Discovery of a Small-Molecule Inhibitor Targeting the Biofilm Regulator BrpT in .

, an opportunistic human pathogen, employs biofilm formation as a key survival and virulence mechanism. BrpT, a transcriptional regulator, is essential for biofilm development by regulating the expression of biofilm-related genes. In this study, we aimed to identify a small molecule inhibitor of BrpT to combat biofilm formation. High-throughput screening of 7,251 compounds using an reporter strain carrying the arabinose-inducible gene and a BrpT-activated promoter fused to the operon identified a hit compound, BTI (BrpT Inhibitor). BTI potently inhibited BrpT activity in (EC of 6.48 μM) without affecting bacterial growth or host cell viability. Treatment with BTI significantly reduced the expression of the BrpT regulon and impaired biofilm formation and colony rugosity in , thus increasing its susceptibility to antibiotics. In vitro biochemical analyses revealed that BTI directly binds to BrpT and inhibits its transcriptional regulatory activity. The identification of BTI as a specific inhibitor of BrpT that effectively diminishes biofilm formation provides a promising foundation for the development of novel anti-biofilm strategies, with the potential to address the growing challenge of antibiotic resistance and improve the treatment of biofilm-associated infections.