Further studies on nociceptin-related peptides: discovery of a new chemical template with antagonist activity on the nociceptin receptor

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 (CHO NCR ) were investigated. The compounds of the first series (a series) have an ordinary Xaa 1 -Gly 2 bond, those of the second series (b series) have a Xaa 1 Ψ(CH 2 -NH)Gly 2 pseudopeptide bond, and those of the third series (c series) have a peptoid (Nxaa 1 -Gly 2 ) 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 Phe 1 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, [Phe 1 Ψ(CH 2 -NH)Gly 2 ]NC(1-17)NH 2 and [Phe 1 Ψ(CH 2 -NH)Gly 2 ]NC(1-13)NH 2 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 [Nphe 1 ]NC(1-17)NH 2 and [Nphe 1 ]NC(1-13)NH 2 , which behaved as NC receptor antagonists both in the isolated tissue and in CHO NCR cells (pK B 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 Phe 1 in the NC sequence as obtained with the pseudopeptide bond between Phe 1 and Gly 2 or with the displacement of the benzyl side chain by one atom, as in Nphe 1 , lead respectively to reduction or elimination of efficacy. Indeed, in contrast to [Phe 1 Ψ-(CH2-NH)Gly 2 ]NC(1-13)NH2 which has been reported to exhibit agonist activity in several assays involving either central or recombinant NC receptors, [Nphe 1 ]NC(1-13)NH 2 antagonizes the effect of NC at human recombinant NC receptors and in the mouse tail withdrawal assay.