Fig. 1. Whole cell phenotypic screening and target identification. A) Drug-to-target drug discovery generally exploits whole cell phenotypic screening, which involves the incubation of a mid-log culture of mycobacteria with a library of compounds at a specified concentration (usually 10–20 μM), in a multi-well plate format to allow for high-throughput. Cell viability can be established by the microplate alamar blue assay, where a non-fluorescent resazurin (blue) is reduced to a fluorescent resorufin (pink) by living cells. B) Following the discovery of an anti-tubercular compound, various methods can be used to establish the target. These include spontaneous resistant mutant generation against the compound followed by (i) WGS to identify resistance conferring mutations, or (ii) changes in gene expression profiles, such as the up-regulation of the target gene. Alternatively, mass spectrometry can be exploited: (iii) in a chemoproteomics approach, inhibitor-bound matrices can be used to pull down competitively binding proteins from a cell lysate; (iv) metabolomic analysis of drug-treated cell culture can be used to identify changes to the metabolome, for example the reduction of a product from an inhibited pathway. In morphological profiling (v), cells are treated with drugs that have known and unknown modes of action and the morphological features analysed. Comparisons of the two groups can reveal target pathways. This list of target identification methods is not exhaustive.