*3.5. Inhibitors of the F1F0 ATP Synthase*

The F1F0 ATP synthase is a clinically-validated drug target in *M. tb*, since the approval of BDQ by the US FDA for the treatment of multi-drug resistant TB. The mechanism of action of BDQ has been extensively studied in mycobacteria [20,83,162–164]. Briefly, BDQ binds to the c-subunit and the ε-subunit of the ATP synthase by mimicking key residues in the proton transfer chain and blocking the rotary movement of the c-subunit during the catalysis of ATP [165,166], disrupting a fundamental process for bacterial survival in both actively growing and non-growing states [20,162]. The inhibition of the ATP synthase leads to ATP depletion and also dissipation of the membrane potential, as BDQ was shown to have uncoupling properties mediated by the H+/K+ antiporter [167]. Unfortunately, drug resistance to BDQ has already emerged in MDR-TB patients, with several

mechanisms of resistance having been reported [27,168–170]. Predominantly, mutations in atpE, the transcriptional repressor of the MmpL5-MmpS5, and the coding region of this efflux pump have been identified in humans [170–173]. In light of the emergence of resistance and the cardiotoxicity (QT prolongation) associated with BDQ treatment [174], efforts are currently ongoing to develop optimised diarylquinoline analogues (Table 4). The development of BDQ analogues 3,5-dialkoxypyridines is particularly interesting [175–180]. The preclinical candidate of this series, TBAJ-876, is less cardiotoxic than BDQ and 10 times more potent [180]. Through the isolation of resistant mutants and NMR studies, TBAJ-876 was shown to target the c- and ε-subunits of the ATP synthase at the same binding site as BDQ, indicating that this new analogue retains the mechanism of action of BDQ [180]. Kumar et al. screened a set of 700 compounds from the CSIR-IIIM repository for inhibition of mycobacterial ATP synthase activity using IMVs of *M. smeg*, leading to the identification of two compounds, the thiazolidine 5228485 and the cyclohexanediones 5220632. Both compounds inhibited ATP synthesis in IMVs and had MICs in the low micromolar range [181]. These bactericidal compounds were active against drug-resistant *M. tb* strains and non-replicating *M. tb* without apparent cytotoxicity [181]. Mutants selected against each compound were cross-resistant to BDQ, although resistance studies additionally suggest that these compounds may inhibit a secondary target besides the ATP synthase [181]. Further SAR and mechanistic studies are needed to fully understand the mechanism of action and for the development of these two new classes of ATP synthase inhibitors.


**Table 4.** Inhibitors of the ATP synthase discussed in this review.


**Table 4.** *Cont*.

Squaramides were identified in the same screen as imidazo[1,2-a]pyridine ethers (IPE) (see section inhibitors of the Cyt-bcc-aa3) [127], with both compounds identified as inhibitors of ATP synthesis. Isolation of mutants resistant to squaramides revealed the presence of mutations in the αand c- subunits of the ATP synthase. Interestingly, these mutants showed no cross-resistance to BDQ, suggesting a different mode of interaction with the ATP synthase than BDQ. Docking analysis of the most potent squaramide derivative, 31f (MIC *M. tb* 0.8 μM), showed that squaramides bind at the interface of the α and c -subunits of the ATP synthase. Compound 31f shows good accumulation in the serum in mice at a concentration above the MIC for over 15 h when administered with 100 mg/kg ABT, and was bacteriostatic in an acute model of TB. Further optimisation of the PK properties and in vivo safety is needed to advance this compound as a potential pre-clinical candidate for TB.
