[ 3 H]ac-RYYRWK-NH 2 , a novel specific radioligand for the nociceptin/orphanin FQ receptor

Abstract

The hexapeptide ac-RYYRWK-NH2 has been described as a potent partial agonist at the nociceptin (NC)/orphanin FQ receptor which has no affinity for mu-, kappa- or delta-opioid receptors. However, it is not clear whether ac-RYYRWK-NH2 is truly selective for the NC receptor, and ac-RYYRWK-NH2 has therefore been radiolabelled and characterised in receptor-binding experiments. Saturation experiments with [3H]ac-RYYRWK-NH2 binding to rat cortical membranes revealed a single high affinity site for [3H]ac-RYYRWK-NH2 (Kd=0.071 +/- 0.018 nM; Bmax=22+/-2 fmol/mg protein). Uncoupling of the G-proteins resulted in a significant 45% increase in Kd and no change in Bmax. [3H]ac-RYYRWK-NH2 binding to rat cortical membranes or to membranes from baby hamster kidney cells expressing human orphan opioid receptor-like (ORL1) was displaced by NC and ac-RYYRWK-NH2 to the same extent. The following rank order of potency was observed: ac-RYYRWK-NH2 > [Tyr14]NC-OH = NC-OH = NC-NH2 > NC, H-(1-13)-NH2 > NC(1-12)-NH2 >> NC(1-11)-NH2 and, thus, displayed a typical NC receptor pharmacology. Novel cyclic analogues of ac-RYYRWK-NH2 were prepared but these structures were much less active when compared to ac-RYYRWK-NH2. In vitro receptor autoradiography with [3H]ac-RYYRWK-NH2 to rat brain sections revealed high levels of binding in the cerebral cortex, amygdala, hypothalamus and superior colliculus, but low levels in the cerebellum and striatum. Overall, the regional distribution was very similar to that of [3H]NC. Ac-RYYRWK-NH2 seems indeed to be selective for the NC receptor and [3H]ac-RYYRWK-NH2 is a novel radioligand which may be useful for further exploring the pharmacology and receptor-ligand interaction of the NC receptor.

In vitro Binding and Functional Studies of Ac-RYYRIK-ol and Its Derivatives, Novel Partial Agonists of the Nociceptin/Orphanin F/Q Receptor

“…Structure activity studies were performed by using the Ac-RYYR-I/W-K-NH 2 sequence as a template. The binding affinity was decreased by the head-to-tail cyclization of Ac-RYYRWK-NH 2 [17] and increased by N-terminal acylation with a pentanoyl group [18] or the replacement of the Tyr 2,3 residues with (pF)Phe [19] . A NOP receptor agonist was generated by N-terminal alkylation of the central core YYRW with groups bearing a guanidine function [20] .…”

Development of nociceptin receptor (NOP) agonists and antagonists

“…Cyclic analogues of 3 like cyclo-(-RYYRWK-) had only a scarce affinity. 59 A SAR study on 4 showed that the removal of acetyl or Ala substitution of Arg1, Tyr2, or Tyr3 almost suppressed affinity, whereas this effect was much smaller for the same substitution of one of the other three residues or the removal of Lys6. Moreover, the removal of the terminal amide group had almost no effect.…”

“…53 Whereas the introduction of helix structure in 1 is generally favorable to activity, a conformational constraint to a b-strain diminishes both affinity and activity, as in Other compounds derived from 1, which also showed an increased activity due to conformational restraints, are some cyclic analogues, such as the agonists cyclo 59 (see Table XII for cyclopeptides and Table IX for some acyclic counterparts). Another ligand derived from N/OFQ is retronociceptin methyl ester, with poor affinity in vitro (IC 50 5 480 mM) but active on mice by i.c.v.…”

“…62 They resulted to be inactive in vivo because of their instability to metabolism, 63 but were the lead compounds for the development of many other NOP ligands; moreover, tritiated 3 is a selective radioligand. 59 The partial agonist Ac-RYYRWKKKKKKK-NH 2 (ZP120, developed by Zealand Pharma) 64 is interesting because of its inability to penetrate the CNS, after iv administration, thus its action is limited to peripheral NOP receptors, producing prolonged diuretic and antinatriuretic effects. 65,66 Many SAR studies have been performed on hexapeptides 2-6 (Table XIII).…”

High Affinity Conformationally Constrained Nociceptin/Orphanin FQ(1−13) Amide Analogues

“…21 Also potent cyclic analogues may provide information on possible biologically active conformations of the peptide. Only a limited number of cyclic analogues of N/OFQ have been reported to date, [22][23][24][25] and these have all involved cyclization via a disulfide bond; cyclo[Cys 10 ,Cys 14 ]N/OFQ(1-14)NH 2 23,24…”