## 3 Overview of the Inhibitors Targeting the OxPhos Pathway
### Inhibitors of the F\({}_{1}\)F\({}_{0}\) ATP Synthase  
The F\({}_{1}\)F\({}_{0}\) 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; 163; 164]. Briefly, BDQ binds to the c-subunit and the \(\epsilon\)-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; 169; 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 [171; 172; 173; 170]. 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; 176; 177; 178; 179; 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 \(\varepsilon\)-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]. [PERSON] 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 thiazolite 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. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Chemical Class** & **Represented by** & **Structure** & **Ref** \\ \hline Diarylquinoline & BDQ & & \\ TBAJ-587 & B\({}_{\text{N}}\) & & \\ TBAJ-876 & B\({}_{\text{N}}\) & & \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Inhibitors of the ATP synthase discussed in this review. Squaramides were identified in the same screen as imidazole[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 \(\alpha\)- and 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 \(\upmu\)M), showed that squaramides bind at the interface of the \(\alpha\) 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.