RESUMEN
Previous structure-activity studies on nociceptin/orphanin FQ (N/OFQ) identified [Phe(1)Psi(CH(2)NH)Gly(2)]N/OFQ(1-13)-NH(2) and [Nphe(1)]N/OFQ(1-13)-NH(2) as a N/OFQ peptide receptor (NOP) partial agonist and pure antagonist, respectively. The addition of fluorine to the Phe(4) or the insertion of a further pair of basic amino acids Arg(14)-Lys(15) generate potent agonists. On the basis of these findings, we combined in the N/OFQ-NH(2) template the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) that increase the agonist potency with those conferring partial agonist (Phe(1)Psi(CH(2)NH)Gly(2)) or pure antagonist (Nphe(1)) properties. Twelve peptides were synthesized and pharmacologically evaluated in Chinese hamster ovary cells expressing the human recombinant NOP and in electrically stimulated mouse vas deferens and guinea pig ileum assays. All peptides behaved as NOP ligands; the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) increased ligand affinity/potency. Peptides with the normal Phe(1)-Gly(2) peptide bond behaved as full agonists, and those with the Phe(1)Psi(CH(2)NH)Gly(2) modification behaved as partial agonists, while those with the Nphe(1) modification behaved as partial agonists or pure antagonists depending on the presence or absence of the (pF)Phe(4) modification, respectively. The full agonist [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), the partial agonist [Phe(1)Psi(CH(2)NH)Gly(2),(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), and the pure antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) represent the most potent peptide ligands for NOP.
Asunto(s)
Antagonistas de Narcóticos , Péptidos Opioides/química , Péptidos Opioides/síntesis química , Receptores Opioides/agonistas , Animales , Unión Competitiva , Células CHO , Cricetinae , Cricetulus , Estimulación Eléctrica , Cobayas , Humanos , Íleon/fisiología , Técnicas In Vitro , Ligandos , Masculino , Ratones , Péptidos Opioides/farmacología , Ensayo de Unión Radioligante , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Relación Estructura-Actividad , Conducto Deferente/fisiología , Receptor de Nociceptina , NociceptinaRESUMEN
A novel ligand for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-102), has been generated by combining in the N/OFQ-NH(2) sequence two chemical modifications, [Arg(14),Lys(15)] and [(pF)Phe(4)], that have been previously demonstrated to increase potency. In vitro, UFP-102 bound with high affinity to the human NOP receptor, showed at least 200-fold selectivity over classical opioid receptors, and mimicked N/OFQ effects in CHO(hNOP) cells, isolated tissues from various species, and mouse cortical synaptosomes releasing 5-hydroxytryptamine. UFP-102 showed similar maximal effects but higher potency (2- to 48-fold) relative to N/OFQ. The effects of UFP-102 were sensitive to NOP-selective antagonists J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (pA(2) = 7.75-8.12) and UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2))(pA(2) = 6.91-7.33) but not to naloxone, and no longer observed in tissues taken from NOP receptor knockout mice (NOP(-/-)). In vivo, UFP-102 (0.01-0.3 nmol i.c.v.) mimicked the pronociceptive action of N/OFQ (0.1-10 nmol i.c.v.) in the mouse tail withdrawal assay, displaying higher potency and longer lasting effects. The action of UFP-102 was not apparent in NOP(-/-) mice. Similar results were obtained measuring locomotor activity in mice. In conscious rats, UFP-102 (0.05 nmol i.c.v.) produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. These effects were comparable with those evoked by N/OFQ at 5 nmol. Collectively, these findings demonstrate that UFP-102 behaves as a highly potent and selective NOP receptor agonist that produces long-lasting effects in vivo.
Asunto(s)
Péptidos Opioides/farmacología , Receptores Opioides/agonistas , Animales , Unión Competitiva , Presión Sanguínea/efectos de los fármacos , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cobayas , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Riñón/efectos de los fármacos , Riñón/fisiología , Masculino , Actividad Motora/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Serotonina/metabolismo , Transmisión Sináptica , Conducto Deferente/efectos de los fármacos , Receptor de NociceptinaRESUMEN
The synthesis and pharmacological activity of novel nociceptin/orphanin FQ (N/OFQ) analogues modified in the Phe(1)-Gly(2) peptide bond are reported. The aim of the present work was to elucidate the importance of this peptide bond for the N/OFQ receptor (NOP) interaction. Our study indicates that the first peptide bond in N/OFQ is important but not crucial for interaction with the N/OFQ receptor; for instance, substitution with a methyleneoxy bond generates an agonist derivative just 3-fold less potent than the reference compound.
Asunto(s)
Péptidos Opioides/síntesis química , Péptidos Opioides/farmacología , Animales , Estimulación Eléctrica , Glicina/química , Técnicas In Vitro , Ligandos , Masculino , Ratones , Relajación Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Péptidos/química , Fenilalanina/química , Relación Estructura-Actividad , NociceptinaRESUMEN
UNLABELLED: Complex regional pain syndrome is often treated with the sympatholytic guanethidine and a local anesthetic in a Bier's block. The efficacy of this treatment has been questioned. Because local anesthetics inhibit the norepinephrine uptake transporter, we hypothesized that this variable efficacy results from the local inhibiting the uptake of guanethidine. In this study, we tested this hypothesis by using a sympathetically innervated mouse vas deferens preparation. Organ bath-mounted mouse vasa deferentia were electrically stimulated in the absence and presence of guanethidine, prilocaine, procaine, and cocaine in various combinations. Prilocaine (1 mM) induced an immediate inhibition of twitch response (maximum 100% after 2 min) that fully reversed after washing. Guanethidine (3 microM) also inhibited twitching by 95% +/- 3% in 15 min, but this effect was only partially reversed after 1 h of washing (33% +/- 12% of control). When prilocaine and guanethidine were added in combination, a reversal of 80% +/- 13% (at 1 h) was observed. Procaine (300 micro M) produced a transient increase (152% +/- 14%) in response. When co-incubated with guanethidine (3 microM), the twitch was reduced to 24% +/- 4% of control and was reversed to 77% +/- 7% after 1 h. Cocaine (30 microM) inhibited the twitch response to 53% +/- 8%, which was fully reversed by 1 h of washing. When co-incubated with guanethidine, the response was reduced to 39% +/- 6% of control and was reversed to 86% +/- 10% after 1 h. In all cases, the reversal produced by the combination was significantly more intense (P < 0.05) than that produced by guanethidine alone. Local anesthetics reduce the sympatholytic actions of guanethidine, and this may explain the variable efficacy of guanethidine in the treatment of complex regional pain syndrome. IMPLICATIONS: In this study, with a sympathetically innervated vas deferens preparation, local anesthetics reduced the efficacy of the sympatholytic guanethidine, questioning its co-administration in the pain clinic.
Asunto(s)
Adrenérgicos/farmacología , Anestésicos Locales/farmacología , Guanetidina/farmacología , Músculo Liso/efectos de los fármacos , Conducto Deferente/efectos de los fármacos , Antagonistas Adrenérgicos alfa/farmacología , Animales , Cocaína/farmacología , Estimulación Eléctrica , Técnicas In Vitro , Masculino , Ratones , Contracción Muscular/efectos de los fármacos , Prazosina/farmacología , Prilocaína/farmacología , Procaína/farmacología , Tetrodotoxina/farmacologíaRESUMEN
1 This study reports on the pharmacological characterization of ZP120, a novel ligand of the nociceptin/orphanin FQ (N/OFQ) peptide receptor, NOP. ZP120 is a structure inducing probes modified NOP ligand: Zealand Pharma proprietary SIP technology was used to increase the enzymatic stability and half-life of peptide. 2 In vitro, ZP120 mimicked the inhibitory effects of N/OFQ in the electrically stimulated mouse vas deferens, showing however higher potency (pEC(50) 8.88 vs 7.74), lower maximal effects (E(max) 69+/-5% vs 91+/-2%), and slower onset of action. Like N/OFQ, the effects of ZP120 were not modified by 1 micro M naloxone, but they were antagonized by the NOP receptor selective antagonist J-113397 (pA(2) 7.80 vs ZP120, 7.81 vs N/OFQ). 3 In vivo, ZP120 mimicked the effects of N/OFQ, producing pronociceptive effects in the tail withdrawal assay and decreased locomotor activity after i.c.v., but not after i.v. administration in mice. ZP120 elicited similar maximal effects as N/OFQ, but it was about 10 fold more potent and its effects lasted longer. 4 In conclusion, the novel NOP receptor ligand ZP120 is a highly potent and selective partial agonist of the NOP receptor with prolonged effects in vivo.
Asunto(s)
Antagonistas de Narcóticos , Oligopéptidos/farmacología , Péptidos Opioides/metabolismo , Animales , Bencimidazoles/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Técnicas In Vitro , Inyecciones Intravenosas , Ligandos , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Péptidos Opioides/farmacología , Piperidinas/farmacología , Receptores Opioides/metabolismo , Conducto Deferente/efectos de los fármacos , Conducto Deferente/fisiología , Receptor de Nociceptina , NociceptinaRESUMEN
Phe(4) in the nociceptin (NC) sequence has been identified as the most critical residue for receptor interaction. In the present study, we investigated the pharmacological activity of a series of NC(1-13)NH(2) analogues, in which the hydrogen atom in the para position of Phe(4) was substituted with F, NO(2), CN, Cl, Br, I, CH(3), OH or NH(2). In receptor binding studies, performed using CHO cells expressing the recombinant human NC receptor (CHO(hOP4)) and in rat cerebral cortex membranes, [(pF)Phe(4)]NC(1-13)NH(2), [(pNO(2))Phe(4)]NC(1-13)NH(2), and [(pCN)Phe(4)]NC(1-13)NH(2) displayed higher affinity than NC(1-13)NH(2). The affinity of [(pCl)Phe(4)]NC(1-13)NH(2) was essentially identical to that of NC(1-13)NH(2), while the remaining compounds displayed reduced affinity. In a series of functional assays (stimulation of GTPgammaS binding in CHO(hOP4)cells and rat cerebral cortex membranes and inhibition of cAMP accumulation in CHO(hOP4) cells), the para substituted analogues behaved as full agonists (with the exception of [(pOH)Phe(4)]NC(1-13)NH(2) which acted as a partial agonist in the GTPgammaS binding assays) with the following rank order potency:[(pF)Phe(4)]NC(1-13)NH(2) and [(pNO(2))Phe(4)]NC(1-13)NH(2) were either inactive or displayed micromolar potencies in cAMP accumulation experiments performed on cells expressing classical opioid receptors. All compounds were full agonists in isolated tissues from various species (guinea pig ileum, mouse colon and mouse/rat vas deferens) with the exception of [(pOH)Phe(4)]NC(1-13)NH(2) which displayed partial agonist/weak antagonist activities. The rank order of potency was similar to that found in the other assays. The effects of all analogues were not modified by naloxone. The selective OP(4) receptor antagonist [Nphe(1)]NC(1-13)NH(2), tested in all preparations against one or both of the highly potent derivatives [(pF)Phe(4)]NC(1-13)NH(2) and [(pNO(2))Phe(4)]NC(1-13)NH(2), showed pA(2) values similar to those found against NC, the pA(2) in the GTPgammaS binding/rat cerebral cortex assay being much higher (ca. 7.5) than in the other functional assays (ca. 6). This study further supports the notion that Phe(4) of NC is the critical residue for receptor occupation and activation. Moreover, as part of this study, we have identified two novel, highly potent and selective agonists for the OP(4) receptor, [(pF)Phe(4)]NC(1-13)NH(2) and [(pNO(2))Phe(4)]NC(1-13)NH(2).
Asunto(s)
Péptidos Opioides/química , Péptidos Opioides/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Receptores Opioides/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Cobayas , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Receptor de NociceptinaRESUMEN
1. Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions by activating a specific G-protein coupled receptor (NOP). Few molecules are available that selectively activate or block the NOP receptor. Here we describe the in vitro and in vivo pharmacological profile of a novel NOP receptor ligand, [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101). 2. UFP-101 binds to the human recombinant NOP receptor expressed in Chinese hamster ovary (CHO) cells with high affinity (pK(i) 10.2) and shows more than 3000 fold selectivity over classical opioid receptors. UFP-101 competitively antagonizes the effects of N/OFQ on GTPgamma(35)S binding in CHO(hNOP) cell membranes (pA(2) 9.1) and on cyclic AMP accumulation in CHO(hNOP) cells (pA(2) 7.1), being per se inactive at concentrations up to 10 microM. 3. In isolated peripheral tissues of mice, rats and guinea-pigs, and in rat cerebral cortex synaptosomes preloaded with [(3)H]-5-HT, UFP-101 competitively antagonized the effects of N/OFQ with pA(2) values in the range of 7.3 - 7.7. In the same preparations, the peptide was inactive alone and did not modify the effects of classical opioid receptor agonists. 4. UFP-101 is also active in vivo where it prevented the depressant action on locomotor activity and the pronociceptive effect induced by 1 nmol N/OFQ i.c.v. in the mouse. In the tail withdrawal assay, UFP-101 at 10 nmol produces per se a robust and long lasting antinociceptive effect. 5. UFP-101 is a novel, potent and selective NOP receptor antagonist which appears to be a useful tool for future investigations of the N/OFQ-NOP receptor system.
Asunto(s)
Antagonistas de Narcóticos , Péptidos Opioides/metabolismo , Péptidos Opioides/farmacología , Fragmentos de Péptidos/farmacología , Receptores Opioides/metabolismo , Animales , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Cobayas , Técnicas In Vitro , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Péptidos Opioides/química , Dimensión del Dolor/efectos de los fármacos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley , Receptor de NociceptinaRESUMEN
Analogues of the 2',6'-dimethyl-L-tyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) pharmacophore were prepared to test the hypothesis that a "spacer" and a third aromatic center in opioid peptides are required to convert a delta-antagonist into ligands with delta-agonist or with mixed delta-antagonist/mu-agonist properties. Potent delta-agonists and bifunctional compounds with high delta- and mu-opioid receptor affinities were obtained by varying the spacer length [none, NH-CH(2), NH-CH(2)-CH(2), Gly-NH-CH(2)] and C-terminal aromatic nucleus [1H-benzimidazole-2-yl, phenyl (Ph) and benzyl groups]. C-terminal modification primarily affected mu-opioid receptor affinities, which increased maximally 1700-fold relative to the prototype delta-antagonist H-Dmt-Tic-NH(2) and differentially modified bioactivity. In the absence of a spacer (1), the analogue exhibited dual delta-agonism (pEC(50), 7.28) and delta-antagonism (pA(2), 7.90). H-Dmt-Tic-NH-CH(2)-1H-benzimidazole-2-yl (Bid) (2) became a highly potent delta-agonist (pEC(50), 9.90), slightly greater than deltorphin C (pEC(50), 9.56), with mu-agonism (pE(50), 7.57), while H-Dmt-Tic-Gly-NH-CH(2)-Bid (4) retained potent delta-antagonism (pA(2), 9.0) but with an order of magnitude less mu-agonism. Similarly, H-Dmt-Tic-Gly-NH-Ph (5) had nearly equivalent high delta-agonism (pEC(50), 8.52) and mu-agonism (pEC(50), 8.59), while H-Dmt-Tic-Gly-NH-CH(2)-Ph (6) whose spacer was longer by a single methylene group exhibited potent delta-antagonism (pA(2), 9.25) and very high mu-agonism (pEC(50), 8.57). These data confirm that the distance between the Dmt-Tic pharmacophore and a third aromatic nucleus is an important criterion in converting Dmt-Tic from a highly potent delta-antagonist into a potent delta-agonist or into ligands with mixed delta- and mu-opioid properties.
Asunto(s)
Dipéptidos/química , Oligopéptidos/síntesis química , Receptores Opioides delta/antagonistas & inhibidores , Tetrahidroisoquinolinas , Animales , Encéfalo/metabolismo , Técnicas In Vitro , Ligandos , Masculino , Ratones , Oligopéptidos/química , Oligopéptidos/farmacología , Ensayo de Unión Radioligante , Ratas , Receptores Opioides delta/agonistas , Receptores Opioides mu/agonistas , Relación Estructura-Actividad , Sinaptosomas/metabolismo , Conducto Deferente/efectos de los fármacos , Conducto Deferente/fisiologíaRESUMEN
The nociceptin (NC)/orphanin FQ analog, [Arg(14),Lys(15)]NC, has been recently demonstrated to behave as a potent agonist at the human recombinant NC receptors (OP(4)). In this study, we evaluated the pharmacological profile of [Arg(14),Lys(15)]NC in vitro on the native OP(4) receptors expressed in isolated tissues and in vivo in the locomotor activity and the tail-withdrawal assays in mice. On isolated tissues, [Arg(14),Lys(15)]NC mimicked the effects of NC, showing similar maximal effects but higher potencies (17-fold in the mouse vas deferens, 10-fold in the rat vas deferens, and about 5-fold in the guinea pig ileum and mouse colon). In these preparations, the effects of [Arg(14),Lys(15)]NC were not modified by 1 microM naloxone, although antagonized by the OP(4) receptor antagonists [Nphe(1)]NC(1-13)NH(2) (pA(2) congruent with 6) and (+/-)trans-1-[1-cyclooctylmethyl-3hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) (pA(2) congruent with 8). In the rat vas deferens, a cocktail of peptidase inhibitors increased the maximal effects of NC, its analog, and the pEC(50) of NC (by 4-fold); the potency of [Arg(14),Lys(15)]NC was not significantly modified by peptidase inhibitors. In in vivo experiments, [Arg(14),Lys(15)]NC mimicked the effects of NC, producing, after intracerebroventricular administration, pronociceptive effects in the tail-withdrawal assay and inhibiting the locomotor activity of the mice. In both assays, [Arg(14),Lys(15)]NC was about 30-fold more potent than NC and produced longer lasting effects. Taken together, the present data demonstrate that [Arg(14),Lys(15)]NC behaves as a highly potent agonist of the OP(4) receptor and is able to produce long-lasting effects in vivo, compared with the natural ligand NC.