*2.2. Succinate Dehydrogenase*

Succinate dehydrogenase (SDH), also known as complex II of the ETC, couples the oxidation of succinate to fumarate with the reduction of MK to menaquinol (MKH2) in *M. tb* [36]. This complex directly connects the tricarboxylic acid cycle and the OxPhos pathway, thereby playing a critical role in bacterial carbon metabolism and respiration [37]. Despite its significance, there has been a general reluctance to develop inhibitors against this complex due to the presence of a mammalian counterpart and the presence of two isotypes [38]. *M. tb* possesses two canonical SDH enzymes, Sdh1 and Sdh2, and an additional reversible fumarate reductase that can catalyse the reaction in both directions [39]. Independent transposon-site hybridization screens in *M. tb* suggest that Sdh1 is essential for optimal growth under aerobic conditions [32], whereas Sdh2 seems to play a more significant role in the growth arrest and dormancy of *M. tb* during its transition from aerobic to hypoxic conditions [40]. SDH enzymes also appear to function as an important regulator of respiration in *M. tb*. The deletion of sdh1 results in perturbation of respiration, leading to an increased rate of oxygen consumption which prevented recovery of *M. tb* from the stationary phase [41]. While the functional redundancy of these enzymes remains to be fully clarified, it is established that the SDH enzymes in *M. tb* are phylogenetically and biochemically distinct from each other, as well as from the mammalian complex [14,42,43]. On this basis, it would be theoretically possible to selectively inhibit the mycobacterial SDHs without off-target effects, although functional redundancy and structural differences between the mycobacterial SDHs would pose a challenge.

#### *2.3. Menaquinone (MK) Biosynthesis*

As the sole lipoquinone in mycobacteria, MK has a central and essential role in OxPhos [44,45]. Its biosynthetic pathway is a highly attractive drug target space as it is absent in humans, and has been demonstrated to be critical for maintaining mycobacterial survival under aerobic and anaerobic conditions [46]. In *M. tb*, the biosynthesis of MK mirrors that of *Escherichia coli*, in which the pathway has been better characterised [45]. Eight enzymes (MenF, MenD, MenH, MenC, MenE, MenB, MenA and MenG) are involved in the pathway, with MenF catalysing the first committed step of the conversion of chorismate [14]. Not all of the genes encoding for the MK synthesis pathway seem to be essential. Through initial transposon studies and confirmation by high-resolution phenotypic profiling experiments, most MK biosynthesis enzymes have been reported to be essential via transposon mutagenesis in H37Rv for *M. tb* growth, except for MenF, whose deletion resulted in a growth defect in *M. tb* [31–33].

Small molecules inhibiting MenD, MenE, MenB, MenA and MenG, and have been reported [47–51]. MenD is a thiamine diphosphate-dependent enzyme which catalyses the second step of MK biosynthesis. It uses 2-oxoglutarate and isochorismate for the synthesis of 2-Succinyl-5-enolpyruvyl-6 hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC). MenE encodes for an o-succinylbenzoate-CoA ligase that converts o-succinylbenzoate (OSB) to OSB-CoA, and is the fifth step of MK biosynthesis in *M. tb*. It is responsible for the addition of a CoA group via an OSB-AMP intermediate [50]. MenB encodes for a 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA) synthase that catalyses the formation of DHNA-CoA from o-succinylbenzoate-CoA and is the sixth step of the MK biosynthesis [52]. MenA encodes for a DHNA-octaprenyltransferase, which is involved in the seventh step of the MK biosynthesis. It catalyses the conversion of 1,4-dihydroxy-2-naphthoate (DHNA) to demethylmenaquinone (DMK), and uses a variety of allylic isoprenyl diphosphates as substrates, requiring at least three isoprene units [46,49]. Bacterial MenA was reported to be homologous to the eukaryotic UbiA [53]. Additionally known as UbiE, MenG (rv0558) catalyses the methylation of DMK-9 using S-adenosylmethionine to form MK [54]. This reaction is the last step of MK biosynthesis in *M. tb*. That these enzymes of the MK biosynthesis pathway can be pharmacologically inhibited demonstrate their essentiality and highlight their potential as drug targets.
