*6.2. Mode of Action*

Sharma et al. demonstrated the role for lipidomics in mechanism of action studies by mapping the impact of the natural antimycobacterial compound vanillin in *M. smegmatis*. The authors observed that vanillin changes the composition of fatty acids, glycolipids, glycerophospholipids and saccharolipids causing disruption of cell membrane homeostasis [108]. Similarly, lipidomic analysis has been instrumental in investigating the consequences of *M.tb* drug-resistance conferring mutations to well-characterized anti-TB drugs [109]. Howard et al. demonstrated that rifampicin-resistant *M.tb* isolates with mutations in *rpoB* exhibited altered lipid profiles dependent on the specific location of the SNPs [109]. Lipidomic analysis showed that different *rpoB* SNPs resulted in distinct relative abundances of short-chain and long-chain fatty acid phthiocerol dimycocerosates (PDIM), which induced alternative macrophage activation pathways and altered macrophage metabolism. Such analyses are useful not only to understand the mode of action of a compound, but to deconvolute the consequences of drug resistance-conferring mutations. Lipidomics is often used as an unbiased approach to complement findings from other methodologies. An example is the evaluation of the mycolic acid transporter, MmpL3, as a druggable target [110]. MmpL3 is an integral inner membrane transporter, with a role in the export of mycolic acids to the periplasmic space in the biosynthesis of the mycobacterial cell wall. Lipidomic analysis of *mmpL3 M.tb* knockdown mutants using thin layer chromatography of total lipids revealed a fast decline in cell wall-bound mycolic acids and trehalose dimycolates. Combined with confirmation of in vitro and in vivo essentiality, this study confirmed the mycolic acid transporter MmpL3, as a validated druggable target for *M.tb* drug discovery. Similarly, lipidomics, alongside genomics and transcriptomics, characterized the mode of action of HC2091, a novel compound that likely targets MmpL3. *M.tb* HC2091-resistant mutants were shown to have SNPs in the *mmpL3* gene, likely conferring drug resistance. Thin layer chromatography of the total extractable lipids from HC2091-treated *M.tb* cultures supplemented with radiolabelled-sodium acetate demonstrated a dose-dependent reduction in trehalose dimycolate accumulation with increasing concentration of HC2091. In combination with transcriptional profiling of HC2091-treated bacilli, the authors confirmed that HC2091 targets MmpL3 through a mechanism distinct from other MmpL3 inhibitors [66]. Lipidomics, therefore, often in combination with other omics approaches, is an effective tool, especially in the interrogation of cell wall biosynthetic pathways, a rich source of druggable *M.tb* targets.
