Hederagenin is a Highly Selective Antagonist of the Neuropeptide FF Receptor 1 that Reveals Mechanisms for Subtype Selectivity.

RF-amide peptide receptors including the neuropeptide FF receptor 1 (NPFFR1) are G protein-coupled receptors (GPCRs) that modulate diverse physiological functions. High conservation of endogenous ligands and receptors makes the identification of selective ligands challenging. Previously identified antagonists mimic the C-terminus of peptide ligands and lack selectivity towards the closely related neuropeptide FF receptor 2 (NPFFR2) or the neuropeptide Y receptor (YR). In a high-throughput screening, we identified the pentacyclic triterpenoid hederagenin (1) as a novel selective antagonist for the NPFFR1. Hederagenin (1) is a natural product isolated from Hedera helix (ivy). We characterized its mode of activity using in vitro and in silico methods, revealing an overlapping binding site of the small molecule with the orthosteric peptide agonists. Despite the high similarity of the orthosteric binding pockets of NPFFR1 and NPFFR2, hederagenin (1) shows strong subtype selectivity, particularly caused by slight differences in the shape of the binding pockets and the rigidity of the small molecule. Several residues inhibiting the activity of hederagenin (1) at the NPFFR2 were identified. As NPFFR1 antagonists are discussed as potential candidates for the treatment of chronic pain, these insights into the structural determinants governing subtype specificity will facilitate the development of next-generation analgesics with improved safety and efficacy.