Quorum sensing (QS) is widespread in bacteria and plays a pivotal role in their interactions with eukaryotic hosts. This intercellular signaling mechanism is based on small molecule ligands and their cognate protein receptors, and allows bacteria to assess their local population densities and function as a group. The long-term objective of the proposed research is to design, synthesize, and characterize non-native small molecules capable of intercepting native QS signals for use as tools to dissect the myriad roles of QS in bacterial populations and in bacteria-host associations. The potential impact of such chemical probes is enormous, and ranges from applications in basic research to therapeutic and biomaterials development. As many of the most notorious human pathogens use QS to activate virulence pathways that are the origin of acute and chronic infections, including biofilm formation, the application of QS antagonists holds significant promise as a novel antimicrobial strategy. Such anti-virulence agents differ from current antibiotics because they target infectivity as opposed to growth, and represent a paradigm shift for the treatment of bacteria-mediated disease. QS in Gram-negative bacteria is the best characterized to date and the focus of this project. These QS circuits are based on N-acyl L-homoserine lactone (AHL) signals and LuxR-type transcription factors, and binding of the AHL to its target LuxR-type receptor triggers QS-controlled gene expression at high cell densities. In earlier work, we studied the structures of LuxR-type receptor ligand-binding sites, designed non-native ligands capable of targeting these sites, and developed efficient synthetic routes to libraries of these compounds. Evaluation of the libraries in model bacterial strains revealed several of the most potent synthetic antagonists and agonists of LuxR-type QS reported to date. These results validate our overall research strategy. Our intent now is to develop new small molecules scaffolds capable of intercepting LuxR-type QS with improved potencies and stabilities, determine the mechanisms by which these compounds exert their QS modulatory activities, and examine their ability to attenuate QS phenotypes in wild-type human pathogens. During the grant period, these objectives will be pursued in three Specific Aims. These are: (1) Design and Structural Optimization of New Synthetic QS Antagonists and Agonists, (2) Mechanistic Analysis of LuxR-type Receptor Antagonism and Agonism by Synthetic Ligands, and (3) Cell-Based Virulence Assays and Studies of Resistance Development to QS Antagonists. The results of the interdisciplinary research proposed herein will provide fundamental insights into the mechanisms of QS and ultimately could provide an approach for the development of next- generation, anti-virulence treatments for bacterial infection.