Screening of FDA-approved small molecules to discover inhibitors of the Pseudomonas aeruginosa quorum-sensing enzyme, PqsE

Pseudomonas aeruginosa is a notorious pathogen that is a leading cause of hospital-acquired infections. Due to its heightened resistance to a broad range of antibiotics, there is great need for new antimicrobial agents that are effective in treating P. aeruginosa infections. One attractive option for exploring new antibiotic targets is the quorum sensing (QS) pathway, as it governs many of the pathogenic behaviors that allow P. aeruginosa to stage and maintain infections. Within the QS pathway, there is a key protein-protein interaction between an enzyme, PqsE, and one of the master QS regulators, RhlR. Although its catalytic function is dispensable for its interaction with RhlR, previous mutagenic work characterizing the active site of PqsE identified active site mutations that induce a conformational change in PqsE, preventing it from forming a complex with RhlR. These active site mutations, when introduced stably into the genome of P. aeruginosa , also lead to a significant decrease in production of a key toxin, pyocyanin, and prevent colonization in the lungs of a murine host. These findings encourage drug discovery efforts to identify small molecules that bind in the active site of PqsE and induce a conformational change similar to the ones induced by particular amino acid substitutions in the active site. Here, we performed a fluorescence polarization screen to identify molecules that bind in the active site of PqsE. We screened a library of FDA-approved drugs, with the intention of potentially repurposing a known molecule for the treatment of P. aeruginosa infections. Three molecules were identified, two of which showed inhibitory activity that was consistent with a competitive mode of inhibition. One hit molecule, Apomorphine, had a distinctly different inhibitory profile, and is potentially binding outside of the active site to allosterically inhibit enzyme activity of PqsE. All three hit molecules were tested in an in vivo enzyme activity assay, and one of the competitive inhibitors, Vorinostat, was found to inhibit intracellular PqsE. Vorinostat is now being explored as a candidate for synthetic derivatization to inhibit the PqsE-RhlR protein-protein interaction via binding in the PqsE active site.