*5.1. Target Identification*

Metabolomics, the analysis of the metabolite network within a biological system, is an indispensable omics approach for drug discovery, providing information on the potentially druggable processes occurring in a cell. Metabolomics in a discovery biology setting has elucidated *M.tb* metabolic pathways in use in different microenvironments. Carvalho et al. supplemented cultures grown aerobically at 37 ◦C with different 13C-labelled carbon substrates followed by LC-MS to separate and identify metabolites, demonstrating that *M.tb* co-catabolizes multiple carbon sources simultaneously, through glycolytic, pentose phosphate and tricarboxylic acid pathways [92]. For example, during co-catabolism of dextrose and acetate, dextrose was preferentially metabolized into intermediates of glycolysis and the pentose phosphate pathway, whereas acetate was preferentially used for tricarboxylic acid cycle (TCA cycle) intermediates. This understanding of the *M.tb* metabolic network will help to delineate key pathways and essential metabolites that could be exploited as therapeutic targets. More recently, Serafini et al. used a similar approach to elucidate the metabolic pathways involved in the assimilation of pyruvate and lactate in *M.tb*. Although it is well-established that lipids are important carbon sources for *M.tb* during infection, the authors demonstrated a novel function for the methylcitrate cycle, highlighting that it could be reversed for the biosynthesis of propionyl-CoA and the metabolism of pyruvate and lactate, identifying new targets for drug discovery efforts [93]. This study is an excellent example of a multi-omics approach, combining transposon-directed insertion site sequencing, RNAseq transcriptomics, proteomics and metabolomics, enabling an multi-analyte functional overview of the carbon metabolism network in *M.tb*.

Agapova et al. coupled stable isotope tracing of labelled amino acids with mass spectrometry to elucidate the use of amino acids as a nitrogen source in *M.tb* [94]. The authors showed that the co-metabolism of multiple amino acids as nitrogen sources did not improve growth compared to metabolism of a single source. In addition, several amino acids were utilized as sole nitrogen sources much faster than ammonium, suggesting that *M.tb* preferentially metabolizes specific host amino acids as sources of nitrogen. As such, metabolomics provided insight into the potential for targeting specific pathways in the *M.tb* nitrogen metabolic network. The authors also suggested that greater emphasis should be placed on amino acids as sole carbon sources to better mimic physiologically relevant conditions found in the host. Borah et al. used 15N-flux spectral ratio analysis to demonstrate that *M.tb* in macrophages has access to multiple amino acids for nitrogen metabolism and identified serine as an amino acid not available to intracellular bacilli [95]. The proteinogenic amino acid serine that provides the nitrogen backbone for glycine and cysteine synthesis must be synthesized by intracellular *M.tb*, highlighting this pathway, and phosphoserine transaminase in particular, as a novel target for drug discovery. In a similar target identification application, Dutta et al. used metabolomics to confirm the role of the stringent response regulator Rel in controlling transition to non-replicating states by comparing wild type *M.tb* with an *M.tb* knockout strain lacking RelMtb, verifying Rel as a potential anti-TB drug target. The authors then screened a library of compounds against this target, identifying lead compounds that killed nutrient-starved *M.tb* [96].
