Structure-activity relationships for inhibitors of Pseudomonas aeruginosa exoenzyme S ADP-ribosyltransferase activity

During infection, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa employs its type III secretion system to translocate the toxin exoenzyme S (ExoS) into the eukaryotic host cell cytoplasm. ExoS is an essential in vivo virulence factor that enables P. aeruginosa to avoid phagocytosis and eventually kill the host cell. ExoS elicits its pathogenicity mainly via ADP-ribosyltransferase (ADPRT) activity. We recently identified a new class of ExoS ADPRT inhibitors with in vitro IC₅₀ of around 20 μM in an enzymatic assay using a recombinant ExoS ADPRT domain. Herein, we report structure-activity relationships of this compound class by comparing a total of 51 compounds based on a thieno [2,3-d]pyrimidin-4(3H)-one and 4-oxo-3,4-dihydroquinazoline scaffolds. Improved inhibitors with in vitro IC₅₀ values of 6 μM were identified. Importantly, we demonstrated that the most potent inhibitors block ADPRT activity of native full-length ExoS secreted by viable P. aeruginosa with an IC₅₀ value of 1.3 μM in an enzymatic assay. This compound class holds promise as starting point for development of novel antibacterial agents.