3.2.3. MenA

Several inhibitors have been identified to target MenA. Aurachin RE is a natural product from *Rhodococcus erythropolis* JCM6824, and harbours potent activity against a wide range of Gram-positive and Gram-negative bacteria, including *Corynebacterium glutamicum* [121]. Using the aurachin RE scaffold, Debnath et al. synthesised a series of more than 400 derivatives that were assessed for their ability to specifically inhibit the *M. tb* MenA enzyme [49]. Compounds with IC50 lower than 20 μM against *M. tb* and *M. smeg*, and without activity against *Staphylococcus aureus* MenA enzyme, were identified [49]. The lead compound of this series, (R)-13, had a MIC of 2.3 μg/mL against *M. tb*. The compound is also bactericidal against hypoxic, non-replicating mycobacteria, suggesting a high MK turnover rate during persistence.

Ro 48-8071, a (4-bromophenyl)[2-fluoro-4-[[6-(methyl-2-propenylamino)hexyl]oxy] phenyl] methanone developed by Hoffman La Roche Inc is a known inhibitor of oxidosqualene cyclase, which is involved in cholesterol biosynthesis in mammalians. It is effective against *M. tb*, *M. bovis* BCG and *M. smeg* [46,53]. The target of Ro 48-8071 was inferred from metabolic labelling experiment in *M. bovis BCG* [46]. The synthesis of a neutral, apolar liquid, which was biochemically identified as MK through the incorporation of radiolabelled isoprenoid precursors and methionine, was inhibited in the presence of Ro 48-8071 [46]. Moreover, when *M. smeg* was treated with Ro 48-8071, a reduction of 2.5–3.3-fold in the concentrations of DMK-9 and MK-9 was observed, suggesting that the compound inhibits the late steps of MK biosynthesis in mycobacteria. Ro 48-8071 decreased the oxygen consumption in *M. smeg* and *M. tb*, a phenotype that was rescued by MK supplementation. To validate MenA as the direct target of Ro 48-8071, MenA activity was assessed in membranes prepared from a recombinant *E.coli* strain expressing the *M. tb* MenA [46]. An eight-fold decrease in MenA activity was observed upon treatment with Ro 48-8071, indicating that MenA is the target of this inhibitor. Structure-activity relationship (SAR) studies led to the derivative CSU-20 which has improved MIC against *M. tb* and was bactericidal against hypoxic, non-replicating mycobacteria [46]. Dhiman et al. showed that Ro 48-8071 has dual activity against MenA by behaving as a non-competitive inhibitor of the substrate DHNA, but also as a competitive inhibitor of isoprenyl diphosphate [122].

The bicyclic long-chain fatty acids 7-methoxy-2-naphthol-based inhibitors also target MenA [117,123,124]. Berube et al. tested four compounds of this series (NM-1 to NM-4) for their ability to inhibit MenA and *M. tb* growth. The most active compound, NM-4, had an MIC90 of 4.5 μM and was bactericidal against replicating and non-replicating *M. tb* [117]. NM-4 was synergistic with BDQ, CFZ and imidazopyridine ND-10885 in vitro against *M. tb*, with culture sterilisation observed after 21 days of incubation. This new lead is non-cytotoxic, specific for Gram-positive bacteria, and amenable to chemical optimisation, properties that could be exploited to optimise the chemical series [124]. Further characterisation work on the PK/PD properties and in vivo activity remains to be undertaken.
