The small molecule regacin binds a conserved pocket in RegA to inhibit the expression of bacterial virulence factors

Antivirulence therapeutics are a promising strategy to combat rising antimicrobial resistance. Transcription factor proteins have shown merit as antivirulence drug targets, as demonstrated by the small molecule Savirin. Regacin was identified as a potent inhibitor of the AraC-family virulence regulator (AraC-VR) RegA of Citrobacter rodentium, providing proof-of-concept for targeting AraC-VR family members in related human enteric pathogens. The structural basis of RegA inhibition by regacin was previously undefined and could prove essential for future drug design campaigns targeting AraC-VR family members. This work reveals that RegA is structurally similar to its homologs ToxT and Rns, including a conserved ligand-binding pocket in the N-terminal domain. A regacin-RegA co-crystal structure shows regacin binds within this pocket, with some heterogeneity in binding pose. Finally, structure-guided mutagenesis of key pocket residues, validated using a {beta}-galactosidase reporter assay, confirmed their role in regacin-mediated inhibition. Together, these findings define the regacin binding site, suggest a molecular basis of RegA inhibition, and provide a framework for future inhibitor design targeting this conserved pocket in other virulence regulators in human enteric pathogens.