*3.1. NADH Dehydrogenases*

NADH dehydrogenases play a major role in maintaining mycobacterial respiratory chain energization by using MK as an electron carrier. NDH-1 is non-essential for mycobacterial growth and for persistence [19,34]. Therefore, no drug development is currently in progress for inhibitors of mycobacterial NDH-1 [39]. NDH-2 is widespread in bacteria and the mitochondria of fungi, plants and some protists. In some organisms, more than one copy is present [91]. Notably, NDH-2 (Ndh and NdhA) are absent in mammalian genomes, suggesting a potential leading target for anti-mycobacterial drug development.

Several inhibitors have been found to target NDH-2 in mycobacteria, including the phenothiazines, quinolinyl pyrimidines and 2-mercapto-quinazolinones (Table 1). It is also thought that NDH-2 plays a role in the reduction of the anti-leprosy drug clofazimine (CFZ), leading to the production of reactive oxygen species (ROS) and, consequently, its bactericidal activity against *M. tb* [92]. However, CFZ is still potent against a *M. tb* Δndh-2 mutant strain, indicating that CFZ does not require activation by NDH-2 to exert its anti-mycobacterial potency [35]. Further studies will be needed to understand the mechanism of action of CFZ against mycobacteria. The CFZ analogue TBI-166 is more potent than CFZ against *M. tb in vitro,* and it is at least as potent as CFZ in acute and chronic murine models of TB while being potentially associated with less skin discoloration [93]. A study by Beites et al. demonstrated that the inactivation of both NDH-2 (Δndh-2) can be achieved in the absence of fatty acids in the growth media. Interestingly, rotenone, an inhibitor of NDH-1, has bactericidal activity against Δndh-2, which suggests that NDH-1 and NDH-2 have a redundant role in *M. tb* [35].

The phenothiazines, a class of antipsychotic drugs, including thioridazine, chlorpromazine and trifluoperazine, have been widely studied against *M. tb*. They have activity in vitro against *M. tb* with minimum inhibitory concentration (MIC) of thioridazine, chlorpromazine and trifluoperazine ranging between 5 to 32 μg/mL [94–98]. However, their limited potency coupled with an unfavourable toxicological profile preclude clinical use for TB treatment [99]. However, phenothiazines accumulate in macrophages and are particularly potent against intracellular *M. tb* [100,101]. Current efforts aim at developing new phenothiazine derivatives with improved activity against *M. tb* and reduced toxicity [102–105].

Another class of NDH-2 inhibitors is the quinolinyl pyrimidines [106]. This class of compounds was identified during an NDH-2 target-based high-throughput screening of more than 100 000 compounds. The series showed a good inhibitory range against *M. tb* in vitro, with MIC50 below 1 μg/mL [106]. In contrast to the phenothiazines, the quinolinyl pyrimidines series is not hemolytic.

CBR-1825 and CBR-4032 are two representatives compounds of the thioquinazoline (TQZ) and tetrahydroindazole scaffolds, respectively, that were identified in a high-throughput screen of over 800 000 compounds in mycobacterial inverted membrane vesicles (IMVs) [29]. The assay was designed to identify small-molecules interfering with ATP production. CBR-1825 and CBR-4032 had an MIC50 of 0.43 μM and 6.6 μM, respectively, when tested, and no apparent cytotoxicity [29]. Both compounds are bactericidal against *M. tb* in vitro and seem to perturb NADH turnover. Sequencing of three escape mutants resistant to TQZ revealed a non-synonymous mutation in the promoter region of ndhA (rv0392c), suggesting that these compounds target NDH-2.

A 2-mercapto-quinazolinones cluster of hits (1, 2, and 3) was identified in a screening of a commercial diversity library [30]. Compound 1 potently inhibiting *M. tb* growth in vitro in the low micromolar range without cytotoxicity against HepG2 cells [30]. Bioenergetic analyses conducted in cells in which the membrane potential was uncoupled from ATP production revealed a decrease in oxygen consumption rates (OCR) in response to the inhibitor, while IMVs showed mercapto-quinazolinonedependent inhibition of ATP production when NADH was the primary electron donor of the respiratory chain. Enzyme kinetic studies further demonstrated non-competitive inhibition of recombinant *M. tb* Ndh protein [30]. Resistance to the compounds in *M. tb* was conferred by promoter mutations in ndhA. Interestingly, hypersusceptibility to this class of compounds was observed when ndhA was deleted, suggesting that NDH-1 and other electron donors cannot compensate for the inhibition of NDH-2 by 2-mercapto-quinazolinones [30]. Chemical optimisation is required to improve the pharmacokinetics (PK) properties of this interesting series [30].

Quinolinequinones (QQ) exert mycobactericidal activity through NDH-2 inhibition. Interestingly, QQ prevent the emergence of persistent cells in a time- and dose-dependant manner in *Mycobacterium bovis* BCG in vitro [107]. This scaffold is known for its anti-cancer and anti-inflammatory properties, and can be modified to give rise to derivatives with anti-mycobacterial activity [108–110]. Mulchin BJ et al. synthesised a range of 6,7-substituted-5,8-quinolinequinones, and their anti-mycobacterial activities were assessed against *M. bovis* BCG, in addition to their anti-tumour and anti-inflammatory properties [111]. Several compounds containing amine and halogen functionality exhibited tuberculostatic activity in the range 3–12.5 μg/mL [111]. The derivative QQ8c stimulated NADH oxidation in *M. tb* and *M. smeg* IMVs. Ndh overexpression enhanced the stimulation of NADH oxidation [107]. This increase in NDH-2 catalytic activity has been associated with the production of lethal concentrations of ROS. Recently, Santoso et al. synthesised a library of 32 new QQ derivatives, with one derivative (16b) showing enhanced in vitro activity against *M. tb* [112]. Activation of NADH-dependent oxygen consumption in *M. smeg* IMVs in the presence of 16b suggests that this new inhibitor has the same anti-mycobacterial mechanism of action than QQ8c [112].


**Table 1.** Structures of the NADH dehydrogenase inhibitors discussed in this review.


**Table 1.** *Cont*.

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

The essentiality of this pathway makes it an attractive target for anti-TB drug development. To date, chemical inhibitors of MenA, MenB, MenG, MenD and MenE have proven efficacious in inhibiting *M. tb* growth. Inhibitors of the MK biosynthesis discussed in this review are listed in Table 2.


**Table 2.** Menaquinone (MK) biosynthesis inhibitors discussed in this review.
