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The F1 FO -ATP synthase is required for growth and viability of Mycobacterium tuberculosis and is a validated clinical target. A mycobacterium-specific loop of the enzyme's rotary γ subunit plays a role in the coupling of ATP synthesis within the enzyme complex. We report the discovery of a novel antimycobacterial, termed GaMF1, that targets this γ subunit loop. Biochemical and NMR studies show that GaMF1 inhibits ATP synthase activity by binding to the loop. GaMF1 is bactericidal and is active against multidrug- as well as bedaquiline-resistant strains. Chemistry efforts on the scaffold revealed a dynamic structure activity relationship and delivered analogues with nanomolar potencies. Combining GaMF1 with bedaquiline or novel diarylquinoline analogues showed potentiation without inducing genotoxicity or phenotypic changes in a human embryonic stem cell reporter assay. These results suggest that GaMF1 presents an attractive lead for the discovery of a novel class of anti-tuberculosis F-ATP synthase inhibitors. National Research Foundation (NRF) Accepted version This research was supported by the National ResearchFoundation (NRF) Singapore,NRF Competitive Research Programme (CRP), Grant Award Number NRF-CRP18-2017-01. A. Hotra is grateful to receive an IGS PremiumScholarship,Institute of Technology in Health and Medicineat NTU,and J. P. Sarathy received aPhD scholarship from theSchool of Medicine,NUS,Singapore.Wethank Dr. YongxinLi for the X-ray crystallographic structure determinations.

The F1FO-ATP synthase is one of the enzymes that is essential to meet the energy requirement of both the proliferating aerobic and hypoxic dormant stages of the life cycle of mycobacteria. Most F-ATP synthases consume ATP in the α3:β3 headpiece to drive the γ subunit, which couples ATP cleavage with proton pumping in the c ring of FO via the bottom of the γ subunit. ATPase-driven H+ pumping is latent in mycobacteria. The presence of a unique 14 amino acid residue loop of the mycobacterial γ subunit has been described and aligned in close vicinity to the c-ring loop Priya R et al. (2013) J Bioenerg Biomembr 45, 121-129 Here, we used inverted membrane vesicles (IMVs) of fast-growing Mycobacterium smegmatis and a variety of covalent and non-covalent inhibitors to characterize the ATP hydrolysis activity of the F-ATP synthase inside IMVs. These vesicles formed a platform to investigate the function of the unique mycobaterial γ loop by deleting the respective loop-encoding sequence (γ166–179) in the genome of M. smegmatis. ATP hydrolysis-driven H+ pumping was observed in IMVs containing the Δγ166–179 mutant protein but not for IMVs containing the wild-type F-ATP synthase. In addition, when compared to the wild-type enzyme, IMVs containing the Δγ166–179 mutant protein showed increased ATP cleavage and lower levels of ATP synthesis, demonstrating that the loop affects ATPase activity, ATPase-driven H+ pumping and ATP synthesis. These results further indicate that the loop may affect coupling of ATP hydrolysis and synthesis in a different mode. NMRC (Natl Medical Research Council, S’pore) MOH (Min. of Health, S’pore) Accepted version