Dual targeting of cytochrome bd and bc1-aa3 terminal oxidases eradicates phenotypically diverse population of Mycobacterium tuberculosis.

Bedaquiline approval paved the way for the development of drug candidates against energy metabolism pathways of TB-causing organisms, and oxidative phosphorylation was proven to be a relevant target domain. While bedaquiline acts on the F1F0 ATP synthase, Q203 targets the cytochrome bc1-aa3 terminal oxidase, which mediates electron transport to O2 in Mycobacterium tuberculosis. However, due to functional redundancies between cytochrome bc1-aa3 and a second terminal oxidase, the cytochrome-bd, the sterilization effect of Q203 is impaired. Here, we describe the identification of BD-2, a novel small molecule inhibitor of the cytochrome-bd in a cell-based screening campaign. Alone, BD-2 does not exhibit any significant activity. When paired with Q203, however, BD-2 strongly inhibits Mycobacterium tuberculosis respiratory chain function and ATP synthesis. Moreover, BD-2 combined with Q203 was shown to possess potent bactericidal effect on both replicating and nonreplicating Mycobacterium tuberculosis, including those living under nutrient starved and hypoxic conditions, representing the persistent forms found in granulomas of patients. Mechanistic analysis included molecular docking studies using the known structure of Mycobacterium tuberculosis cytochrome-bd, in vitro activity testing on purified enzyme, all of which confirmed that BD-2 is a direct cytochrome-bd inhibitor. Furthermore, the BD-2/Q203 combination was effective against intracellular bacteria in an in vitro macrophage infection model based on THP-1 cells, with over 90% reduction in bacterial load achieved at clinically relevant concentrations. This study supports the hypothesis of combining a cytochrome-bd inhibitor and Q203 in tuberculosis therapy as a rational, mechanistically driven approach that might result in more efficient treatment and overcome antibiotic tolerance.