Development of a whole‐cell high‐throughput phenotypic screen to identify inhibitors of mycobacterial amino acid biosynthesis

Anti‐tubercular drug discovery continues to be dominated by whole‐cell high‐throughput screening campaigns, enabling the rapid discovery of new inhibitory chemical scaffolds. Target‐based screening is a popular approach to direct inhibitor discovery with a specified mode of action, eliminating the discovery of anti‐tubercular agents against unsuitable targets. Herein, a screening method has been developed using Mycobacterium bovis BCG to identify inhibitors of amino acid biosynthesis. The methodology was initially optimized using the known branched‐chain amino acid biosynthetic inhibitors metsulfuron‐methyl (MSM) and sulfometuron‐methyl (SMM), and subsequently, whole genome sequencing of resistant mutants and the use of over‐expressor strains confirming their mode of action. The GlaxoSmithKline compound library of small molecule inhibitors with known activity against Mycobacterium tuberculosis was then used to validate the screen. In this paper, we have shown that media supplementation with amino acids can rescue M bovis BCG from known amino acid synthesis inhibitors, MSM and SMM, in a pathway specific manner. The therapeutic potential of amino acid biosynthesis inhibitors emphasizes the importance of this innovative screen, enabling the discovery of compounds targeting a multitude of related essential biochemical pathways, without limiting drug discovery toward a single target.