Design, Synthesis, and Antimicrobial Activity of Novel Cyt- <i>bd</i> Inhibitors Targeting Energy Compensation in <i>Mycobacterium tuberculosis</i>

Abstract Drug-resistant Mycobacterium tuberculosis (Mtb) poses a growing threat, necessitating new therapeutic strategies. The cytochrome bcc-aa3 oxidase (Cyt-bcc) is an attractive target, yet single-agent inhibition fails owing to compensation by the alternative cytochrome bd terminal oxidase (Cyt-bd). Herein, we describe a structure–activity relationship (SAR) study starting from the previously identified Cyt-bd inhibitor 12, leading to the discovery of 17aa (YH-685). YH-685 potently inhibits Cyt-bcc deficient Mycobacterium smegmatis (Δbcc) with a MIC of 0.8 μM and suppresses Cyt-bd dependent oxygen consumption in inverted membrane vesicles with an IC50 of 0.45 μM. When combined with the Cyt-bcc inhibitor TB47, YH-685 enhanced inhibition of virulent Mtb H37Rv, reducing both planktonic growth and intracellular bacterial burdens in human macrophages, as assessed by high-content imaging. Under the tested in vitro conditions, this combination achieved growth inhibition comparable to standard regimens rifampicin–isoniazid and bedaquiline. Our study supports co-targeting Cyt-bd and Cyt-bcc as a therapeutic strategy for drug-resistant tuberculosis.