*5.2. Mode of Action*

The utility of metabolomics is demonstrated in studies to elucidate the mode of action of novel compounds. In a high-throughput metabolomics approach, Zampieri et al. evaluated mass spectra of supernatants from drug-treated *M. smegmatis* cultures to profile a library of 212 antimycobacterial compounds with unknown modes of action [97]. The metabolomic signatures were first established for 62 reference compounds with 17 known targets, before assessing similarity to the test compound profiles. Over 70% of the 212 compounds could be classified with a known mechanism of action, whilst 16 compounds resulted in metabolomic profiles dissimilar to the reference compounds. Of these 16 compounds, 6 exhibited a similar metabolomic response suggestive of the inhibition of lipid and trehalose metabolism. This approach revealed new druggable pathways in *M.tb*, and importantly enables drug discovery programs to diversify target pathways, discarding molecules that likely inhibit targets of existing drugs. In this study, the compounds with unknown mechanisms of action exhibited modest inhibitory activity against *M. smegmatis* with unique metabolic patterns that likely reflect specificity in their underlying modes of action. Whilst further studies should be directed at assessing the activity and mechanism of action of these compounds in *M.tb*, this study clearly demonstrates the utility of this approach to recognize compounds that target novel pathways. In a similar approach, untargeted metabolite profiling using flow infusion electrospray ion high resolution mass spectrometry was used to explore the mode of action of pretomanid [98]. The *M. smegmatis* metabolite profile after exposure to pretomanid was distinct when compared to ampicillin, ethambutol, ethionamide, isoniazid, kanamycin, linezolid, rifampicin and streptomycin-treated cultures. Mapping of differentially abundant metabolites onto pathways highlighted the pentose phosphate pathway, suggesting that accumulation of the toxic metabolite methylglyoxal may contribute to the antibacterial activity of pretomanid. A recent LC-MS-based metabolic linkage analysis of bedaquiline-treated *M.tb* revealed that, alongside inhibition of ATP synthase, glutamine metabolism was also impacted. Since glutamine synthesis inhibitors were synergistic in combination with bedaquiline, an indirect secondary effect of bedaquiline on glutamine biosynthesis was distinguished that could be targeted therapeutically [99]. These approaches demonstrate how metabolomics may be used to elucidate the action of unknown drugs, and reveal fundamental information about the physiology of *M.tb*.
