A chemical strategy for inhibiting Vibrio quorum sensing and virulence.

PROJECT SUMMARY Rates of the human disease vibriosis caused by Vibrio bacterial infections are steadily increasing world- wide due to rising ocean temperatures, surges in Vibrio abundance, and broader bacterial distribution in aquatic ecosystems. Due to multi-drug resistance, there is an urgent need to develop alternative and innovative strategies beyond standard antibiotics for treating Vibrio infections. A major pathway that drives pathogenesis in Vibrios is quorum sensing (QS): bacterial cell-cell chemical communication. QS signaling culminates in the production of a TetR-type master transcription factor collectively called the SmcR family, which regulates genes required for infection of host organisms. Thus, it is postulated that SmcR-type proteins are optimal targets for drug development to inhibit QS and treat vibriosis. Previous work by the van Kessel group showed that thiophenesulfonamide compounds such as PTSP (3-phenyl-1-(thiophen-2-ylsulfonyl)-1H- pyrazole) are potent and specific inhibitors that bind in the ligand binding pocket of SmcR-type proteins in multiple Vibrio pathogens. The long-term goal of this work is to develop PTSP and its derivatives into potent anti-QS molecules that treat vibriosis infections in humans. The van Kessel and Paczkowski groups collaborated on structure-function analyses to identify PTSP-interacting residues in the ligand binding pocket required for inhibition of SmcR activity in vivo. The results show that thiophenesulfonamides specifically bind SmcR proteins in multiple Vibrios, alter the conformation of the DNA binding domain, and promote protein degradation, thereby suppressing downstream gene expression. Collectively, these substantial preliminary data implicate ligand binding as a mediator of SmcR protein stability to govern virulence gene expression in Vibrio pathogens. However, the ligand binding pocket of SmcR proteins differ widely among Vibrios and the native ligand for the SmcR family of proteins is unknown. Thus, optimal inhibitor design for multiple Vibrio species will require a thorough understanding of SmcR-ligand interactions. The central hypothesis is that the active conformation of SmcR is driven by native ligand binding. The objective of this proposal is to use computational, biochemical, genetic, and biophysical assays to determine the interactions that drive SmcR- ligand specificity via three aims: 1) perform structure-activity relationship analyses of a thiophenesulfonamide library against the SmcR family, 2) determine the structural basis of ligand-induced conformational changes in SmcR proteins, and 3) identify the native ligand(s) of SmcR. These experiments will determine the molecular changes to Vibrio SmcR proteins induced by ligand binding, which will facilitate inhibitor optimization to enhance efficacy, stability, and pharmacokinetic properties for drug design treatments for vibriosis disease.