## 3 Overview of the Inhibitors Targeting the OxPhos Pathway
### Inhibitors of the Cyt-bcc-aa3 Complex
The morpholino-thiophenes were identified from the Lilly corporate library that was screened in an aerobic whole-cell phenotypic setting against _M. tb_. The series was extensively profiled and optimised, with the lead of this series having an MIC\({}_{90}\) of 0.24 uM against _M. tb_, a high-selective index and improved microsomal stability [134]. In an acute model of TB infection, the lead compound reduced the bacterial burden by 0.8 log CFUs in mouse lungs. Cross-resistance studies of the compounds to _M. tb_ strains harbouring QcrB T313I and M342T mutations indicated that the compounds interact with the quinol binding site of QcrB. 4-amino-thieno[2,3-d]pyrimidines were found to inhibit the growth of _M. smeg_ in a screen of small-molecule nucleotide mimetics from ChemBridge Corporation. The most potent compound of the series, CWHM-1023, had an IC\({}_{50}\) of 0.083 uM against _M. tb_[135]. Resistant mutants raised to CWHM-1023 in _M. tb_ had mutations A178T, A178V, V338G, G175S or G315S in QcrB, and this target was further confirmed by the increased susceptibility of a _M. tb_ strain lacking Cyt-bd oxidase to the compounds. The most recent class of Cyt-bcc-aa3 inhibitors identified are the quinazoline derivatives, 2-ethylthio-4-methylaminoquinazolines [77]. The most potent derivatives have activity against in vitro and intracellular _M. tb_ in the micromolar range, and have low cytotoxicity in human hepatocytes. The lead compound 11726148 has a low clearance in human microsomes and was active in an acute TB infection model. Whole-genome sequencing of _M. tb_ resistant mutants to the quinazoline derivatives revealed mutations Trp312 Gly and Gly175 Ser in QcrB. Interestingly, one escape mutant has a SNP in qcrA leading to the substitution of Leu356 Val in QcrA, the Rieske iron-sulphur protein of the Cyt-bcc-aa3. Mapping of the mutated residues on a model of the _M. smeg_ Cyt-bcc-aa3 complex [58] indicated that all of the mutated residues, including Leu356 Val in QcrA, map to the Qp site of QcrB. This is in line with the Q-cycle model, in which quinol oxidation occurs at the interface of cytochrome b and the 2 Fe-2S cluster domain of the Rieske protein, which make up the catalytic Qp site [57; 157]. Cross-resistance studies confirmed the target of QcrB, and also reveal the role of Leu356 Val in QcrA in its interaction with other QcrB inhibitors, namely Q203, AX-35, and LPZS. With the implication of QcrA in the pharmacological inhibition of Cyt-bcc-aa3, novel inhibitors can be generated which target both QcrB and QcrA subunits using structure-assisted drug-design to decrease the likelihood of resistance to QcrB inhibitors. Within this novel class, lead compound 11726148 appears the most amenable for further optimisation, as its phenyl moiety can be changed on different heterocycles to improve pharmacological properties. The Qp site of the Cyt-bcc-aa3 complex is particularly susceptible to chemical inhibition, as evident from the multitude of structurally diverse and distinct compounds detailed above. It has been consistently observed across several of the studies that characteristic consequences of cytochrome bcc inhibition include a general depletion of intracellular bacterial ATP levels, an upregulation of Cyt-bd oxidase, and an increase in OCR and bacterial respiration due to Cyt-bd [12; 76; 77; 134; 135; 136; 137; 138; 139; 140; 135; 155]. This compensation by the alternate terminal oxidase leads to an incomplete respiratory shutdown in _M. tb_, resulting in the bacteriostatic nature of cytochrome bcc inhibitors alone. However, with the deletion of Cyt-bd, mycobacterial respiration is effectively blocked, and cytochrome bcc inhibitors become bactericidal [65; 76; 77]. Taken altogether, these concerted efforts have generated a potential pool of backup cytochrome bcc inhibitors as Q203 continues in clinical trials. Additionally, these findings have highlighted the extreme vulnerability of _M. tb_ without both of its terminal oxidases, and the immense potential of both respiratory branches as drug targets.