The goal of this project is to develop and test small-molecule antagonist probes of the relaxin-3/RXFP3 system for behavioral studies in alcohol addiction and relapse. Alcohol addiction is a heterogeneous, chronic relapsing disorder. Current therapies are inadequate, and therefore new medications based on novel targets are needed. The recently deorphanized relaxin-3/RXFP3 system comprises the endogenous neuropeptide relaxin-3 and its cognate G protein-coupled receptor RXFP3. Multiple lines of evidence suggest that RXFP3 antagonism is a novel target for therapeutics to treat alcohol addiction and relapse. Although RXFP3 antagonist peptides are available, there are unmet needs for non-peptide small-molecule antagonists, which are systemically bioavailable and can penetrate the blood-brain barrier, to further validate the relaxin-3/RXFP3 system as a novel drug target. To date, our group has made significant progress in this regard. We have developed a stable RXFP3 cell-based cAMP high-throughput screening assay and completed a screening campaign to identify antagonist hits. Focused structure-activity relationship studies of the hit compound have resulted in the first series of small-molecule antagonists that have Ke <500 nM and are highly selective for RXFP3 over another receptor subtype RXFP1 and can penetrate into the brain. In this application, we propose to further refine our early lead-like compounds to produce antagonist probes for in vivo studies through three iterative specific aims. In Aim 1, we will optimize potency, receptor selectivity, and drug-like properties of RXFP3 antagonists using medicinal chemistry. In Aim 2, we will characterize compounds using an RXFP3 functional cAMP assay and a radioligand binding assay. Select compounds will be assessed for receptor selectivity against RXFP1 and RXFP4, two subtypes of the relaxin family, and further evaluated in a target profiling screen. Potent and selective compounds will then be characterized using a battery of ADME and pharmacokinetic assays. In Aim 3, we will test the best compounds, developed in Aims 1 and 2, in animal models of alcohol reinforcement and stress-induced reinstatement. Overall, completion of this project will provide in vivo antagonist probes to pharmacologically validate the relaxin-3/RXFP3 system as a novel target for treatment of alcoholism.