Ac-RYYRIK-NH2

Structural requirements of nociceptin antagonist Ac-RYYRIK-NH2 for receptor binding.[Pubmed:12450325]

J Pept Sci. 2002 Oct;8(10):561-9.

Ac-RYYRIK-NH2 is a peptide isolated from the peptide library as an antagonist that inhibits the biological activities of nociceptin, a hyperalgesic neuropeptide. In order to clarify the structural requirements of this peptide for binding to the nociceptin receptor ORL1, systematic structure-activity studies were carried out. The result of Ala-scanning indicated that the N-terminal tripeptide RYY(= Arg-Tyr-Tyr) is crucially important for binding to the ORL1 receptor. Residual truncations from the N- or C-terminus revealed the special importance of the N-terminal Arg residue. The removal of protecting groups indicated that the N-terminal acetyl group is essential, but the C-terminal amide group is insignificant. These results indicated the conspicuous importance of acetyl-Arg at position 1 of Ac-RYYRIK-NH2 as a key structure allowing binding to the receptor. To investigate the binding site of this peptide in the ORL1 receptor, we synthesized and assayed a series of analogues of the nociceptin dibasic repeat region, residues 8-13 of RKSARK. None of the derivatives were active. Ac-RYYRIK-NH2 was inactive for the mu opioid receptor to which nociceptin binds with considerable strength. All the results suggested that the mode of binding between Ac-RYYRIK-NH2 and the ORL1 receptor is different to that between the ORL1 receptor and nociceptin, and that it may consist of interaction with the receptor site to which nociceptin(1-7) or -(14-17) binds.

Antagonism by acetyl-RYYRIK-NH2 of G protein activation in rat brain preparations and of chronotropic effect on rat cardiomyocytes evoked by nociceptin/orphanin FQ.[Pubmed:10188961]

Br J Pharmacol. 1999 Feb;126(3):555-8.

For the further elucidation of the central functions of nociceptin/orphanin FQ (noc/OFQ), the endogenous ligand of the G protein-coupled opioid receptor-like receptor ORL1, centrally acting specific antagonists will be most helpful. In this study it was found that the hexapeptide acetyl-RYYRIK-NH2 (Ac-RYYRIK-NH2), described in literature as partial agonist on ORL1 transfected in CHO cells, antagonizes the stimulation of [35S]-GTPgammaS binding to G proteins by noc/OFQ in membranes and sections of rat brain. The antagonism of the peptide was competitive, of high affinity (Schild constant 6.58 nM), and specific for noc/OFQ in that the stimulation of GTP binding by agonists for the mu-, delta-, and kappa-opioid receptor was not inhibited. The hexapeptide also fully inhibited the chronotropic effect of noc/OFQ on neonatal rat cardiomyocytes. It is suggested that Ac-RYYRIK-NH2 may provide a promising starting point for in vivo tests for antagonism of the action of noc/OFQ and for the further development of highly active and specific antagonists.

The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties.[Pubmed:10998548]

Peptides. 2000 Jul;21(7):1131-9.

The hexapeptide acetyl-RYYRIK-amide (Ac-RYYRIK-NH(2)) has recently been reported to act as partial agonist of the nociceptin/orphanin FQ (noc/OFQ) receptor expressed in CHO cells. In addition, this peptide acts as a competitive antagonist of noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes as well as of the noc/OFQ-evoked chronotropic effect in rat cardiomyocytes. In contrast to this antagonism, in the present study, Ac-RYYRIK-NH(2) was found to behave as an agonist at noc/OFQ receptors, affecting spontaneous locomotor activity. When administered intracerebroventricularly (i.c.v.), noc/OFQ and Ac-RYYRIK-NH(2) inhibited spontaneous locomotor activity in mice with ID(50) of 1.1 and 0.07 nmol, respectively. Co-administration of both peptides lead to additive effects. The higher potency of Ac-RYYRIK-NH(2) could not be clearly explained by differential metabolism, because in vivo microdialysis in rat striatum and in vitro metabolic inactivation by rat and mouse brain membranes revealed extensive inactivation of both peptides. Similar to Ac-RYYRIK-NH(2), [Phe(1)psi(CH(2)-NH)Gly(2)]noc/OFQ(1-13)-NH(2) ([F/G]NC(1-13)NH(2)) inhibited the noc/OFQ-stimulated GTPgamma(35)S binding in rat brain membranes (Schild constant 3.83 nM) and mouse brain sections, although several reports have shown that this peptide exhibits agonist activity of noc/OFQ in the CNS. Changes in the optimum conditions of the in vitro assay for GTP binding increased low partial agonism of Ac-RYYRIK-NH(2) in GTP binding response. To explain the discrepancy between the in vitro antagonism of G protein coupling of the noc/OFQ receptor and in vivo agonism of Ac-RYYRIK-NH(2) and of [F/G]NC(1-13)NH(2), it is suggested that low partial agonism of receptor/G protein coupling in native systems may be sufficient to evoke full biologic responses. The extent of partial agonism for GTP binding and of coupling reserve may vary in different systems, thus explaining why [F/G]NC(1-13)NH(2) and Ac-RYYRIK-NH(2) were reported to exhibit antagonist, partial agonist, or even full agonist properties, depending on the system studied.

Binding and in vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor, ORL1.[Pubmed:9353393]

J Pharmacol Exp Ther. 1997 Nov;283(2):735-41.

Fifteen hexapeptides having high affinity for the opioid-like receptor ORL1 were identified from a combinatorial library containing more than 52 million different hexapeptides. The five compounds with the highest affinity were characterized further by use of a variety of in vitro models. Binding studies indicated that these five peptides have affinity for ORL1 in the nanomolar range, similar to the recently discovered endogenous ligand called nociceptin and orphanin FQ (N/OFQ). The activity of these compounds was investigated in three different assays: stimulation of [35S]GTPgammaS binding and inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells transfected with ORL1, and inhibition of electrically induced contractions in the mouse vas deferens. In each assay, the five hexapeptides acted as partial agonists. The EC50 values for stimulation of [35S]GTPgammaS binding and inhibition of cAMP accumulation were in the range of that for N/OFQ, but maximal effects ranged from 70 to 90% of N/OFQ in the cAMP assay, and 30 to 60% of N/OFQ in the GTPgammaS assay. The positive hexapeptides identified were found to have minimal structural similarity to N/OFQ. The peptides are positively charged, which could enable them to bind to the negatively charged second extracellular loop thought to be a likely binding site for N/OFQ.