Discovery and Mechanistic Study of PafA Inhibitors.

Tuberculosis is caused by the bacterium () and is ranked as the second killer infectious disease after COVID-19. Proteasome accessory factor A (PafA) is considered an attractive target because of its low sequence conservation in humans and its role in virulence. In this study, we designed a mutant of PafA that enabled large-scale purification of active PafA. Using a devised high-throughput screening assay, two PafA inhibitors were discovered. ST1926 inhibited PafA by binding in the Pup binding groove, but it was less active against PafA because the ST1926-binding residues are not conserved. Bithionol bound to the conserved ATP-binding pocket, thereby, inhibits PafA in an ATP-competitive manner. Both ST1926 and bithionol inhibited the growth of an attenuated strain (H37Ra) at micromolar concentrations. Our work thus provides new tools for tuberculosis research and a foundation for future PafA-targeted drug development for treating tuberculosis.