Design and application of small molecules for the interception of quorum sensing in Staphylococcus aureus - PROJECT SUMMARY Quorum sensing (QS) is the process by which bacteria of the same species coordinate behavior at a high population density. In many pathogenic bacteria, QS systems are used to regulate virulence. This proposal focuses on the accessory gene regulator (agr) QS system found in the Gram-positive pathogen Staphylococcus aureus, which uses agr to regulate an arsenal of toxins and biofilm formation. Small molecules that inhibit agr QS have the potential to be used as therapeutics to combat S. aureus infection. Previous small molecules that have been reported to inhibit agr QS suffer from low potencies, poorly characterized mechanisms of action, and/or associated toxicities that render them impractical for use as chemical probes. Herein I propose to develop a new class of small molecule agr inhibitors, study their biochemical mechanisms of agr inhibition, and investigate their effects on S. aureus growth/invasion in human cells. In Aim 1, I will develop a class of potent agr inhibitors through structure activity relationship (SAR) analyses on small molecule agr modulators recently identified in our lab. I will design new synthetic routes to access the core structure of two best-in-class small molecule inhibitors that represent the most potent small molecule inhibitors of S. aureus agr QS to date. These redesigned syntheses will enable facile functional group variation for the delineation of SARs and allow for installation of reactive groups for use in photoaffinity probes. My primary goals will be to improve compound potency, membrane permeability, solubility, and stability. In Aim 2, I will characterize the mechanisms of agr inhibition by these new small molecule scaffolds. Almost all known agr inhibitors target autoinducer (AIP) binding to the transmembrane histidine kinase AgrC or intracellular response regulator AgrA:DNA binding. Elucidating the mechanisms of agr inhibition by these new small molecules may provide access to chemical probes that target other components of the agr QS system. I will use a series of in vitro and cell-based assays to determine the target protein/pathway of these compounds, and photoreactive probes to characterize ligand binding sites on these targets. In Aim 3, I will explore the effects of these small molecule agr inhibitors on S. aureus growth/invasion in mammalian cells. S. aureus can use agr to evade the immune system and cause persistent infection by infiltrating a wide range of human cell types. Macrophage invasion assays will be used to evaluate the cell permeability of the lead small molecule agr inhibitors developed in Aims 1 and 2, their ability to be phagocytosed, and whether inhibition of agr QS by these compounds decreases macrophage invasion by S. aureus. Lastly, the inhibition/dysfunction of agr QS may enable the development of S. aureus small colony variants (SCVs) that are less likely to evoke an immune response. The ability of these small molecules to induce transition to SCVs by agr inhibition will be investigated in Aim 3. Such experiments are yet to be explored and could yield a new route to study infection progression. Together these three Aims will provide valuable chemical tools and new insights for the study of agr QS.
