Structure-Function Analyses of the N-Butanoyl L-Homoserine Lactone Quorum-Sensing Signal Define Features Critical to Activity in RhIR

Pseudomonas aeruginosa is an opportunistic pathogen that coordinates the production of many virulence phenotypes at high population density via quorum sensing (QS). The LuxR-type receptor Rh1R plays an important role in the P. aeruginosa QS process, and there is considerable interest in the development of chemical approaches to modulate the activity of this protein. Rh1R is activated by a simple, low molecular weight N-acyl L-homoserine lactone signal, N-butanoylL-homoserine lactone (BHL). Despite the emerging prominence of Rh1R in QS pathways, there has been limited exploration of the chemical features of the BHL scaffold that are critical to its function. In the current study, we sought to systematically delineate the structure-activity relationships (SARs) driving BHL activity for the first time. A focused library of BHL analogues was designed, synthesized, and evaluated in cell-based reporter gene assays for Rh1R agonism and antagonism. These investigations allowed us to define a series of SARs for BHL-type ligands and identify structural motifs critical for both activation and inhibition of the Rh1R receptor. Notably, we identified agonists that have similar to 10-fold higher potencies in Rh1R relative to BHL, are highly selective for Rh1R agonism over LasR, and are active in the P. aeruginosa background. These compounds and the SARs reported herein should pave a route toward new chemical strategies to study Rh1R in P. aeruginosa.