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

Abstract

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 antibiotictolerant, 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.

The cryo-EM structure of the bd oxidase from M. tuberculosis reveals a unique structural framework and enables rational drug design to combat TB

“…IMVs of the indicated strains were prepared using a cell lysis protocol as described in the purification section. Oxygen consumption was measured using an Oroboros O2k fluorespirometer as previously described 11 . Briefly, IMVs were resuspended in buffer (50 mM Tris, 100 mM KCl, 5 mM MgCl 2 , pH 7.5) and 100 mM NADH was used to initiate respiration.…”

“…This quinol-oxidizing terminal oxygen reductase is encoded by the cydAB gene cluster and is a central component of the branched respiratory chain of M. tuberculosis . The clinical relevance of the cytochrome bd oxidase is highlighted by deletion mutants that show dramatically increased susceptibility to the bc 1 : aa 3 supercomplex inhibitor Q203 5 , 11 . This synergistic lethal interaction between the two respiratory branches of M. tuberculosis demonstrates that cytochrome bd oxidase is a major target for combating multi and extensive drug-resistant (MDR/XDR) tuberculosis infections 12 .…”

“…Targeting simultaneously multiple respiratory enzyme complexes in Mycobacterium tuberculosis is regarded as one of the most effective options to treat TB, shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance 5,6 . In this light, new lines of evidence point to a crucial role of the cytochrome bd oxidase for mycobacterial pathophysiology [7][8][9][10][11] . This quinol-oxidizing terminal oxygen reductase is encoded by the cydAB gene cluster and is a central component of the branched respiratory chain of M. tuberculosis.…”

Impaired succinate oxidation prevents growth and influences drug susceptibility in Mycobacterium tuberculosis

“…To further investigate the contribution of succinate oxidation to total respiratory flux, we used Q203 and ND-011992 to inhibit the activity of both terminal oxidases and abolish respiration (46, 47). The complete inhibition of aerobic respiration highlighted an additional ∼30% respiratory capacity in the endogenous respiration rate of M. tuberculosis in the absence of succinate oxidation (Figure 3D).…”

“…To address this, we considered whether depleting cells of CRISPRi target enzymes prior to antibiotic challenge affected the interactions observed. We exploited the known synthetically lethality between the two terminal oxidases (the cytochrome bc 1: aa 3 complex and cytochrome bd ) (13, 46, 62, 63) as a positive control. Killing of a M. tuberculosis strain expressing a sgRNA targeting cytochrome bd by the bc 1 inhibitors, Q203 and TB47 (64), was only observed when cells were pre-depleted of cytochrome bd , and not when knockdown was induced simultaneously with antibiotic challenge (Figure S2).…”