*3.4. Inhibitors of Cyt-bd Oxidase*

Aurachin D is a quinone analogue of the aurachin class that inhibits Cyt-bd oxidase of *E. coli* [136] (Table 3). When tested against membrane vesicles of *M. smeg*, aurachin D demonstrated dose-dependent inhibition of oxygen consumption of up to 50% [159]. Oxygen consumption was further inhibited by 90% in membrane vesicles without QcrCAB, indicating the likelihood of aurachin D targeting Cyt-bd in mycobacteria as well. On its own, aurachin D did not display activity against replicating *M. smeg* nor *M. tb* [159,160]. In spite of the lack of observable activity alone, aurachin D in combination with Q203 resulted in a ~10-fold decrease of the MIC of Q203 against *M. tb*, as well as enhanced bactericidal killing of *M. tb* by >2 log10 CFUs, demonstrating that aurachin D does indeed potentiate the activity of Q203 [160]. However, it remains to be demonstrated that aurachin D is a specific Cyt-bd inhibitor in mycobacteria as given its structural relation with MK, it may interfere with other respiratory complexes. This is particularly important since it was recently demonstrated that small-molecules are able to enhance the bactericidal potency of QcrB inhibitors without targeting the Cyt-bd [161].

It was shown that genetically inactivating both terminal oxidases completely abolished the in vitro growth of *M. tb* [35]. This also dramatically impacted bacterial fitness in vivo in mice, with a strain having a knockdown of Cyt-bcc-aa3 and a knockout of Cyt-bd being unable to establish an initial infection [35]. Even if an initial infection could be first achieved by regulating the expression of one of the terminal oxidases, the absence of both terminal oxidases led to severe persistence defects, with a decrease of five orders of magnitude in bacterial load in 35 days [35]. These findings are consistent with previous reports, whereby the joint inactivation, either pharmacologically or genetically, of both cytochrome bcc and bd oxidases resulted in bactericidal effects on *M. tb* in vitro and in vivo [65,76,77,160]. Exploiting the synthetic lethal interaction between these two terminal oxidases is an extremely attractive approach to eradicate *M. tb*. Inhibitors of this alternate terminal oxidase may have been missed in previous screens, which may be due to the fact that many screens have been performed under conditions in which the Cyt-bd is non-essential. In light of this, screening under stressed conditions may lead to the identification of Cyt-bd inhibitors amenable to chemical optimisation.
