N- and C-terminal modifications of nociceptin/orphanin FQ generate highly potent NOP receptor ligands

Previous structure−activity studies on nociceptin/orphanin FQ (N/OFQ) identified [Phe1Ψ(CH2NH)Gly2]N/OFQ(1-13)-NH2 and [Nphe1]N/OFQ(1-13)-NH2 as a N/OFQ peptide receptor (NOP) partial agonist and pure antagonist, respectively. The addition of fluorine to the Phe4 or the insertion of a further pair of basic amino acids Arg14-Lys15 generate potent agonists. On the basis of these findings, we combined in the N/OFQ-NH2 template the chemical modifications Arg14-Lys15 and (pF)Phe4 that increase the agonist potency with those conferring partial agonist (Phe1Ψ(CH2NH)Gly2) or pure antagonist (Nphe1) properties. Twelve peptides were synthesized and pharmacologically evaluated in Chinese hamster ovary cells expressing the human recombinant NOP and in electrically stimulated mouse vas deferens and guinea pig ileum assays. All peptides behaved as NOP ligands; the chemical modifications Arg14-Lys15 and (pF)Phe4 increased ligand affinity/potency. Peptides with the normal Phe1-Gly2 peptide bond behaved as full agonists, and those with the Phe1Ψ(CH2NH)Gly2 modification behaved as partial agonists, while those with the Nphe1 modification behaved as partial agonists or pure antagonists depending on the presence or absence of the (pF)Phe4 modification, respectively. The full agonist [(pF)Phe4,Arg14,Lys15]N/OFQ-NH2, the partial agonist [Phe1Ψ(CH2NH)Gly2,(pF)Phe4,Arg14,Lys15]N/OFQ-NH2, and the pure antagonist [Nphe1,Arg14,Lys15]N/OFQ-NH2 represent the most potent peptide ligands for NOP.