RESUMEN
In the present article, we summarize the preclinical pharmacology of 4-{(R)-(3-aminophenyl)[4-(4-fluorobenzyl)-piperazin-1-yl]methyl}-N,N-diethylbenzamide (AZD2327), a highly potent and selective agonist of the δ-opioid receptor. AZD2327 binds with sub-nanomolar affinity to the human opioid receptor (K(i) = 0.49 and 0.75 nM at the C27 and F27 isoforms, respectively) and is highly selective (>1000-fold) over the human µ- and κ-opioid receptor subtypes as well as >130 other receptors and channels. In functional assays, AZD2327 shows full agonism at human δ-opioid receptors ([(35)S]GTPγ EC(50) = 24 and 9.2 nM at C27 and F27 isoforms, respectively) and also at the rat and mouse δ-opioid receptors. AZD2327 is active in a wide range of models predictive of anxiolytic activity, including a modified Geller-Seifter conflict test and social interaction test, as well as in antidepressant models, including learned helplessness. In animals implanted with microdialysis probes and then given an acute stressor by pairing electric shock delivery with a flashing light, there is an increase in norepinephrine release into the prefrontal cortex associated with this acute anxiety state. Both the benzodiazepine anxiolytic standard diazepam and AZD2327 blocked this norepinephrine release equally well, and there was no evidence of tolerance to these effects of AZD2327. Overall, these data support the role of the δ-opioid receptor in the regulation of mood, and data suggest that AZD2327 may possess unique antidepressant and anxiolytic activities that could make a novel contribution to the pharmacotherapy of psychiatric disorders.
Asunto(s)
Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacología , Benzamidas/farmacología , Benzamidas/uso terapéutico , Desamparo Adquirido , Piperazinas/farmacología , Piperazinas/uso terapéutico , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismo , Analgésicos Opioides/química , Animales , Benzamidas/química , Condicionamiento Operante/efectos de los fármacos , Condicionamiento Operante/fisiología , Evaluación Preclínica de Medicamentos/métodos , Cobayas , Células HEK293 , Humanos , Masculino , Ratones , Piperazinas/química , Unión Proteica/fisiología , Ratas , Ratas Long-Evans , Ratas Sprague-Dawley , Ratas WistarRESUMEN
Galanin exerts an inhibitory effect on locus coeruleus (LC) neurons via a postsynaptic, as yet unidentified galanin receptor. Using an in vitro intracellular recording technique the effect of two galanin receptor agonists on LC neurons was investigated. Bath application of [Sar(1), D-Ala(12)]gal(1-16)-NH(2) (AR-M961), an agonist both at galanin R1 and R2 (GALR1, GALR2) receptors, evoked a reversible membrane hyperpolarization and inhibition of spike discharge in all LC neurons tested (n=42). The action of AR-M961 was blocked by tetraethylammonium chloride. Hyperpolarizing responses induced by AR-M961 were retained in the presence of tetrodotoxin and high Mg(2+)/low Ca(2+) media. The selective GALR2 agonist Gal(2-11)-NH(2) (AR-M1896) only caused inhibition of spike discharge and a slight hyperpolarization in 26 of 34 LC neurons tested, and was on a molar basis much weaker than AR-M961. These results suggest that it mainly is the GALR1 receptor that mediates hyperpolarization of LC neurons.
Asunto(s)
Galanina/metabolismo , Locus Coeruleus/efectos de los fármacos , Neuronas/efectos de los fármacos , Receptores de Neuropéptido/agonistas , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Relación Dosis-Respuesta a Droga , Galanina/farmacología , Locus Coeruleus/citología , Masculino , Neuronas/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de Galanina , Receptores de Neuropéptido/fisiologíaRESUMEN
Galanin is a 29-aa neuropeptide with a complex role in pain processing. Several galanin receptor subtypes are present in dorsal root ganglia and spinal cord with a differential distribution. Here, we describe a generation of a specific galanin R2 (GalR2) agonist, AR-M1896, and its application in studies of a rat neuropathic pain model (Bennett). The results show that in normal rats mechanical and cold allodynia of the hindpaw are induced after intrathecal infusion of low-dose galanin (25 ng per 0.5 microl/h). The same effect is seen with equimolar doses of AR-M1896 or AR-M961, an agonist both at GalR1 and GalR2 receptors. In allodynic Bennett model rats, the mechanical threshold increased dose-dependently after intrathecal injection of a high dose of AR-M961, whereas no effect was observed in the control or AR-M1896 group. No effect of either of the two compounds was observed in nonallodynic Bennett model rats. These data indicate that a low dose of galanin has a nociceptive role at the spinal cord level mediated by GalR2 receptors, whereas the antiallodynic effect of high-dose galanin on neuropathic pain is mediated by the GalR1 receptors. Thus, a selective GalR1 agonist may be used to treat neuropathic pain.
Asunto(s)
Analgésicos no Narcóticos/uso terapéutico , Causalgia/fisiopatología , Galanina/farmacología , Galanina/fisiología , Hiperestesia/fisiopatología , Proteínas del Tejido Nervioso/fisiología , Fragmentos de Péptidos/farmacología , Isoformas de Proteínas/fisiología , Receptores de Neuropéptido/fisiología , Ciática/fisiopatología , Médula Espinal/fisiopatología , Analgésicos no Narcóticos/administración & dosificación , Analgésicos no Narcóticos/farmacología , Animales , Causalgia/inducido químicamente , Causalgia/tratamiento farmacológico , Frío/efectos adversos , Relación Dosis-Respuesta a Droga , Galanina/administración & dosificación , Galanina/química , Galanina/uso terapéutico , Galanina/toxicidad , Ganglios Espinales/química , Ganglios Espinales/efectos de los fármacos , Miembro Posterior/inervación , Hiperestesia/inducido químicamente , Hiperestesia/tratamiento farmacológico , Hiperestesia/etiología , Bombas de Infusión Implantables , Masculino , Modelos Animales , Proteínas del Tejido Nervioso/agonistas , Umbral del Dolor/efectos de los fármacos , Fragmentos de Péptidos/química , Isoformas de Proteínas/agonistas , Ratas , Ratas Sprague-Dawley , Receptores de Galanina , Receptores de Neuropéptido/agonistas , Nervio Ciático/lesiones , Ciática/tratamiento farmacológico , Ciática/etiología , Médula Espinal/química , Estrés Mecánico , Especificidad por SustratoRESUMEN
The design, synthesis and pharmacological evaluation of a novel class of Dmt-Tic dipeptide analogues are described. These resulting analogues bearing different C-terminal functionalities were found to bind to the human delta receptor with high affinity. One specific class of dipeptides bearing urea/thiourea functionalities showed partial to full activation of the delta receptor. Several dipeptides also showed good binding affinities with full activation of the human kappa receptor, a novel property for those ligands.
Asunto(s)
Dipéptidos/síntesis química , Isoquinolinas/química , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Tetrahidroisoquinolinas , Tirosina/química , Dipéptidos/química , Dipéptidos/metabolismo , Humanos , Receptores Opioides delta/agonistas , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Tirosina/análogos & derivadosRESUMEN
Nonpeptide delta opioid agonists are analgesics with a potentially improved side-effect and abuse liability profile, compared to classical opioids. Andrews analysis of the NIH nonpeptide lead SNC-80 suggested the removal of substituents not predicted to contribute to binding. This approach led to a simplified lead, N, N-diethyl-4-[phenyl(1-piperazinyl)methyl]benzamide (1), which retained potent binding affinity and selectivity to the human delta receptor (IC(50) = 11 nM, mu/delta = 740, kappa/delta > 900) and potency as a full agonist (EC(50) = 36 nM) but had a markedly reduced molecular weight, only one chiral center, and increased in vitro metabolic stability. From this lead, the key pharmacophore groups for delta receptor affinity and activation were more clearly defined by SAR and mutagenesis studies. Further structural modifications on the basis of 1 confirmed the importance of the N, N-diethylbenzamide group and the piperazine lower basic nitrogen for delta binding, in agreement with mutagenesis data. A number of piperazine N-alkyl substituents were tolerated. In contrast, modifications of the phenyl group led to the discovery of a series of diarylmethylpiperazines exemplified by N, N-diethyl-4-[1-piperazinyl(8-quinolinyl)methyl]benzamide (56) which had an improved in vitro binding profile (IC(50) = 0.5 nM, mu/delta = 1239, EC(50) = 3.6 nM) and increased in vitro metabolic stability compared to SNC-80.
Asunto(s)
Benzamidas/síntesis química , Piperazinas/síntesis química , Quinolinas/síntesis química , Receptores Opioides delta/agonistas , Animales , Benzamidas/química , Benzamidas/metabolismo , Disponibilidad Biológica , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Técnicas In Vitro , Espectrometría de Masas , Microsomas Hepáticos/metabolismo , Piperazinas/química , Piperazinas/metabolismo , Quinolinas/química , Quinolinas/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Termodinámica , TransfecciónRESUMEN
The design, synthesis, and pharmacological evaluation of a novel class of delta opioid receptor agonists, N, N-diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide (6a) and its analogues, are described. These compounds, formally derived from SNC-80 (2) by replacing the piperazine ring with a piperidine ring containing an exocyclic carbon carbon double bond, were found to bind with high affinity and exhibit excellent selectivity for the delta opioid receptor as full agonists. 6a, the simplest structure in the class, exhibited an IC(50) = 0.87 nM for the delta opioid receptors and extremely high selectivity over the mu receptors (mu/delta = 4370) and the kappa receptors (kappa/delta = 8590). Rat liver microsome studies on a selected number of compounds show these olefinic piperidine compounds (6) to be considerably more stable than SNC-80. This novel series of compounds appear to interact with delta opioid receptors in a similar way to SNC-80 since they demonstrate similar SAR. Two general approaches have been established for the synthesis of these compounds, based on dehydration of benzhydryl alcohols (7) and Suzuki coupling reactions of vinyl bromide (8), and are herewith reported.
Asunto(s)
Benzamidas/síntesis química , Piperidinas/síntesis química , Receptores Opioides delta/agonistas , Administración Oral , Animales , Benzamidas/química , Benzamidas/metabolismo , Benzamidas/farmacología , Disponibilidad Biológica , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Técnicas In Vitro , Espectrometría de Masas , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Piperazinas/metabolismo , Piperidinas/química , Piperidinas/metabolismo , Piperidinas/farmacología , Ensayo de Unión Radioligante , Ratas , Receptores Opioides delta/metabolismo , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Estereoisomerismo , Relación Estructura-Actividad , Termodinámica , TransfecciónRESUMEN
The potent delta-opioid receptor antagonist H-2',6-L-tyrosine(Dmt)-1, 2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic-OH) exhibited partial inverse agonism (EC(50)=6.35 nM, E(max)=-18.87%) for [35S]GTPgammaS binding and H-Dmt-Tic-NH(2) was a neutral antagonist (no effect up to 30 microM). In contrast N,N(CH(3))(2)-Dmt-Tic-NH(2) was a full inverse agonist (EC(50)=2.66 nM, E(max)=-35.95%) similar to ICI 174864 ([N,N-diallyl-Tyr(1),Aib(2,3),Leu(5)]enkephaline) but with a 3.5-fold higher EC(50). In comparison, naltrindole was a neutral antagonist while its analogue HS 378 was a partial inverse agonist (E(max)=-12.99%).
Asunto(s)
Bencenoacetamidas , Dipéptidos/farmacología , Isoquinolinas/farmacología , Naltrexona/análogos & derivados , Naltrexona/farmacología , Antagonistas de Narcóticos/farmacología , Receptores Opioides delta/agonistas , Tetrahidroisoquinolinas , Benzamidas/farmacología , Unión Competitiva/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Dipéptidos/química , Relación Dosis-Respuesta a Droga , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Isoquinolinas/química , Naltrexona/química , Piperazinas/farmacología , Pirrolidinas/farmacología , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Radioisótopos de AzufreRESUMEN
delta-Opioid receptors, present in very high concentrations in striatum and overlying cortex, are thought to be involved in a number of processes, including analgesia, mood, reward, modulation of neuronal excitability, and alterations in neurotransmitter release. Given the localization of the receptors in motor circuits in brain, we thought it of interest to study the antiparkinson potential of delta-opioid receptor agonists. Rats were given unilateral 6-hydroxydopamine lesions of the nigrostriatal tract, and following recovery, were tested for rotational activity. Tonazocine mesylate is a nonpeptide, partial delta-opioid receptor agonist with mu-receptor antagonist properties. Tonazocine (0.1-10 mg/kg) evoked a dose-related, ipsilateral rotation, consistent with augmentation of dopaminergic function on the unlesioned side. The rotation evoked by tonazocine was blocked by the selective delta-opioid receptor antagonist naltrindole, suggesting that the effect was mediated by delta-opioid receptors. The full delta-opioid receptor agonist (+)-4-¿9-alpha-R)-alpha-(2S,5RO-4-allyl-2, 5-dimethyl-1-piperaziny l)-3-methoxybenzyl-N,N-diethylbenzamide (SNC-80) produced both contralateral and ipsilateral rotation. Tonazocine additionally augmented the effects of L-3,4 dihydroxyphenylalanine (L-DOPA) on reserpine-induced suppression of motor activity. Binding affinities and efficacies of tonazocine and SNC-80 against mu-, kappa-, and delta-opioid receptors were also confirmed and compared to standards. These data suggest therapeutic potential of agents interacting with delta-opioid receptors, and indicate some differences in the activities of tonazocine and SNC-80.
Asunto(s)
Antiparkinsonianos/farmacología , Azocinas/farmacología , Benzamidas/farmacología , Piperazinas/farmacología , Receptores Opioides delta/agonistas , Animales , Masculino , Actividad Motora/efectos de los fármacos , Ratas , Ratas Long-Evans , Reserpina/farmacología , RotaciónRESUMEN
A series of Dmt-Tic analogues with substitution on the Tic aromatic ring has been synthesized and evaluated for opioid receptor affinity and activation. Incorporation of large hydrophobic groups at position 7 of Tic did not greatly alter the delta opioid receptor binding affinities of the dipeptides whereas substitution at position 6 substantially diminished their affinity. These modified Dmt-Tic peptides showed binding affinities as low as 2.5 nM with up to 500-fold selectivity for the delta versus mu opioid receptor and proved to be delta receptor antagonists.
Asunto(s)
Dipéptidos/síntesis química , Isoquinolinas/química , Receptores Opioides delta/antagonistas & inhibidores , Tetrahidroisoquinolinas , Tirosina/análogos & derivados , Benzamidas/metabolismo , Unión Competitiva , Dipéptidos/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Piperazinas/metabolismo , Receptores Opioides delta/agonistas , Tirosina/químicaRESUMEN
AR-M100613 ([I]-Dmt-c[-D-Orn-2-Nal-D-Pro-D-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the delta opioid receptor. Specific [125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (Kd = 0.080 +/- 0.008 nM, Bmax = 45.2 +/- 4.4 fmol/mg protein). [125I]AR-M100613 binding was displaced with high affinity by the delta opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the mu or kappa-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [125I]AR-M 100613 to mu opioid receptors is blocked by the addition of CTOP to the assay buffer. [35S]GTPgammaS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for delta opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [125I]AR-M100613 favor its use as a radiochemical probe for delta opioid receptors.
Asunto(s)
Receptores Opioides delta/metabolismo , Animales , Encéfalo/metabolismo , Radioisótopos de Yodo , Cinética , Ligandos , Masculino , Naltrexona/análogos & derivados , Naltrexona/metabolismo , Oligopéptidos/metabolismo , Ensayo de Unión Radioligante , Ratas , Ratas Sprague-DawleyRESUMEN
Enantiopure heterocyclic Boc-protected Phe-Gly dipeptidomimetics containing 1,3,4-oxadiazole, 1,2,4-oxadiazole, and 1,2,4-triazole ring systems have been synthesized as building blocks in the synthesis of pseudopeptides. Three derivatives (1-3) have the carboxylic acid function directly bound to the heterocyclic ring, and three derivatives (4-6) have an extra methylene group between the heterocyclic ring and the acid function to allow for an increased conformational flexibility. The mimetics were used as Phe-Gly replacements in the biologically active peptides dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) and substance P (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-MetNH(2), SP). The pseudopeptide synthesis was performed using solid-phase methodology on a MBHA-resin using Boc-chemistry. The biological evaluation was performed by testing the micro- and delta-opioid receptor affinities of the dermorphin pseudopeptides and the NK(1) receptor affinities of the SP pseudopeptides. The results showed that all mimetics except 3 were excellent replacements of Phe-Gly in dermorphin since they displayed affinities for the micro-receptor (IC(50) = 12-31 nM) in the same range as dermorphin itself (IC(50) = 6.2 nM). The agonist activity of three pseudopeptides at human micro-receptors was also evaluated. It was shown that the tested compounds retained their agonist activity. The SP pseudopeptides showed considerably lower affinities (IC(50) > 1 microM) for the NK(1) receptor than SP itself (IC(50) = 1.5 nM) indicating that the Phe-Gly replacements prevent the pseudopeptides from adopting bioactive conformations.
Asunto(s)
Dipéptidos/química , Oligopéptidos/síntesis química , Oxadiazoles/síntesis química , Triazoles/síntesis química , Animales , Encéfalo/metabolismo , Línea Celular , Diseño de Fármacos , Humanos , Técnicas In Vitro , Modelos Moleculares , Conformación Molecular , Imitación Molecular , Oligopéptidos/química , Oligopéptidos/metabolismo , Oxadiazoles/química , Oxadiazoles/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptores de Neuroquinina-1/metabolismo , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Triazoles/química , Triazoles/metabolismoRESUMEN
The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.
Asunto(s)
Galanina/química , Glicina/química , Fragmentos de Péptidos/química , Péptidos Cíclicos/química , Alanina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Línea Celular , Dicroismo Circular , Transferencia de Energía , Galanina/síntesis química , Humanos , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/síntesis química , Péptidos Cíclicos/síntesis química , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Triptófano/química , Células Tumorales Cultivadas , Tirosina/químicaRESUMEN
Gene-knockout studies of melanin-concentrating hormone (MCH) and its effect on feeding and energy balance have firmly established MCH as an orexigenic (appetite-stimulating) peptide hormone. Here we identify MCH as the ligand for the orphan receptor SLC-1. The rat SLC-1 is activated by nanomolar concentrations of MCH and is coupled to the G protein G alpha i/o. The pattern of SLC-1 messenger RNA expression coincides with the distribution of MCH-containing nerve terminals and is consistent with the known central effects of MCH. Our identification of an MCH receptor could have implications for the development of new anti-obesity therapies.
Asunto(s)
Encéfalo/metabolismo , Regulación de la Expresión Génica , Hormonas Hipotalámicas/farmacología , Melaninas/farmacología , Hormonas Hipofisarias/farmacología , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Transcripción Genética , Animales , Calcio/metabolismo , Línea Celular , Clonación Molecular , AMP Cíclico/metabolismo , Proteínas Fluorescentes Verdes , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/genética , Especificidad de Órganos , ARN Mensajero/genética , Ratas , Receptores de Somatostatina/efectos de los fármacos , Proteínas Recombinantes de Fusión/biosíntesis , Transfección , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.
Asunto(s)
Encéfalo/metabolismo , Hipófisis/metabolismo , Receptores de Hormona Liberadora de Tirotropina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Clonación Molecular , ADN Complementario/metabolismo , Humanos , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Ensayo de Unión Radioligante , Ratas , Receptores de Hormona Liberadora de Tirotropina/química , Receptores de Hormona Liberadora de Tirotropina/fisiología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Hormona Liberadora de Tirotropina/farmacología , Hormona Liberadora de Tirotropina/fisiología , TransfecciónRESUMEN
Pharmacological study of Phe-Met-Leu-Phe-amide (FMRFa) receptors is hindered by the lack of selective ligands. The classification of these selective ligands is further hampered by the limited availability of functional assays. In this study, we evaluated several synthetic FMRFa analogs for agonist and antagonist activity by measuring their abilities to produce [35-S]-GTP-gamma-S stimulation or to inhibit FMRFa-induced [35S]-GTP-gamma-S binding in squid optic lobes. Analogs included acetyl-Phe-norLeu-Arg-Phe-amide (acFnLRFa), desamino-Tyr-Phe-Leu-Arg-amide (daYFLRa), desamino Tyr-Phe-norLeu-Arg-Phe-amide (daYFnLRFa), desamino Tyr-Phe-norLeu-Arg-[TIC]-amide (daYFnLR[TIC]a), desamino Tyr-Trp-norLeu-Arg-amide (daYWnLRa), (D)-Tyr-Phe-norLeu-Arg-Phe-amide (D)-YFnLRFa), Phe-Leu-Arg-Phe-amide (FLRFa), and the D-amino acid analogs of FMRFa (D-FMRFa, F-(D)-MRFa and FM-(D)-RFa). For agonist studies, full dose-response curves were generated and analyzed for potency and efficacy (maximal percent effect). FMRFamide as well as analogs ac-FnLRFa, daYFnLRFa, daYFnLR[TIC]a, D-YFnLRFa, FLRFa, and (D)-FMRFa stimulated [35S]-GTP-gamma-S binding. Analogs daYWnLRa, daYFLRa, F-(D)-MRFa, and FM-(D)-RFa failed to stimulate either [35S]-GTP-gamma-S binding or to inhibit FMRFa-induced [35S]-GTP-gamma-S binding. The rank order of potency was daYFnLRFa > or = daYFnLRF[TIC]a > acFnLRFa > (D)YFnLRFa > FLRFa > or = FMRFa >> (D)-FMRFa. The order of efficacy was daYFnLRFa = acFnLRFa = (D)-YFnLRFa > FLRFa = FMRFa > or = (D)-FMRFa > or = daYFnLRF[TIC]a. Peptide analog daYFnLR[TIC]a was less efficacious (59% maximal stimulation) than analogs daYFnLRFa, acFnLRFa, and (D)-YFnLRFa (113-146% maximal stimulation). A maximal concentration of daYFnLR[TIC]a (10 microM) reduced daYFnLRFa, acFnLRFa, and (D)-YFnLRFa induced [35S]-GTP-gamma-S stimulation, indicating that daYFnLR[TIC]a is a partial agonist at the receptor stimulated by the FMRFamide analogs. Analysis of the structural requirements needed for promoting [35S]-GTP-gamma-S binding show that elongation (i.e., daYFnLRFa, D-YFnLRFa) or modification of Phe1 (ac-FnLRFa) leads to increased efficacy and potency. Moreover, elimination of the C-terminal Phe (daYWnLRa, daYFLRa,) leads to a loss of biological activity. However, substitution with L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid, a rigid analog of the C-terminal Phe (daYFnLR[TIC]a), leads to decreased efficacy but not loss of potency. The data suggest that immobilization or modification of the C-terminal Phe may produce highly selective and potent FMRFamide antagonists. These results agree with published receptor radioligand studies and indicate that the [35S]GTP-gamma-S assay may be useful in classifying novel FMRFamide-selective ligands.
Asunto(s)
FMRFamida/análogos & derivados , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Lóbulo Óptico de Animales no Mamíferos/metabolismo , Receptores de Péptidos de Invertebrados/agonistas , Receptores de Péptidos de Invertebrados/antagonistas & inhibidores , Animales , Decapodiformes , Evaluación Preclínica de Medicamentos/métodos , FMRFamida/metabolismo , Membranas/metabolismo , Oligopéptidos/metabolismo , Ensayo de Unión Radioligante , Fracciones Subcelulares/metabolismoRESUMEN
We have identified a novel subtype of galanin receptor (GALR-2) in rat dorsal root ganglia and spinal cord. The open reading frame of GALR-2 is 1116 nucleotides long, encoding a protein of 372 amino acids with a theoretical molecular mass of 40.7 kD. Membranes prepared from stable pools of 293 cells expressing GALR-2, but not wild-type 293 cells, demonstrated high affinity galanin binding sites. Rat galanin and galanin-related peptides M40, C7, M15, and galanin effectively competed for binding; peptide C7 demonstrated a lower affinity for rGALR-2, and all these peptides were agonists at rGALR-2 when assessed on a microphysiometer. Studies on the expression of GALR-2 in various tissues by Northern and in situ hybridization analyses suggest a low abundance but wide distribution of GALR-2 mRNA, including several discrete areas in brain and spinal cord and a high abundance in the dorsal root ganglia.
Asunto(s)
Dolor/fisiopatología , Receptores de Neuropéptido/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Ganglios Espinales/fisiología , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Receptores de Galanina , Médula Espinal/fisiologíaRESUMEN
FMRFamide is a molluscan peptide that has shown antiopiate activity in a number of mammalian test systems. Peptidomimetics of FMRFamide substituted with conformationally constrained stereoisomers of Z-2,3-methanomethionine or E-2,3-methanomethionine precipitated abstinence syndrome far more potently than FMRFamide itself. The current study determined the effect on antiopiate potency of an additional rigid substitution. A peptidomimetic containing a stereoisomer of E-2,3-methanomethionine was compared with a peptidomimetic additionally substituted at the C-terminal with E-2,3-methanophenylalanine. Morphine abstinence signs were observed after varying doses (0.125-25.0 micrograms) of these two peptidomimetics were injected into the third ventricle of morphine-dependent rats. The peptidomimetic containing both rigid substitutions was far more potent than the peptidomimetic of FMRFamide containing methanomethionine alone. The increased potency appears to be related to enzyme resistance rather than receptor affinity.
Asunto(s)
Hormonas de Invertebrados/farmacología , Neuropéptidos/farmacología , Péptidos Opioides/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , FMRFamida , Hormonas de Invertebrados/química , Hormonas de Invertebrados/genética , Leucil Aminopeptidasa/metabolismo , Masculino , Metionina/análogos & derivados , Metionina/química , Datos de Secuencia Molecular , Dependencia de Morfina/metabolismo , Neuropéptidos/química , Neuropéptidos/genética , Oligopéptidos/química , Oligopéptidos/genética , Fenilalanina/análogos & derivados , Fenilalanina/química , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido/metabolismo , EstereoisomerismoRESUMEN
In an attempt to attenuate specifically identified receptors through photolysis, a four-step synthesis is of a useful tethered derivative of Azure-B (Az) was developed After characterization, this derivative was covalently attached to CFMRFamide, CFMRF, and CLRFamide (i.e., three different neuropeptide analogues of the putative neurotransmitter FMRFamide. This resulted in the formation of three dye-neuropeptide conjugates: Az-CFMRFamide, Az-CFMRF, and Az-CLRFamide.
Asunto(s)
Colorantes Azulados/química , Cisteína/química , Neuropéptidos/química , Receptores de Péptidos de Invertebrados/antagonistas & inhibidores , FMRFamidaRESUMEN
Three neuropeptide analogues of FMRFamide (FMRFa) were covalently attached to a tethered derivative of methylene blue to form dye-neuropeptide conjugates. The comparative binding of the latter to FMRFa receptors was subsequently examined in both Helix aspersa (circumesophageal ganglia) and squid (optic lobe membrane). In Helix, the FMRFa analogue CFMRFamide (CFMRFa) inhibited the specific binding of the FMRFa ligand [125I]daYFnLRFa in a dose-dependent manner. Az-CFMRFa, one of the dye-neuropeptide conjugates, also dose-dependently inhibited the specific binding of [125I]daYFnLRFa. Moreover, their potencies equaled or exceeded that of FMRFamide. In squid, the binding of CFMRFa and FMRFa was similar. However, the dye-neuropeptide conjugate (IC50 of 14 nM) was about 44-fold less potent than FMRFa. The conjugates were synthesized as part of a study seeking to target and inactivate preselected receptors with heretofore unattainable selectivity and permanence.
Asunto(s)
Colorantes/metabolismo , Decapodiformes/metabolismo , Caracoles Helix/metabolismo , Azul de Metileno/metabolismo , Neuropéptidos/metabolismo , Receptores de Péptidos de Invertebrados/metabolismo , Animales , Encéfalo/metabolismo , Colorantes/farmacología , FMRFamida , Técnicas In Vitro , Azul de Metileno/farmacología , Contracción Miocárdica/efectos de los fármacos , Neuropéptidos/química , Neuropéptidos/farmacología , Lóbulo Óptico de Animales no Mamíferos/metabolismo , Relación Estructura-ActividadRESUMEN
There is evidence that neuropeptide FF (NPFF) has antiopiate activity and may play a role in opiate dependence and subsequent abstinence syndrome. A fragment of NPFF was modified at the C-terminal in an effort to convert it to an NPFF antagonist. It was also dansylated at the N-terminal in an effort to render it more lipophilic and increase its penetration of the blood-brain barrier. Third ventricle administration of the resulting compound, dansyl-PQRamide (0.75 microgram and 1 microgram), dose-dependently antagonized the quasi-morphine abstinence activity of NPFF (10 micrograms) in opiate-naive rats. Subcutaneous injection of dansyl-PQRamide (13 mg/kg) in chronically morphine-infused rats attenuated opiate dependence as indicated by prevention of naloxone-precipitated abstinence syndrome. Dansyl-PQRamide displaced radiolabelled ligand from NPFF receptors in a concentration-dependent manner with a Ki of 13 microM, and had a half-life over 300 times longer than NPFF under aminopeptidase digestion.