Mechanisms of Action of LuxO and its Small Molecule Inhibitors

Abstract

LuxO is a hexameric quorum sensing response regulator that controls the virulence of human pathogens including Vibrio cholerae, V. vulnificus, and V. parahaemolyticus. Previously identified small molecule inhibitors of LuxO, which prevent the virulence of Vibrios, are promising drug candidates due to their broad spectrum applicability and the reduced pressure they place on bacteria to develop resistance. This paper elucidates the mechanisms of action of LuxO and its small molecule inhibitors. A high resolution crystal structure of a LuxO construct showed that LuxO is negatively regulated by an N-terminal receiver domain that occupies the LuxO active site when unphosphorylated and leaves the active site upon phosphorylation. Through enzymatic assays and crystallography competitive and uncompetitive inhibitors of LuxO were identified. Models that can quantitatively predict the behavior of different types of inhibitors of oligomeric enzymes were derived. These models in conjunction with enzymatic assays further showed that inhibitor binding to one subunit in a LuxO oligomer renders the entire oligomer inactive – explaining the high inhibitor potencies. The inhibitors were found to inhibit LuxO from pathogenic Vibrios but not from other closely related species, demonstrating that the broad spectrum and potent inhibitors are also specific – making them ideal drug candidates.