Relaxin/insulin-like family peptide receptor 4 (RXFP4) is a class A G protein-coupled receptor (GPCR),and insulin-like peptide 5 (INSL5) is its endogenous ligand.Although the precise physiological role of INSL5/RXFP4 remains elusive,a number of studies have suggested it to be a potential therapeutic target for obesity and other metabolic disorders.Since selective agonists of RXFP4 are scarcely available and peptidic analogs of INSL5 are hard to make,we conducted a high-throughput screening campaign against 52,000 synthetic and natural compounds targeting RXFP4.Of the 109 initial hits discovered,only 3 compounds were confirmed in secondary screening,with JK0621-D008 displaying the best agonism at human RXFP4.Its S-configuration stereoisomer (JK1) was subsequently isolated and validated by a series of bioassays,demonstrating a consistent agonistic effect in cells overexpressing RXFP4.This scaffold may provide a valuable tool to further explore the biological functions of RXFP4.