Further Studies on Nociceptin-Related Peptides:  Discovery of a New Chemical Template with Antagonist Activity on the Nociceptin Receptor

Abstract

Three series of nociceptin (NC)-related peptides were synthesized and their abilities (i) to bind to the NC sites expressed in mouse forebrain membranes, (ii) to inhibit the electrically evoked contraction of the mouse vas deferens, and (iii) to inhibit forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing the human recombinant NC receptor (CHONCR) were investigated. The compounds of the first series (a series) have an ordinary Xaa1-Gly2 bond, those of the second series (b series) have a Xaa1psi(CH2-NH)Gly2 pseudopeptide bond, and those of the third series (c series) have a peptoid (Nxaa1-Gly2) structure. The affinity values measured in the binding assay and in the two functional assays with the compounds of the three series showed high levels of correlation. Thus, (I) the compounds of the a series in which Phe1 was substituted with Tyr, Cha, or Leu acted as potent NC receptor agonists; (II) the b series compounds behaved as NC receptor antagonists in the mouse vas deferens and as full agonists in CHO(NCR) cells with different potencies depending on the first amino acid residue, [Phe1psi(CH2-NH)Gly2]NC(1-17)NH2 and [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 being the most potent compounds; (III) the compounds of the third series were all inactive both as agonists and as antagonists with the exception of [Nphe1]NC(1-17)NH2 and [Nphe1]NC(1-13)NH2, which behaved as NC receptor antagonists both in the isolated tissue and in CHO(NCR) cells (pKB 6.1-6.4). In conclusion, this study demonstrates that chemical requirements for NC receptor agonists are different from those of antagonists. Moreover, modifications of the steric orientation of the aromatic residue Phe1 in the NC sequence as obtained with the pseudopeptide bond between Phe1 and Gly2 or with the displacement of the benzyl side chain by one atom, as in Nphe1, lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 which has been reported to exhibit agonist activity in several assays involving either central or recombinant NC receptors, [Nphe1]NC(1-13)NH2 antagonizes the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.

The ORL₁ Receptor: Molecular Pharmacology and Signalling Mechanisms

“…[Nphe 1 ]NC(1-13)NH 2 inhibits the binding of a tritiated nociceptin analogue to mouse brain membranes with a K i = 100 nM [51]. Despite this low affinity, [Nphe 1 ]NC(1-13)NH 2 was able to antagonize forskolinstimulated cAMP accumulation in CHO cells transfected with human ORL 1 with a pK b of 6.1 and to antagonize the nociceptin-induced contraction in electrically stimulated mouse vas deferens with a pK b of 6.4 [51].…”

“…[Nphe 1 ]NC(1-13)NH 2 inhibits the binding of a tritiated nociceptin analogue to mouse brain membranes with a K i = 100 nM [51]. Despite this low affinity, [Nphe 1 ]NC(1-13)NH 2 was able to antagonize forskolinstimulated cAMP accumulation in CHO cells transfected with human ORL 1 with a pK b of 6.1 and to antagonize the nociceptin-induced contraction in electrically stimulated mouse vas deferens with a pK b of 6.4 [51]. Most promisingly, the ligand did not show any residual agonist activity and was able to prevent the effects of nociceptin in the mouse tail withdrawal assay and to potentiate morphineinduced analgesia [52].…”

In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor

“…Details about these three classes of NOP ligands can be found in recent review articles such as [53] and [3]. Our research group has substantially contributed to the identification of N/OFQ related NOP ligands by performing a series of structure activity studies which allow the identification of the following useful chemical modifications: C-terminus amidation that increases agonist potency and reduces susceptibility to peptidases [23], reduction of the first peptide bond of N/OFQ which reduces ligand efficacy [8], shift of the Phe 1 side chain to the nitrogen atom that eliminates efficacy [25], and substitutions on the Phe 4 side chain (e.g. (pF)Phe 4 ) that increase peptide potency [24].…”

[Arg14,Lys15]Nociceptin, a Highly Potent Agonist of the Nociceptin/Orphanin FQ Receptor: in Vitro and in Vivo Studies

“…Identification of such new molecules (at least of peptide nature) should be facilitated by the knowledge of the amino acid residues of the NC sequence that are critical for receptor occupation and activation. Over the last 5 years, several structure-activity relationship studies have been performed on NC (Dooley and Houghten, 1996;Reinscheid et al, 1996;Butour et al, 1997;Guerrini et al, 1997;Calo' et al, 1998a;Guerrini et al, 2000a). These studies suggested that Phe 1 and Phe 4 represent the critical residues of the message domain of NC (Phe 1 -Gly 2 -Gly 3 -Phe 4 ), which should be involved in receptor binding and activation, whereas the positively charged residues that are present in the address domain of the molecule (Arg 8,12 , Lys 9,13 ) appear to be crucial for receptor occupation.…”

“…In Guerrini et al, 2000a), J-113397 (Kawamoto et al, 1999), and the nonselective opioid receptor antagonist naloxone. The effects of [Arg 14 ,Lys 15 ]NC were also evaluated in vivo, in the locomotor activity and in the tail-withdrawal assays after i.c.v.…”