In search for effective antagonist structures for the nociceptin (NOP) receptor, a number of N-acylated oligopeptides, including N-acyl tetra- and pentapeptides selective for the κ-opioid receptor, as well as N-acyl hexapeptides bearing the Ac-Arg-Tyr-Tyr-Arg-Ile-Lys (Ac-RYYRIK) core sequence originally isolated from combinatorial chemical libraries, were synthesized and studied in radioreceptor binding assays, [35S]GTPγS functional tests and in mouse vas deferens (MVD) bioassays. The properties of the novel antagonist candidates were compared to known antagonists. A new antagonist structure with a reduced, primer alcohol C-terminus, Ac-Arg-Tyr-Tyr-Arg-Ile- lysinol (Ac-RYYRIK-ol) was described in the mouse vas deferens tests, showing an equilibrium inhibitory constant value (Ke) of 2.44 nM, and no agonist effect at 10 μM ligand concentration. Schild-analysis indicated a clearly competitive interaction at the NOP receptor, whereas the peptide did not affect the action of the δ-opioid receptor agonist [D-Ala 2,D-Leu5]enkephalin. Ac-RYYRIK-ol also exhibited a high affinity in [3H]nociceptin-NH2 binding competition assays using rat brain membranes. Agonist-induced G-protein activation via NOP receptors was studied in [35S]GTPγS binding stimulation assays by the use of both native brain tissue preparations and membranes from cultured CHO cells expressing recombinant nociceptin receptors. Ac-RYYRIK-ol displayed only weak intrinsic agonist activity, whereas it effectively inhibited the stimulation generated by nociceptin. The results support the high potency and antagonist nature of Ac-RYYRIK-ol and reveal important roles for both the N- and the C-terminal region of the molecule.
ASJC Scopus subject areas
- Clinical Biochemistry
- Cellular and Molecular Neuroscience