Identification of key Y ₄ R residues enables the discovery of selective non-peptide small-molecule agonists

G protein-coupled receptors (GPCRs) are central regulators of human physiology and disease, classifying them as relevant targets for therapeutic interventions. As transmembrane proteins, they convert extracellular signals into intracellular responses through agonist-induced conformational changes. Understanding how agonists stabilize active receptor conformations is decisive for rational drug design. In this study, we used the endogenous ligand pancreatic polypeptide (PP) and the cyclic hexapeptides UR-AK95c and UR-AK86c as molecular tools to determine key interactions critical for Y ₄ R activation, which plays a crucial role in metabolic diseases. Guided by molecular docking, we systematically replaced Y ₄ R residues and assessed activation. The in vitro and in silico studies delineated a key Y ₄ R activation interface centered around the conserved C-terminal RXRY-NH ₂ motif of the peptides and, separately, identified receptor residues with distinct peptide-specific functional effects. Next, we performed an ultra-large library screening (ULLS) and experimentally validated three predicted hits as selective Y ₄ R agonists that engage in a substantial subset of the identified critical receptor contacts. This study demonstrates how GPCR activation interface knowledge can be translated into the discovery of novel small-molecule agonists and outlines a general strategy for advanced GPCR drug discovery.