The nociceptin/orphanin FQ receptor ligand acetyl-RYYRIK-amide exhibits antagonistic and agonistic properties

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.     

Pharmacological characterization of the nociceptin receptor, ORL1

A novel opioid receptor-like orphan receptor (ORL1) was cloned and identified to be homologous to classical opioid receptors but insensitive to traditional opioids. A heptadecapeptide, termed orphanin FQ or nociceptin (OFQ/N), was identified as its endogenous ligand. OFQ/N shares overlapping distribution sites in pain-processing areas and common cellular mechanisms with opioids but exerts diverse effects on nociceptive responses. Of the two reported ORL1 antagonists, [Phe(1)psi(CH(2)-NH)- Gly(2)] nociceptin-(1-13)-NH(2) (Phepsi) and naloxone benzoylhydrazone (NBZ), antagonisms were validated in the activation of inward rectifying K channels induced by OFQ/N, using the patch clamp technique in ventrolateral periaqueductal gray slices. Results showed that Phepsi acted as a partial agonist and NBZ was a weak nonselective antagonist of ORL1. It is comparable with most but not all of the findings from other tissues. Comparing all the reports supports the above inference for these two antagonists. The possible causes for the discrepancy were discussed. A brief review on the putative ORL1 antagonists, acetyl-RYYRIK-NH2, some sigma-ligands and the functional antagonist, nocistatin, is also included. It indicates that a potent and selective ORL1 antagonist is expecting to elucidate the physiological role of OFQ/N.     

Nociceptin/orphanin FQ and related peptides reduce the increase in plasma corticosterone elicited in mice by an intracerebroventricular injection

Nociceptin/orphanin FQ (=N/OFQ), the endogenous ligand of ORL1 receptor (=NOP), has been reported to induce, in rodents, after intracerebroventricular (i.c.v.) administration, anti-stress and anxiolytic effects. We have observed that the handling of mice followed by an i.c.v. injection of saline, induced a marked increase in the plasma corticosterone level (+250%) measured 30 minutes later. When N/OFQ was injected intracerebroventricularly, using a 1 microg dose, the increase in plasma corticosterone was significantly lower than in saline injected mice. N/OFQ(1-13)NH(2), known as a NOP receptor agonist, at the same 1 microg dose, also induced a lesser increase in plasma corticosterone level than a saline i.c.v. injection. The pseudopeptide [Phe(1)-psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2), defined either as an agonist or an antagonist of NOP receptor, at the 0.1 microg dose, behaved in a similar manner as N/OFQ, by decreasing the plasma corticosterone level. Finally, [Nphe(1)]N/OFQ(1-13)NH(2), although presumed to be a selective NOP receptor antagonist, also decreased the corticosterone level at the 0.1 microg dose. These observations suggest the implication of N/OFQ in the regulation of response to stress, through an action on the hypothalamo-pituitary-adrenocortical axis. Moreover, they evidence a similar effect of N/OFQ and N/OFQ(1-13)NH(2), but also of two other related peptides displaying antagonist properties on NOP receptors. These data suggest that several subtypes of N/OFQ receptors could exist.     

Nociceptin, OP4 receptor ligand in different models of experimental epilepsy

The anticonvulsive activity of nociceptin, endogenous OP4 receptors agonist was investigated in pentylenetetrazole (PTZ), N-methyl D-aspartic acid (NMDA), bicucculine (BCC) and electrically evoked seizure models of experimental epilepsy. Nociceptin, at the dose of 10 nmol, suppressed the clonic seizures induced by PTZ, NMDA and BCC. [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 which has been proposed to be selective antagonist OP4 receptors, did not prevent the action of nociceptin. The effect of [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 on seizures induced by PTZ, NMDA and BCC was very similar to that of nociceptin. These data support the hypothesis that it possesses agonistic properties. Naloxone did not reverse the anticonvulsive action of nociceptin as well as [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 which excludes the participation of opioid receptor in this action. On the other hand in the electroconvulsive model of generalized seizures, nociceptin as well as [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 influenced neither the electroconvulsive threshold nor the maximal electroshock test. The data suggest that nociceptin and [Phe1(psi)(CH2-NH)Gly2]nociceptin-(1-13)-NH2 can exert anticonvulsive action. These properties depend on OP4 but not opioid receptors activation.     

Nociceptin/orphanin FQ and neurotransmitter release in the central nervous system

In this article, the effect of nociceptin (orphanin FQ) on transmitter release in the central nervous system in vitro and in vivo is reviewed. Nociceptin inhibits the electrically or K(+)-evoked noradrenaline, dopamine, serotonin, and glutamate release in brain slices from guinea-pig, rat, and mouse. This effect is usually naloxone-resistant but antagonized by OP(4) receptor antagonists like [Phe(1)psi(CH(2)-NH)Gly(2)]-nociceptin(1-13)NH(2). In the rat in vivo, nociceptin diminishes acetylcholine release in the striatum, reduces dopamine release, and prevents the stimulatory effect of morphine on this transmitter in the nucleus accumbens and also elevates extracellular glutamate and gamma-aminobutyric acid levels in mesencephalic dopaminergic areas. The effect of nociceptin on the mesencephalic dopaminergic system might explain its actions on motor behavior.     

Synthesis and biological activity of nociceptin/orphanin FQ analogues substituted in position 7 or 11 with Calpha,alpha-dialkylated amino acids

Previous structure-activity and NMR studies on nociceptin/orphanin FQ (N/OFQ) demonstrated that Aib substitution of Ala(7) and/or Ala(11) increases the peptide potency through an alpha helix structure induction mechanism. On these bases we synthesised and evaluated pharmacologically in the mouse vas deferens assay a series of N/OFQ-NH(2) analogues substituted in position 7 and 11 with Calpha,alpha-disubstituted cyclic, linear and branched amino acids. None of the 20 novel N/OFQ analogues produced better results than [Aib(7)]N/OFQ-NH(2). Thus, this substitution was combined with other chemical modifications known to modulate peptide potency and/or efficacy generating compound 21 [Nphe(1)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (coded as UFP-111), compound 22 [(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) and compound 23 [Phe(1)Psi(CH(2)-NH)Gly(2)(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-113). These novel peptides behaved as highly potent NOP receptor ligands showing full (UFP-112) and partial (UFP-113) agonist and pure antagonist (UFP-111) activities in a series of in vitro functional assays performed on pharmacological preparations expressing native as well as recombinant NOP receptors.     

Supraspinal and spinal effects of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 on nociception in the rat

A new derivative of the neuropeptide nociceptin (NC) has recently been developed. This molecule, the pseudopeptide [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was found to antagonize NC inhibitory effects in peripheral smooth muscle preparations in vitro. However, contrasting results have appeared as regards its pharmacodynamic profile in the CNS. Here, we investigated the pseudopeptide effects, in vivo, on nociceptive responses in the rat. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 was administered intracerebroventricularly (i.c.v.) or intrathecally (i.t.) (alone or in combination with NC), and tail-flick latencies (TFL) to radiant heat were assessed. I.c.v. [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 (1-10 nmol/rat) caused a short-lasting decrease (5 min) of TFL and did not antagonize the threshold lowering effect of i.c.v. NC (1 nmol/rat). At the spinal level, the i.t. administration (0.2-10 nmol/rat) of [Phe1psi(CH2-NH)Gly2]-nociceptin(1-13)-NH2 produced a dose-dependent and long-lasting antinociceptive effect that was not modified by the administration of a high dose (30 nmol/rat i.t.) of the opioid antagonist naloxone. The i.t. co-administration of the pseudopeptide (10 nmol/rat) did not block the antinociceptive effect of i.t. NC (10 nmol/rat). These data indicate that the pseudopeptide behaves as an NC agonist at supraspinal and spinal levels in the rat tail-flick test of nociception. These different profiles in the periphery and the CNS could suggest differences between central and peripheral NC receptor/s and provide a basis for further development of antagonist molecules suitable for their characterization.     

Nociceptin/orphanin FQ increases ANP secretion in neonatal cardiac myocytes

Nociceptin (N/OFQ) is a novel heptadecapeptide with an amino acid sequence similar to that of endogenous opioid peptide dynorphin A. Dynorphin have been reported to increase the secretion of atrial natriuretic peptide (ANP) via selective activation of kappa-opioid receptor in cultured atrial cardiocytes. The present study was designed to investigate the direct effect of N/OFQ on the ANP secretion in cultured neonatal rat cardiac myocytes via N/OFQ receptor (NOP) activation. The secretion of ANP from cultured neonatal cardiac myocytes was increased in terms of incubation time. N/OFQ, at a dose of 0.3, 1, 3, and 10 microM, caused increases in ANP secretion in a dose-dependent manner. The N/OFQ-induced ANP secretion was completely antagonized by antagonists of NOP, 1 microM each of [Phe1 (CH2-NH) Gly2] nociceptin (1-13)-NH2 ([FG]N/OFQ(1-13)NH2) or naloxone benzoylhydrazone. In contrast, naloxone (1 microM), the non-selective opioid receptor antagonist, did not alter ANP response to N/OFQ. N/OFQ at 3 microM inhibited basal and forskolin-stimulated cAMP production, which was partially antagonized with the pretreatment of [FG]N/OFQ(1-13)NH2. An increase in ANP secretion by N/OFQ was also partially blocked by the pretreatment of forskolin. Homologous competition studies in neonatal cardiomyocyte membranes revealed the presence of two distinct sites. The high affinity site (10.9 +/- 1.6 nM) was far less abundant than the low affinity site. Therefore, these results suggest that N/OFQ causes an increase in ANP secretion in cultured neonatal cardiac myocytes by decreasing cAMP through its binding sites.     

Structural modelling and mutation analysis of a nociceptin receptor and its ligand complexes

We recently modelled and proposed four ligand-bound conformations for a G-protein-coupled receptor, namely, forms I, II, III and IV, based on the 3D structure and functional evidences for rhodopsin. In this study, the same strategy was applied to a human nociceptin receptor (NR), in order to predict ligand-bound receptor structures. Additionally, site-directed mutagenesis studies were carried out to evaluate these structures. A Thr138Ala mutant demonstrated the same affinity for [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) as the wild-type receptor; however, the affinity of this mutant for nociceptin was 20-fold lower than that of the wild type. A Ser223Ala mutation showed the same characteristics as those of the wild type. On the other hand, a Gln280Ala mutation reduced the affinity to nociceptin by more than 60-folds. These results suggested that a change in the conformation of NR following agonist binding did not accompany the rigid-body rotation of the sixth transmembrane segment that was reported for an adrenergic receptor and a kappa-opioid receptor. NR is potently activated not only by nociceptin but also a synthetic peptide, i.e. Ac-RYYRIK-NH(2), although the amino acid sequences of both these ligands are completely different. The model explains why both the ligands activate NR and shows that their receptor-bound conformations have similar 3D structures.     

Structure-activity relationships of nociceptin and related peptides: comparison with dynorphin A

Nociceptin and its receptor (OP(4)) share sequence homologies with the opioid peptide ligand dynorphin A and its receptor OP(2). Cationic residues in the C-terminal sequence of both peptides seem to be required for selective receptor occupation, but the number and the distribution of these basic residues are different and quite critical. Both receptors are presumably activated by the peptides N-terminal sequence (Xaa-Gly Gly-Phe, where Xaa = Phe or Tyr); however, although OP(4) requires Phe(4) as a determinant pharmacophore, OP(2) requires Tyr(1) as do the other opioid receptors. An extensive structure-activity analysis of the N-terminal tetrapeptide has led to conclude that the presence of aromatic residues in position one and four, preferably Phe, as well as the distance between Phe(1) and Phe(4) are extremely critical for occupation and activation of OP(4) in contrast with other opioid receptors (e.g. OP(1), OP(3), OP(2)). Modification of distance between the side chains of Phe(1) and Phe(4) (as obtained with Nphe(1) substitution in both NC and NC(1-13)-NH(2)) and/or conformational orientation of Phe(1) (as in Phe(1)psi(CH(2)-NH)-Gly(2)) has brought to discovery of pure antagonist ([Nphe(1)]-NC(1-13)-NH(2)) and a partial agonist ([Phe(1) psi(CH(2)-NH)-Gly(2)]-NC(1-13)-NH(2)), which have allowed us to characterize and classify the OP(4) receptor in several species. Thus, although antagonist activities at the OP(4) receptor are obtained by chemical modification of Phe(1)-Gly(2) peptide bond or by a shift of Phe(1) side chain of NC peptides, antagonism at the OP(2) receptor requires the diallylation of the N-terminal amino function, for instance, of dynorphin A. These considerations support the interpretation that the two systems nociceptin/OP(4) and dynorphin A/OP(2) are distinct pharmacological entities that differs in both their active sites (Tyr(1) for Dyn A and Phe(4) for NC) and the number and position of cationic residues in the C-terminal portions of the molecules. The chemical features of novel OP(4) receptor ligands either pseudopeptides obtained by combinatorial library screening or molecules of nonpeptide structure are reported and discussed in comparison with NC and NC related peptides.     

Endomorphin 1[psi] and endomorphin 2[psi], endomorphins analogues containing a reduced (CH2NH) amide bond between Tyr1 and Pro2, display partial agonist potency but significant antinociception

Endomorphin 1 (EM1) and endomorphin 2 (EM2) are highly potent and selective mu-opioid receptor agonists and have significant antinociceptive action. In the mu-selective pocket of endomorphins (EMs), Pro2 residue is a spacer and directs the Tyr1 and Trp3/Phe3 side chains into the required orientation. The present work was designed to substitute the peptide bond between Tyr1 and Pro2 of EMs with a reduced (CH2NH) bond and study the agonist potency and antinociception of EM1[psi] (Tyr[psi(CH2NH)]Pro-Trp-Phe-NH2) and EM2[psi] (Tyr[psi(CH2NH)]Pro-Phe-Phe-NH2). Both EM1[psi] and EM2[psi] are partial mu opioid receptor agonists showing significant loss of agonist potency in GPI assay. However, EMs[psi] exhibited potent supraspinal antinociceptive action in vivo. In the mice tail-flick test, EMs[psi] (1, 5, 10 nmol/mouse, i.c.v.) produced potent and short-lasting antinociception in a dose-dependent and naloxone (1 mg/kg) reversed manner. At the highest dose of 10 nmol, the effect of EM2[psi] was prolonged and more significant than that of EM2. In the rat model of formalin injection induced inflammatory pain, EMs[psi] (0.1, 1, 10 nmol/rat, i.c.v.), like EMs, exerted transient but not dose-dependent antinociception. These results suggested that in the mu-selective pocket of EMs, the rigid conformation induced by the peptide bond between Tyr1 and Pro2 is essential to regulate their agonist properties at the mu opioid receptors. However, the increased conformational flexibility induced by the reduced (CH2NH) bond made less influence on their antinociception.     

beta-Sulfonamido gonadotropin-releasing hormone analogs: synthesis and evaluation of several parent hormone properties.

With the aim of producing long-acting analogs of gonadotropin releasing hormone (GnRH), four analogs, containing -X(6) (aa)psi(CH(2)SO(2)NH)-Leu(7) building unit (X(aa)=Gly, Ala, Val or Phe), and a reduced-size analog [Des-Tyr(5)]-GnRH which includes the unit Phe(5)psi(CH(2)SO(2)NH)-Leu(6), and [beta-Ala(6)]-GnRH were synthesized. The peptides were evaluated for their capacity to induce LH-release from rat pituitary cells and to withstand proteolysis by pituitary-derived enzymes, compared with the parent peptide GnRH. Albeit stable toward enzymatic degradation, the sulfonamido containing peptides were only marginally bioactive. [beta-Ala(6)]-GnRH, however, induced LH-release and bound to pituitary receptors nearly as efficiently as GnRH. This analog was also highly stable toward proteolysis suggesting that it may serve as a long-acting GnRH-analog.     

Study in vitro and in vivo of nociceptin/orphanin FQ(1-13)NH2 analogues substituting N-Me-Gly for Gly2 or Gly3

In the present study, two analogues containing N-Me-Gly (Sarcosine, Sar) were synthesized to further investigate the structural-activity relationships of orphanin FQ/nociceptin (OFQ/NC, NC). The replacement of Gly(2) or Gly(3) with Sar increased the flexibility and decreased the hydrophobicity of the N-terminal tetrapeptide. The activity of the analogues was investigated in a series of assays in vivo and in vitro. [Sar(2)]NC(1-13)NH(2) was found to (1) produce dose-dependent inhibition of the electrically induced contraction in MVD assay (pEC(50) = 6.14); (2) produce significant hyperalgesia effects in a dose-dependent manner when intracerebroventricularly (i.c.v.) injected in mice. The inhibitive effects of [Sar(2)]NC(1-13)NH(2) in MVD assay could be significantly antagonized by [Nphe(1)]NC(1-13)NH(2), and partially antagonized by naloxone; the hyperalgesic effect of [Sar(2)]NC(1-13)NH(2) could be significantly antagonized by naloxone, and partially antagonized by [Nphe(1)]NC(1-13)NH(2). On the contrary, [Sar(3)]NC(1-13)NH(2) showed no effects in these assays. All the findings suggest that the flexibility of the peptide bond between Phe(1) and Gly(2) and between Gly(2) and Gly(3) play an important role in NC-OP(4) receptor interaction, and the hydrophobicity of the N-terminal tetrapeptide showed no significant effect on this interaction. The present work also helps to provide a novel method to elucidate structural and conformational requirements of the opioid peptide-receptor interaction.     

Nociceptin receptor activation inhibits tachykinergic non adrenergic non cholinergic contraction of guinea pig isolated bronchus

We studied the action of nociceptin (NC) on the atropine-resistant contractions of the guinea pig isolated bronchus evoked by the electrical field stimulation (EFS), an effect that is mediated by the activation of excitatory non adrenergic-non cholinergic (eNANC) nerves and the subsequent release of tachykinins. The functional site by which NC acts in this preparation was investigated using few different NC receptor agonists and the newly discovered NC receptor antagonist, [Phe1psi(CH2-NH)Gly2]NC(1-13)NH2 ([F/G]NC(1-13)NH2). NC inhibited in a concentration dependent manner (pEC50 7.14; Em - 87 +/- 3% of control values) EFS induced contractions. NC effect was mimicked by the NC analogues, NCNH2 and NC(1-13)NH2, but not by NC(1-9)NH2. NC (1 microM) did not affect the contractile effects of exogenously applied neurokinin A (1 microM). [F/G]NC(1-13)NH2 (10 microM) completely prevented the inhibition induced by NC (1 microM), whereas naloxone (1 microM) was found inactive. Both naloxone and ([F/G]NC(1-13)NH2 were per se inactive on basal resting tone as well as on the electrically induced contractions. The present findings show that NC inhibits the atropine-resistant EFS-induced contraction in the guinea pig bronchus by inhibiting eNANC nerves, and suggest the presence of NC receptors, distinct from opioid receptors, on the nerves of the guinea pig bronchus.     

Synthesis and biological activities of linear and cyclic enkephalin analogues containing a psi (E,CH = CH) or psi (CH2CH2) isosteric replacement.

The peptide CO-NH function was replaced by a trans carbon-carbon double bond or by a CH2-CH2 isostere in enkephalin analogues of DADLE, DCDCE-NH2 or DPDPE. In DADLE the 2-3 and the 3-4 peptide bond was modified, whereas in the cyclic analogues the Gly3-Phe4 bond was replaced by the isosteres Gly psi (E,CH = CH)Phe [5-amino-2-(phenylmethyl)-3(E)-pentenoic acid] or Gly psi (CH2CH2)Phe [5-amino-2-(phenylmethyl)pentanoic acid]. In general, the modification results in a drop in potency which is the largest for the flexible CH2-CH2 replacement. The Gly3 psi (E,CH = CH)Phe4 DCDCE-NH2 analogue retains considerable potency. These results confirm the importance of the peptide function at the 2-3 and 3-4 position in enkephalin analogues for biological potency.     

Novel potent agonist [(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 and antagonist [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 of nociceptin/orphanin FQ receptor

Two novel ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe4,Aib7, Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-1) and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 (peptide-2), have been generated by combining different modifications of N/OFQ sequence. In the present study, we investigated the actions of two analogues and compared them with those of N/OFQ in four assays. Peptide-1 mimicked N/OFQ effects in mouse vas deferens and mouse colon and showed similar maximal effects but higher potency relative to N/OFQ. The effects of peptide-1 were sensitive to NOP receptor selective antagonist ([Nphe1]N/OFQ(1-13)-NH2) but not to naloxone in vitro. Peptide-1 (25 pmol, i.c.v.) mimicked the pronociceptive action of N/OFQ (2.5 nmol, i.c.v.) in mouse tail withdrawal assay, displaying higher potency and longer lasting effects. In anesthetized rats, peptide-1 (1 nmol/kg, i.v.) produced a marked decrease in mean arterial pressure, which was comparable to that evoked by i.v. N/OFQ (100 nmol/kg). Peptide-2 did not produce any effect per se but antagonized N/OFQ actions in mouse vas deferens and mouse colon assays. Peptide-2 is active in vivo where it prevented the pronociceptive effect induced by 2.5 nmol N/OFQ i.c.v. in the mouse tail withdrawal assay. Furthermore, peptide-2 at 5 nmol produced alone a robust and long lasting antinociceptive effect. Moreover, peptide-2 (10 and 40 nmol/kg i.v.) didn't produce any effect per se but antagonized hypotensive actions produced by i.v. administration of N/OFQ. Collectively, these findings demonstrate that [(pF)Phe4,Aib7,Aib11, Arg14,Lys15]N/OFQ-NH2 behaves as a highly potent NOP receptor agonist which produces long lasting effects in vivo and [Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 acts as a pure and competitive antagonist of the NOP receptor.     

Morphiceptin and beta-casomorphin-5 analogues containing a reduced peptide bond: selective mu-receptor agonists and a novel mu antagonist, H-Tyr-Pro psi (CH2-NH)Phe-Pro-Gly-OH.

In order to prevent enzymatic degradation of beta-casomorphin-5 (1) and morphiceptin, reduced peptide bonds were incorporated at the 2-3 and 3-4 bonds, respectively. The analogues were synthesized by a combination of solid phase methodology and reductive alkylation of resin-bound peptide amines with Boc-amino acid aldehydes (Boc: tert-butyloxycarbonyl) in the presence of NaBH3CN. During reversed phase high pressure liquid chromatography purification, peak shape distortions could be observed. Epimerization was excluded, based on gas chromatography/mass spectroscopy analysis, which indicated acceptable levels of racemization (less than 3%) in the crude product. Instead, the phenomena could be attributed to slow cis/trans isomerizations originating from the Xxx-Pro bonds in the sequence. The presence of different conformational isomers was also established by 1H-nmr spectroscopy in DMSO-d6. All analogues showed high stability in blood plasma, enhanced binding affinity for the mu receptor, and very low binding to the delta receptor. While the Phe 3 psi(CH2-N)Pro4 analogues (3) and (5) displayed agonist activity, the Pro 2 psi(CH2-NH)Phe3 modified analogue (2) showed antagonist activity comparable to D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2.     

Synthesis and receptor binding properties of chimeric peptides containing a mu-opioid receptor ligand and nociceptin/orphanin FQ receptor ligand Ac-RYYRIK-amide

Four chimera peptides composed of ORL1 receptor ligand Ac-RYYRIK-NH2 and a mu-opioid receptor agonist dermorphin YAFGYPS-NH2 or YRFB-NH2, with a spacer linking the two pharmacophores, were synthesized and tested for their receptor binding properties. Chimera peptides with long spacers (a Lys and five or eight Gly residues) showed synergistically improved affinity for both the mu-opioid receptor and ORL1 receptor, while the chimera peptides with short spacers (Lys residue only) showed decreased or similar affinity compared to the monomeric receptor ligands. Chimera peptides containing long spacers may prove to be useful tools for studying ORL1 receptor/mu-opioid receptor heterodimers.     

Designed modification of partial agonist of ORL1 nociceptin receptor for conversion into highly potent antagonist

Nociceptin is an endogenous agonist ligand of the ORL1 (opioid receptor-like 1) receptor, and its antagonist is a potential target of therapeutics for analgesic and antineuropathy drugs. Ac-RYYRIK-NH(2) is a hexapeptide isolated from the peptide library as an antagonist that inhibits the nociceptin activities mediated through ORL1. However, the structural elements required for this antagonist activity are still indeterminate. In the present study, we evaluated the importance of the acetyl-methyl group in receptor binding and activation, examining the peptides acyl-RYYRIK-NH(2), where acyl (R-CO) possesses a series of alkyl groups, R=C(n)H(2n+1) (n=0-5). The isovaleryl derivative with the C(4)H(9) (=(CH(3))(2)CHCH(2)-) group was found to reveal a high receptor-binding affinity and a strong antagonist nature. This peptide achieved a primary goal of eliminating the agonist activity of Ac-RYYRIK-NH(2) and producing pure antagonist activity.     

Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats

Nociceptin/orphanin FQ/(N/OFQ), a novel heptadecapeptide recently isolated from porcine and rat brain, is the endogenous ligand of the N/OFQ peptide receptor (NOP, previously known as ORL-1). In this study we examined the effects of intracerebroventricularly (icv) injected N/OFQ on gastric emptying, gastrointestinal transit, colonic propulsion and gastric acid secretion in rats. N/OFQ (0.01-10 nmol/rat) significantly delayed gastric emptying of a phenol red meal, inhibited transit of a non-absorbable charcoal marker through the small intestine and increased the mean colonic bead expulsion time. These N/OFQ-motor effects were abolished by the NOP receptor selective antagonist [NPhe(1)]N/OFQ(1-13)-NH(2) (50 nmol/rat), but were unaltered by the classical opioid receptor antagonist, naloxone (9.2 micromol/kg). Icv injected N/OFQ (10 nmol/rat) decreased gastric acid secretion in 2-h pylorus ligated rats in a naloxone sensitive manner. [NPhe(1)]N/OFQ(1-13)-NH(2) (100 nmol/rat) icv administered alone stimulated gastric acid secretion. These results indicate that N/OFQ activates via NOP receptor stimulation a central inhibitory pathway modulating gastrointestinal propulsive activity and gastric acid secretion in rats.