Two-way regulation of protein expression for identification and validation of on-target inhibitors of Mycobacterium tuberculosis.

Whole-cell screens are a powerful tool to identify small molecules that inhibit bacterial growth by targeting pharmacologically relevant mechanisms. Identification of the target for many of these whole cell hits could often be a complicated and time-consuming process. To supplement Tuberculosis drug discovery effort in this regard, we developed cell-based assays that utilizes two-way regulation of protein expression to readily identify on-target inhibitors of essential genes in Mycobacterium tuberculosis. We successfully used this approach to identify a novel guanidine-based inhibitor of MmpL3, an essential mycolate transporter in the inner membrane of Mycobacterium tuberculosis from a library of 220 compounds. We next extended this approach to categorize inhibitors that either directly or indirectly impact the activity of Biotin Protein Ligase; BirA. This work highlights the value of target-based phenotypic screens to identify novel antimycobacterial inhibitors. These assays have significant utility in facilitating prompt identification of new lead molecules and target-driven optimization of analogues of more advanced scaffolds that inhibit MmpL3 and BirA activity in the cellular context.