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

Abstract The neuropeptide Y 4 receptor (Y 4 R) 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 Y 4 R positive allosteric modulator C1 that enhances Y 4 R G-protein signaling, ligand binding, and arrestin-3 recruitment to Y 4 R. Comparison with a close analog revealed the structural importance of an ethyl acetate moiety for Y 4 R affinity and PAM activity at the G-protein pathway. C1 shows a high selectivity for the Y 4 R, while signaling of the related subtypes Y 1 R, Y 2 R, and Y 5 R is not affected. Y 4 R G-protein signaling is even potentiated by the low-affinity agonists neuropeptide Y and peptide YY. Binding affinity of the endogenous ligands to Y 4 R is enhanced by C1, indicating a stabilization of the ligand-bound Y 4 R conformation. Using Y 4 R/Y 1 R chimera, important Y 4 R domains for C1 activity were identified. Single point mutagenesis and computational docking pinpointed hot-spot residues at Y 4 R important for stabilizing the active ligand-bound conformation.