-resistant strains of M. tuberculosis. HBAs had a narrow spectrum of activity, since they were not active against the ESKAPEE pathogens. Combination of the key HBA with bedaquiline was synergistic, as expected for a QcrB inhibitor, but there was no strong synergy with other drugs. Exposure of M. tuberculosis to the key HBA led to ATP depletion and boosted the oxygen consumption rate. This effect was specific to M. tuberculosis, since human THP-1 macrophage-like cells were unaffected by exposure to the HBA. HBA did not induce the production of reactive oxygen species or affect membrane potential but did affect pH homeostasis. Taken together, these data provide further evidence to support the identification of QcrB as the target and indicate that they are suitable for further drug development.