Structural basis for inhibition of mycobacterial ATP synthase by squaramides and second generation diarylquinolines

Abstract

Mycobacteria, such asMycobacterium tuberculosis, depend on the activity of adenosine triphosphate (ATP) synthase for growth. The diarylquinoline bedaquiline (BDQ), a mycobacterial ATP synthase inhibitor, is an important medication for treatment of drug-resistant tuberculosis but suffers from off-target effects and is susceptible to resistance mutations. Consequently, both new and improved mycobacterial ATP synthase inhibitors are needed. We used electron cryomicroscopy and biochemical assays to study the interaction ofMycobacterium smegmatisATP synthase with the second generation diarylquinoline TBAJ-876 and the squaramide inhibitor SQ31f. The aryl groups of TBAJ-876 improve binding compared to BDQ, while SQ31f, which blocks ATP synthesis ~10 times more potently than ATP hydrolysis, binds a previously unknown site in the enzyme's proton-conducting channel. Remarkably, BDQ, TBAJ-876, and SQ31f all induce similar conformational changes in ATP synthase, suggesting the resulting conformation is particularly suited for drug binding. Further, high concentrations of the diarylquinolines uncouple the transmembrane proton motive force while for SQ31f they do not, which may explain why high concentrations of diarylquinolines have been reported to kill mycobacteria while SQ31f has not.

A tale of two inhibitors: diarylquinolines and squaramides

“…In their paper, Courbon et al (2023) also examined the activity of squaramide SQ31f on M. smegmatis ATP synthase. SQ31f inhibited ATP hydrolysis without restoring hydrolysis at high concentrations and did not dissipate the PMF in acidified IMVs, contrasting the diarylquinolines compounds.…”

“…The authors noted that this result was surprising since TBAJ‐876 was reported to not induce uncoupling of the PMF (Sarathy et al , 2020). To further probe the activity of these diarylquinoline compounds, Courbon et al (2023) reconstituted M. smegmatis ATP synthase in inverted membrane vesicles (IMVs), which would mimic a lipid bilayer environment. They make two key observations: (i) restoration of hydrolytic activity does not occur when the enzyme is in a lipid bilayer and therefore is an artifact of detergent solubilization, and (ii) TBAJ‐587 and TBAJ‐876 are able to dissipate PMF in acidified IMVs, though at a slower rate compared to BDQ.…”

“…The diarylquinoline bedaquiline (BDQ) is an FDA-approved drug for the treatment of multidrug-resistant tuberculosis that targets the mycobacterial adenosine triphosphate (ATP) synthase, a key enzyme in cellular respiration. In a recent study, Courbon et al (2023) examine the interaction between Mycobacterium smegmatis ATP synthase with the second generation diarylquinoline TBAJ-876 and the squaramide inhibitor SQ31f, showing that both drugs prevent the rotatory motions needed for enzymatic function. M ycobacterium tuberculosis, the causative agent of tuberculosis, is one of the leading causes of death due to infectious disease worldwide.…”

“…In their paper published in this issue of The EMBO Journal, Courbon et al (2023) investigate the biochemical activities of second-generation diarylquinolines TBAJ-587 and TBAJ-876 on Mycobacterium smegmatis ATP synthase. Bactericidal properties of BDQ have been proposed to rely on both its inhibition of ATP synthesis and its uncoupling of the PMF (Hards et al, 2015(Hards et al, , 2018.…”

“…To understand how TBAJ-876 binds ATP synthase, Courbon et al (2023) solved the electron cryomicroscopy (cryo-EM) structure of the M. smegmatis enzyme in the presence of saturating amounts of TBAJ-876. Similar to the structure with BDQ (Guo et al, 2021), this structure reveals that seven molecules of TBAJ-876 bind the F O region: five are bound to "c-only" sites, and two are bound at the "leading" and "lagging" sites at the interface of subunits a and c (Fig 1B -iii).…”