Dual inhibition of the terminal oxidases eradicates antibiotic‐tolerant Mycobacterium tuberculosis

The approval of bedaquiline has placed energy metabolism in the limelight as an attractive target space for tuberculosis antibiotic development. While bedaquiline inhibits the mycobacterial F 1 F 0 ATP synthase, small molecules targeting other components of the oxidative phosphorylation pathway have been identified. Of particular interest is Telacebec (Q203), a phase 2 drug candidate inhibitor of the cytochrome bcc:aa 3 terminal oxidase. A functional redundancy between the cytochrome bcc:aa 3 and the cytochrome bd oxidase protects M . tuberculosis from Q203‐induced death, highlighting the attractiveness of the bd ‐type terminal oxidase for drug development. Here, we employed a facile whole‐cell screen approach to identify the cytochrome bd inhibitor ND‐011992. Although ND‐011992 is ineffective on its own, it inhibits respiration and ATP homeostasis in combination with Q203. The drug combination was bactericidal against replicating and antibiotic‐tolerant, non‐replicating mycobacteria, and increased efficacy relative to that of a single drug in a mouse model. These findings suggest that a cytochrome bd oxidase inhibitor will add value to a drug combination targeting oxidative phosphorylation for tuberculosis treatment. Synopsis The functional redundancy of two terminal oxidases in mycobacteria limits the efficacy of phase 2 clinical candidate Telacebec (Q203). In this study we identified a cytochrome bd oxidase inhibitor ND‐011992 that together with Q203 forms a bactericidal drug combination against Mycobacterium tuberculosis . ND‐011992 was identified and validated as an inhibitor of the Cytochrome bd oxidase. ND‐011992 and Q203 jointly enhanced inhibition of oxygen respiration, intracellular ATP depletion, and bactericidal activity. The drug combination was active against M. tuberculosis clinical isolates of various lineages as well as against MDR‐ and XDR‐TB isolates. The addition of ND‐011992 to Q203 treatment did not significantly alter the frequency of resistance to treatment, suggesting there is limited added risk of rapid emergence of escape mutants. The ND‐011992‐Q203 combination showed enhanced killing effect in the animal study compared to that of single drugs, demonstrating translatability in an in vivo model. Graphical Abstract The functional redundancy of two terminal oxidases in mycobacteria limits the efficacy of phase 2 clinical candidate Telacebec (Q203). In this study we identified a cytochrome bd oxidase inhibitor ND‐011992 that togethe