The pathway-independent positive allosteric modulator C1 allows for the identification of active Y4 receptor relevant positions

The neuropeptide Y₄ receptor (Y₄R) and its endogenous ligand pancreatic polypeptide (PP) are primarily involved in the regulation of satiety and energy balance and present relevant pharmacological targets. We characterized the novel Y₄R positive allosteric modulator C1 that enhances Y₄R G-protein signaling, ligand binding, and arrestin-3 recruitment to Y₄R. Comparison with a close analog revealed the structural importance of an ethyl acetate moiety for Y₄R affinity and PAM activity at the G-protein pathway. C1 shows a high selectivity for the Y₄R, while signaling of the related subtypes Y₁R, Y₂R, and Y₅R is not affected. Y₄R G-protein signaling is even potentiated by the low-affinity agonists neuropeptide Y and peptide YY. Binding affinity of the endogenous ligands to Y₄R is enhanced by C1, indicating a stabilization of the ligand-bound Y₄R conformation. Using Y₄R/Y₁R chimera, important Y₄R domains for C1 activity were identified. Single point mutagenesis and computational docking pinpointed hot-spot residues at Y₄R important for stabilizing the active ligand-bound conformation.