Project Summary Inflammation is a complex process with many lipid and peptide mediators involved. A large number of these mediators elicit either pro-inflammatory or pro-resolving effects through the action on G protein-coupled receptors (GPCRs). My lab is interested in the molecular mechanisms by which pro-inflammatory and pro- resolving GPCRs sense diverse signaling molecules and transduce chemical signals across the cell membrane to regulate inflammation. We employ a combination of research approaches in structural biology including cryo- electron microscopy (cryo-EM), pharmacology, and computational biology. We are also making efforts to develop novel ligands of those GPCRs as potential new drugs through structure-based approaches. In the last funding period, we have determined structures of several members of a chemotactic GPCR family including the C5a receptor (C5aR), the prostaglandin D2 receptor 2 (DP2), and the formylpeptide receptors bound to diverse agonists or antagonists at different conformational states, which allowed us to define the molecular basis for the action of several drugs and drug candidates. In the current proposal, we will study the signaling and pharmacology of three lipid GPCRs that regulate different aspects of inflammation, the chemerin receptor CMLKR1, GPR32, and GPR84. Both CMLKR1 and GPR32 are closely related to the C5aR, DP2 and FPRs. They have been shown to respond to resolvins, which are SPMs derived from ω-3 polyunsaturated fatty acids (PUFAs), to induce the resolution of inflammation. In contrast, GPR84 is a newly characterized pro-inflammatory GPCR that can be activated by endogenous medium-chain fatty acids (MCFAs) to augment inflammatory responses and enhance phagocytosis by macrophages. Through a combination of approaches in structural biology, pharmacology, computational biology, and immunology, we aim to provide a comprehensive molecular understanding of how diverse lipid, peptide, and synthetic small-molecule ligands act on and regulate the activities of these three lipid GPCRs. Our studies will exploit unique properties of CMLKR1, GPR32, and GPR84 to address several fundamental issues of GPCR signaling and pharmacology including lipid recognition, receptor antagonism, allosteric modulation, and biased signaling. Our research strategies developed in this proposal will be applied to our future research on the structure and pharmacology of other fatty acid receptors (FFARs) in immunometabolism. We will also develop new agents for these GPCRs as useful pharmacological tools and potential drugs to control inflammation. The new compounds will serve as the leading compounds for our future drug development effort through structure-based compound optimization and functional characterization.