Comparative biochemical and pharmacological characterization of a novel, NOP receptor selective hexapeptide, Ac-RYYRIR-ol

Nociceptin/orphanin FQ (N/OFQ) is an endogenous neuropeptide, which is widely distributed in central and peripheral nervous system. Some N/OFQ sequence unrelated hexapeptides can effectively bind to the N/OFQ peptide (NOP) receptor and they were used as template for structure-activity studies that lead to discovery of the new NOP selective ligands. In the present study, the pharmacological profile of the novel hexapeptide Ac-RYYRIR-ol was investigated using various in vitro assays including receptor binding and G-protein activation in rat brain membranes, mouse and rat vas deferens, guinea pig ileum, mouse colon and Ca2+ mobilization assay in chinese hamster ovary (CHO) cells co-expressing the human recombinant NOP receptor and the C-terminally modified Gαqi5 protein. In rat brain membranes Ac-RYYRIR-ol displaced both [3H]nociceptin/OFQ and [3H]Ac-RYYRIK-ol with high affinity (pKi 9.35 and 8.81, respectively) and stimulated [35S]GTPγS binding showing however lower maximal effects than N/OFQ (α = 0.28). The stimulatory effect of Ac-RYYRIR-ol was antagonized by the selective NOP receptor antagonist UFP-101. In the electrically stimulated mouse vas deferens Ac-RYYRIR-ol displayed negligible agonist activity while antagonizing in a competitive manner (pA2 7.99) the inhibitory effects of N/OFQ. Similar results were obtained in the rat vas deferens. In the mouse colon Ac-RYYRIR-ol produced concentration dependent contractile effects with similar potency and maximal effects as N/OFQ. Finally, in the Ca2+ mobilization assay performed with CHO-hNOP-Gαqi5 cells Ac-RYYRIR-ol displayed lower potency and maximal effects (α = 0.87) compared with N/OFQ. In conclusion, the novel NOP receptor selective hexapeptide Ac-RYYRIR-ol has been shown to have fine selectivity, high potency, furthermore agonist and antagonist effects toward the NOP receptors were measured in various assays; this is likely due to its partial agonist pharmacological activity.