RESUMEN

In an attempt to explain the relationship between conformations of peptide substrates of thermolysin in natural form and the experimental enzymatic cleavages, five peptides of various length were studied in two solvents H2O and glycerol, which may mimic the catalytic environmental conditions. As NMR failed to define sufficiently rough constraints to ensure a convergence of a refinement process for such short and flexible peptides, the conformational space was first searched using the MCMM method. The generated structures were then clustered in families using a 0.3A rmsd criterion and the derived structural characteristics were compared to the experimental NMR parameters. In a first approach, the NMR consistent conformations were compared with the structure of a thermolysin bound peptidic inhibitor ZG(P)LL to characterize the free-ligand predisposition to be cleaved. Further molecular dynamic calculations were performed at 300 K on the conformations corresponding to families in agreement with the ZG(P)LL structure in order to obtain information on their stability and on the trajectories of the torsion angles involved in the active site recognition. In conclusion, for four studied peptides, some conformations were found to be in agreement with 5 of the 8 cleavages experimentally observed.

Asunto(s)

Glicerol/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Péptidos/química , Termolisina/química , Agua/química , Cinética , Conformación Proteica , Temperatura , Factores de Tiempo

RESUMEN

Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recognition and elucidation of several new criteria affecting enzyme specificity. Nature and position of the recognized residue, role of adjacent amino acids, lateral chain hydrophobicity, and volume and length of peptides were all considered. Hydrolysis reactions were also carried out in the presence of glycerol; the effect of microenvironment modifications was quantitative, for example in inducing variations in catalytic reaction rates, and also qualitative, such as in influencing affinity.

Asunto(s)

Termolisina/metabolismo , Tampones (Química) , Relación Dosis-Respuesta a Droga , Glicerol/farmacología , Hidrólisis , Cinética , Oligopéptidos/síntesis química , Oligopéptidos/metabolismo , Especificidad por Sustrato , Termolisina/efectos de los fármacos

RESUMEN

Three series of highly delta-opioid selective peptides are now available, and each family is used as template to investigate the structural parameters involved in delta-receptor recognition and in the modulation of the selectivity of the parent peptide. The first series includes cyclic derivatives such as Tyr-D-Pen-Gly-Phe-D-Pen(DPDPE) and Tyr-D-Pen-Gly-Phe-Pen(DPLPE); the second are the synthetic linear constrained peptides [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(DSTBULET), Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr (OtBu)(BUBU) and especially Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu) (BUBUC)] and the last one the natural peptides [Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (deltorphin or dermenkephalin) and Tyr-D-Ala-Phe-Asp-Val-Val-GlyNH2 ([D-Ala2] deltorphin I)]. In the present study, the possibly of transposing some of the decisive factors of delta-selectivity evidenced in the two other families, to the linear constrained peptides series was examined. With this aim in view, residues such as Phe3, pClPhe4 or Asp were introduced in the sequence of DSTBULET, BUBU or BUBUC. Direct comparison between the biochemical profiles of the [pClPhe4] analogs of the linear constrained peptides and their parent compounds shows that the addition of an electronegative atom on the Phe4 residue of enkephalin sequences is not an absolute parameter for delta-selectivity improvement. The hydrophobic delta-receptor subsite seems able to receive a range of molecular volumes and electronegativities. By contrast, this subsite cannot interact with a Phe3 aromatic ring introduced in this series of peptides. Moreover, the results obtained with linear peptides including additional negatively charged residues demonstrate that the proposed location of the delta-receptors in a cationic membrane environment is not adequate to explain the selectivity profile of a number of compounds.

Asunto(s)

Encéfalo/metabolismo , Endorfinas/síntesis química , Endorfinas/metabolismo , Receptores Opioides delta/metabolismo , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/química , Encefalinas/metabolismo , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Ratas

RESUMEN

The effect of various doses of the selective delta agonist BUBU (Tyr-D-Ser(O-t-butyl)-Gly-Phe-Leu-Thr(O-t-butyl) on the vocalization threshold to paw pressure were compared in normal and arthritic rats, a suitable clinical model of chronic pain. In both group of rats, the intravenous administration of BUBU (6, 9, 12 mg/kg in normal and 1.5, 3, 6 mg/kg in arthritic rats) led to significant antinociceptive effects. The same dose of BUBU (6 mg/kg i.v.) produced a much more potent antinociceptive effect in arthritic than in normal rats, and a dose as low as 1.5 mg/kg produced a significant analgesic effect in the arthritic animal, whereas at 3 mg/kg BUBU was ineffective in normal rats. The analgesic effects of BUBU (9 mg/kg in normal and 3 mg/kg in arthritic rats) were completely prevented by the selective delta antagonist naltrindole (1 mg/kg i.v. a dose devoid of analgesic potency per se), while they were not affected by the selective mu antagonist naloxone (0.05 mg/kg i.v.). In addition, 3 mg/kg i.v. of BUBU remained effective in morphine tolerant arthritic rats. These results suggest that delta opioid receptor activation can modulate the transmission of cutaneous mechanical nociceptive information in rats, especially in inflammatory pain conditions.

Asunto(s)

Analgésicos/farmacología , Artritis Experimental/fisiopatología , Encefalinas/farmacología , Morfina/farmacología , Oligopéptidos/farmacología , Dolor/fisiopatología , Receptores Opioides delta/fisiología , Animales , Esquema de Medicación , Interacciones Farmacológicas , Masculino , Morfina/administración & dosificación , Naloxona/farmacología , Naltrexona/análogos & derivados , Naltrexona/farmacología , Ratas , Ratas Sprague-Dawley , Receptores Opioides delta/efectos de los fármacos , Valores de Referencia , Vocalización Animal

RESUMEN

TRIMU-5 (Tyr-D-Ala-Gly-NHC2H4CH(CH3)2) is a potent mu 2-opioid agonist/mu 1-opioid antagonist. A supraspinal dose (0.5 micrograms i.c.v.) of TRIMU-5 which is not analgesic when given alone antagonizes the analgesia produced by intracerebroventricular (i.c.v.) morphine, a mu 1 action. In contrast, in a synergy model consisting of the simultaneous administration of intrathecal morphine (0.1 micrograms) with multiple doses of i.c.v. morphine, the same supraspinal TRIMU-5 dose (0.5 micrograms i.c.v.) enhances analgesia. Supraspinal TRIMU-5 also potentiates spinal morphine directly, shifting its dose-response to the left. These results imply that within the brainstem mu 1 receptors mediate supraspinal analgesia while mu 2 receptors mediate the synergy with spinal mu systems.

Asunto(s)

Tronco Encefálico/fisiología , Morfina/farmacología , Oligopéptidos/farmacología , Receptores Opioides mu/fisiología , Analgesia , Animales , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Inyecciones Intraventriculares , Inyecciones Espinales , Masculino , Ratones , Morfina/administración & dosificación , Morfina/antagonistas & inhibidores , Oligopéptidos/administración & dosificación

RESUMEN

TRIMU-5 (Tyr-D-Ala-Gly-NH-(CH2)2CH(CH3)2) is a potent enkephalin analog with analgesic actions. Detailed studies show high affinity for both mu 1 and mu 2 sites, with poor affinity for delta, kappa 1 and kappa 3 receptors. Of all the mu ligands examined in binding assays, TRIMU-5 was the most mu-selective. In mice, TRIMU-5 administered either intracerebroventricularly (i.c.v.) or intrathecally elicited analgesia which was readily reversed by the mu-selective antagonist beta-funaltrexamine (beta-FNA). However, the analgesia observed following i.c.v. injections differed from traditional mu ligands: (1) the dose of drug required for analgesic activity i.c.v. was 100-fold greater than those following intrathecal administration; (2) although sensitive to beta-FNA, the analgesia was not antagonized by naloxonazine; and (3) the analgesia was reversed by an opioid antagonist given intrathecally (i.t.) but not i.c.v. Thus, TRIMU-5 analgesia appeared to be mediated spinally through mu 2 receptors. TRIMU-5 did have supraspinal actions, inhibiting gastrointestinal transit, another mu 2 action. In binding studies TRIMU-5 had high affinity for mu 1 sites, but pharmacological studies revealed antagonist actions at this receptor. In mice, the analgesia produced by morphine given i.c.v. was antagonized by coinjection of a low TRIMU-5 dose which was inactive alone. Similarly, TRIMU-5 coadministered with morphine into the periaqueductal gray of rats reversed the analgesia seen with morphine alone. Thus, TRIMU-5 is a highly selective mixed mu 2 opioid receptor agonist/mu 1 opioid receptor antagonist.

Asunto(s)

Analgésicos/farmacología , Morfina/farmacología , Oligopéptidos/farmacología , Receptores Opioides mu/efectos de los fármacos , Animales , Sitios de Unión/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Tránsito Gastrointestinal/efectos de los fármacos , Masculino , Ratones , Oligopéptidos/administración & dosificación , Ratas , Ratas Sprague-Dawley , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/clasificación

RESUMEN

Previous work examining the binding of [3H]naloxone benzoylhydrazone (NalBzoH) in calf brain has identified a novel binding site, kappa 3. In mice and rats NalBzoH elicits an analgesic response which can clearly be differentiated from classical mu, delta or kappa 1 mechanisms and which is pharmacologically consistent with a kappa 3 receptor mechanism of action. In the current studies we demonstrate the presence of kappa 3 sites in both mouse and rat brains. The selectivity of the kappa 3 sites for opioids clearly discriminated it from traditional mu, kappa 1, kappa 2 or delta receptors. In the rat, the density of kappa 3 sites increased 2.5-fold from age 2 days to 21 days, after which it remained relatively stable. Among a number of brain regions in the rat, the density of kappa 3 sites varied dramatically. Highest levels were present in the hypothalamus, thalamus, striatum and midbrain with very low levels in the cerebellum. Intermediate levels were present in cortex, brain stem and spinal cord. Together, these studies support the presence of kappa 3 receptors in both mouse and rat brain which are very similar to those previously reported in calf brain.

Asunto(s)

Encéfalo/metabolismo , Endorfinas/metabolismo , Naloxona/análogos & derivados , Receptores Opioides kappa/metabolismo , Animales , Sitios de Unión , Bovinos , Cinética , Masculino , Ratones , Morfina/metabolismo , Naloxona/metabolismo , Ratas , Ratas Sprague-Dawley

RESUMEN

Lesioning of dopamine neurons by injection of 6-hydroxydopamine into the nucleus accumbens blocked the increased rearing activity measured in the open-field and induced by injection into the ventral tegmental area of: [R)-3-(N-hydroxylcarboxamido-2-benzyl-propanoyl)-L-alanine), kelatorphan (complete inhibitor of enkephalin catabolism) or by (Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)): BUBU (selective delta agonist) but not the hypolocomotion evoked by the mu agonist (Tyr-D-Ala-Gly-N(Me)-Phe-Glyol): DAMGO. This suggests the involvement of different neuronal pathways in mu and delta effects.

Asunto(s)

Conducta Animal/efectos de los fármacos , Dopamina/fisiología , Encefalinas/antagonistas & inhibidores , Sistema Límbico/fisiología , Neuronas/fisiología , Receptores Opioides/fisiología , Secuencia de Aminoácidos , Analgésicos/farmacología , Animales , Dipéptidos/farmacología , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/farmacología , Sistema Límbico/efectos de los fármacos , Masculino , Datos de Secuencia Molecular , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Oligopéptidos/farmacología , Oxidopamina/farmacología , Ratas , Ratas Endogámicas , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta , Receptores Opioides mu , Tegmento Mesencefálico/efectos de los fármacos , Tegmento Mesencefálico/metabolismo

RESUMEN

The mu opioid receptors are unquestionably implicated both in supraspinal and spinal analgesia, but there is some controversy about the role of delta receptors in the control of pain at the supraspinal level. This could be due, at least in part, to the local or i.c.v. administration of the opioid agonists. It was therefore interesting to reassess the overall contribution of mu and delta opioid receptors in modulating nociceptive thermal stimuli in the hot plate-test in mice after i.v. injections of DAMGO (Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol) and BUBU (Tyr-D-Ser(O-tert-butyl)-Gly-Phe-Leu-Thr(O-tert-butyl), two highly selective mu and delta receptor agonists, respectively, whose passage into the brain has been demonstrated recently. Both agonists induced dose-dependent, short-lasting (less than 30 min), antinociceptive responses that peaked 5 min after the administration of DAMGO and 10 min after the administration of BUBU. At these times, DAMGO [ED50: 1.26 mumols (0.65 mg)/kg] was 34 times more potent than BUBU [ED50: 42.5 mumols (34 mg)/kg] in the jump response and 13 times more potent in the paw lick. Apparent pA2 values of naloxone (0.004-0.1 mg/kg s.c.) antagonism for DAMGO and BUBU did not differ significantly, 6.95 +/- 0.054 and 7.28 +/- 0.030 for paw lick tests and 7.11 +/- 0.045 and 7.25 +/- 0.027 for jump tests, respectively. The slopes of the pA2 plots were close to the theoretical -1 value for competitive antagonism.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Analgesia , Encefalinas/farmacología , Naltrexona/análogos & derivados , Oligopéptidos/farmacología , Receptores Opioides/efectos de los fármacos , Animales , Relación Dosis-Respuesta a Droga , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/administración & dosificación , Indoles/farmacología , Inyecciones Intravenosas , Masculino , Ratones , Morfinanos/farmacología , Antagonistas de Narcóticos/farmacología , Oligopéptidos/administración & dosificación , Receptores Opioides mu

RESUMEN

The peptidase-resistance and bioavailability of BUBU [H-Tyr-D.Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)-OH], a highly selective and potent agonist of the delta opioid receptor, have been investigated in vitro and in vivo. In vitro at 37 degrees C, the peptide was fully resistant to degradation by rat serum and strongly resistant to degradation by rat brain membranes. In vivo 0.065% of the dose of [3H]BUBU injected intravenously to the mouse was present 15 min later in the brain. The percentage determined for [3H]DAGO [H-Tyr-D.Ala-Gly-(NMe)Phe-Gly-ol], a selective ligand for mu sites, was 0.038%. Specific binding to mouse brain membranes, determined after intracerebroventricular injection of [3H]BUBU, was saturable and a high affinity (KDapp = 25 pmol) was evaluated for the delta-agonist. Competition experiments showed that BUBU is a selective ligand for delta receptors in vivo. Comparison of the analgesic potency (hot plate test) of ICV or IV administered increasing doses of BUBU and DAGO with their in vivo binding properties supports the preferential involvement of mu receptors in supraspinal analgesia. BUBU also induced an increase in spontaneous locomotion after IV administration at a dose lower than that which produced analgesia. The quantitative results obtained in the present study demonstrate that BUBU and DAGO could be used to characterize the pharmacological responses induced by selective stimulation of delta and mu receptors after systemic administration.

Asunto(s)

Encéfalo/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Receptores Opioides/fisiología , Analgésicos , Animales , Conducta Animal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina Metionina/metabolismo , Encefalinas/metabolismo , Inyecciones Intravenosas , Inyecciones Intraventriculares , Cinética , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Oligopéptidos/sangre , Oligopéptidos/farmacología , Ratas , Ratas Endogámicas , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta , Receptores Opioides mu

RESUMEN

The possible role of opioid receptor heterogeneity in the biphasic changes in locomotion (activation and inhibition) induced by non-selective opiates such as morphine, has been investigated by measuring the behaviour of rats exposed to different environments after injection into the ventral tegmental area, of selective mu (DAGO) or delta (DTLET, DSTBULET, BUBU) opioid agonists and of kelatorphan, a complete inhibitor of enkephalin metabolism. delta agonists or kelatorphan-induced hyperactivity in a familiar (actimeter), unfamiliar (four-hole box) and a fear inducing (open-field) environment. These effects were suppressed by naloxone and delta selective antagonists (ICI 174, 864 2 mg/kg SC, naltrindole 7 nmol in the ventral tegmental area). Moreover, the delta agonists and endogenous enkephalins protected by kelatorphan did not affect the emotional state of rats measured in an elevated plus maze. Infused into the ventral tegmental area, DAGO also enhanced locomotion in the actimeter but in contrast to delta agonists and kelatorphan, the mu agonist decreased activity in the open-field and the four-hole box. The hypoactivity observed in these tests could be related to an enhanced emotionality produced by mu receptor stimulation, as shown by the significant decrease in the number of visits and time spent in open arms of the elevated plus maze. Naloxone (0.3 mg/kg SC) but not delta selective antagonists, blocked the various responses induced by DAGO.

Asunto(s)

Emociones/efectos de los fármacos , Endorfinas/farmacología , Naltrexona/análogos & derivados , Receptores Opioides/fisiología , Animales , Ansiedad/psicología , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/farmacología , Conducta Exploratoria/efectos de los fármacos , Indoles/farmacología , Inyecciones , Masculino , Morfinanos/farmacología , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Oligopéptidos/farmacología , Ratas , Ratas Endogámicas , Receptores Opioides delta , Receptores Opioides mu , Tegmento Mesencefálico/anatomía & histología

RESUMEN

A recently developed series of highly selective and systemically active delta-agonists such as Tyr-X-Gly-Phe-Leu-Thr(OtBu), with X = D.Ser (OtBu) in BUBU and X = D.Cys(StBu) in BUBUC, and complete inhibitors of enkephalin metabolism (Kelatorphan, RB 38 A, PC 12) have enabled the major role played by mu-opioid receptors in supraspinal analgesia to be demonstrated. This is in agreement with the results of in vivo mu-receptor occupancy measured by taking into account the cross-reactivity of the delta-ligands for mu-sites. In contrast, mu and delta binding sites seem to act independently to control pain at the spinal level. Strong analgesic effects, especially in arthritic rats, can also be obtained by complete protection of tonically or phasically released endogenous enkephalins with mixed inhibitors such as RB38A. Chronic icv administration of the mu agonist DAGO, led to a severe naloxone precipitated withdrawal syndrome whilst a weak dependence was seen with the delta agonist, DSTBULET or with RB 38 A. Moreover, mixed inhibitors did not induce any significant respiratory depression. All these data emphasize the interest in developing delta-agonists and mixed inhibitors with appropriate bioavailability for clinical evaluation.

Asunto(s)

Analgésicos/farmacología , Secuencia de Aminoácidos , Analgésicos/farmacocinética , Animales , Tolerancia a Medicamentos , Encefalinas/metabolismo , Técnicas In Vitro , Datos de Secuencia Molecular , Estructura Molecular , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta , Receptores Opioides mu , Médula Espinal/fisiología , Trastornos Relacionados con Sustancias/fisiopatología

RESUMEN

Based on the results of conformational studies of linear and cyclic delta-opioid peptides such as BUBU [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)] and DPLPE c[Tyr-D-Pen-Gly-Phe-Pen], a new enkephalin-related peptide, Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr(OtBu) (BUBUC) was synthesized and tested for its opioid activity and selectivity at both the peripheral and central levels. Amongst all the synthetic compounds described so far, BUBUC appears to be the most highly delta-selective probe [KI (mu) = to 2980 nM, KI (delta): 2.9 nM, KI (mu)/KI (delta) approximately 1000]. This selectivity was confirmed by the results of pharmacological studies, including measurements of supraspinal analgesia and behavioral changes in mice. In the later test, BUBUC was shown to increase the rearing activity after IV administration at very low concentrations (0.1 mg/kg) and this effect was reversed by the delta-selective antagonist naltrindole. No antinociceptive response was observed at a 10-fold higher concentration. Thanks to its enzymatic stability and its hydrophobicity. BUBUC is the first systemically active, highly selective delta agonist and should therefore be useful to characterize the physiological role of delta-opioid receptors.

Asunto(s)

Analgésicos Opioides/síntesis química , Oligopéptidos/síntesis química , Receptores Opioides/efectos de los fármacos , Secuencia de Aminoácidos , Analgésicos Opioides/metabolismo , Analgésicos Opioides/farmacología , Animales , Unión Competitiva , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Encefalinas/farmacología , Cobayas , Inyecciones Intraventriculares , Masculino , Ratones , Datos de Secuencia Molecular , Actividad Motora/efectos de los fármacos , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Oligopéptidos/metabolismo , Oligopéptidos/farmacología , Ratas , Receptores Opioides delta

RESUMEN

Introduction into the structure of the linear hexapeptide DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) or DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) of tert-butyl groups as constraints different from cyclization leads to a large increase in the selectivity for delta opioid binding site in the case of DSTBULET [Tyr-D-Ser-(OtBu)-Gly-Phe-Leu-Thr] (Ki delta = 6.14 nM; Ki mu = 374 nM) and BUBU [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)] (Ki delta = 4.68 nM; Ki mu = 475 nM) or a loss of affinity for DTTBULET [Tyr-D-Thr(OtBu)-Gly-Phe-Leu-Thr] (Ki delta = 866 nM; Ki mu = 4500 nM). This puzzling behavior is studied here by 400-MHz 1H NMR spectroscopy in DMSO-d6 solution and by theoretical calculations. When DSLET and DTLET are compared, the reduction in energetically accessible phi and psi angles induced by the tert-butyl group in the D-Ser2 residue decreases the degree of freedom in the N-terminal part of the peptides. For DSTBULET and BUBU, the rigidification of the backbone evidenced by the appearance of the large NOE's of Phe4 NH-Gly3 alpha and Gly3 NH-alpha and by the loss of the C7 folding around the D-Ser2 residue found in DSLET could explain the drastic loss of affinity for mu opioid receptors. In DTTBULET, a large change in the spatial orientation around the D-Thr2 (OtBu) residue forces the aromatic rings far from each other.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Encefalina Leucina/análogos & derivados , Encefalinas/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Secuencia de Aminoácidos , Fenómenos Químicos , Química , Dimetilsulfóxido , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Temperatura

RESUMEN

A series of linear conformationally constrained opioid peptides was designed in an attempt to develop highly selective and potent agonists for the delta opioid receptors. These enkephalin analogues corresponding to the general formula Tyr-D-X(OY)-Gly-Phe-Leu-Thr(OZ) were obtained by incorporating bulky residues (X = Ser or Thr; Y = tert-butyl or benzyl; Z = tert-butyl) into the sequence of the previously reported delta specific agonists DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr). In binding studies based on displacement of mu and delta opioid receptor selective radiolabeled ligands from rat brain membranes, the two constrained hexapeptides, Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr (1, DSTBULET) (KI(mu) = 374 nM, Kr(delta) = 6.14 nM, KI(delta)/KI(mu) = 0.016) and in particular Tyr-D-Ser(O-t-Bu)-Gly-Phe-Leu-Thr(O-t-Bu) (7, BUBU) (KI(mu) = 475 nM, KI(delta) = 4.68 nM, KI(delta)/KI(mu) = 0.010) were shown to be among the most potent and selective delta probes reported to date. A roughly similar pattern of selectivity was obtained with the guinea pig ileum and mouse vas deferens bioassays. In addition, the analgesic potency (hot-plate test) of these peptides intracerebroventricularly administered in mice was shown to be significantly related to their mu-receptor affinity.

Asunto(s)

Encefalinas/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Animales , Unión Competitiva , Encéfalo/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Ratones , Oligopéptidos/síntesis química , Conformación Proteica , Receptores Opioides delta

RESUMEN

Insertion of bulky tertiobutyl groups into the sequence of [D-Ser2,Leu5]enkephalyl-Thr6 leads to a conformationally induced large increase in selectivity toward rat brain delta-opioid binding sites, as shown by the ratio of apparent affinities for mu and delta receptors of [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6,KI(mu)/KI(delta) = 130, and [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 (O-tert-butyl),KI(mu)/KI(delta) = 280. In addition to a selectivity similar to that of the cyclic compounds [D-Pen2, D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin, the affinity of [3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 for the delta sites of rat brain membranes is significantly better (KD = 2.2 nM) than that of [3H][D-Pen2,D-Pen5]enkephalin (KD approximately 8.5 nM). Therefore, [3H][D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6 seems to be the most appropriate delta-probe currently available for binding studies. Moreover, the lipophilic and protected peptide [D-Ser2(O-tert-butyl),Leu5]enkephalyl-Thr6(O-tert-butyl) behaves as the most specific ligand for the delta-opioid binding sites and appears appropriate for in vivo investigations. The inactive analogue [D-Thr2(O-tert-butyl),Leu5]enkephalyl-Thr6 might serve as a negative control in biochemical or pharmacological studies.

Asunto(s)

Encefalinas/metabolismo , Oligopéptidos/metabolismo , Receptores Opioides/metabolismo , Animales , Encéfalo/metabolismo , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalina D-Penicilamina (2,5) , Encefalina Metionina/metabolismo , Cobayas , Cinética , Membranas/metabolismo , Ratas , Ratas Endogámicas , Receptores Opioides delta

RESUMEN

The previous rules proposed for selective recognition of mu and delta opioid receptors by modified enkephalins were investigated through an extensive structure-activity study. Thus, modifications of the sequence of TRIMU 4 (Tyr-D-Ala-Gly-NHCH(CH3)CH2CH(CH3)2, a peptide that exhibits mu selectivity close to that of DAGO (Try-D-Ala-Gly-N(Me)Phe-Gly.ol), were performed for two positions, 2 and 4, critical for mu recognition. The drastic loss of potency following introduction of L-Ala or Aib in position 2 emphasizes the importance of the stereochemistry and the steric size of the X2 amino acid for optimal mu binding. The enhancement of the intrinsic flexibility of the C-terminal alkyl chain of TRIMU 4 through removal of a methyl group leads to TRIMU 5 (Tyr-D-Ala-Gly-NHCH2CH2CH(CH3)2), a peptide with a mu selectivity similar to that of DAGO. In contrast, introduction of an O-tert-butyl Ser2 residue increases affinity for delta receptors. In the hexapeptide series derived from DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr), a D-Thr2 moiety was shown to be very efficient in improving delta recognition and delta selectivity appeared also to be modulated by the nature of the sixth residue. The potencies of the 24 peptides studied to inhibit the electrically evoked contractions of the GPI or MVD are relatively well correlated with their affinities for brain mu or delta receptors labeled with [3H]DAGO or [3H]DSLET, respectively. Moreover, the analgesic potency (hot plate test) of the peptides is related to their affinity for rat brain mu receptors. The wide range of receptor affinities exhibited by the compounds reported here could be useful to study the physiological role of mu and delta receptors.

Asunto(s)

Encefalina Leucina/análogos & derivados , Encefalinas/farmacología , Receptores Opioides/efectos de los fármacos , Analgésicos/farmacología , Animales , Encefalina Ala(2)-MeFe(4)-Gli(5) , Encefalinas/metabolismo , Cobayas , Ratones , Contracción Muscular/efectos de los fármacos , Oligopéptidos/metabolismo , Receptores Opioides delta , Receptores Opioides mu , Relación Estructura-Actividad

RESUMEN

In the present investigation the effects of selective agonists for mu (Tyr-D-Ala-Me-Phe-Gly-ol (DAGO)) and delta (Tyr-D-Thr-Gly-Phe-Leu-Thr (DTLET)) opioid receptors on neuronal activities induced by noxious cutaneous stimuli in the rat ventrobasal (VB) thalamus were analyzed. The two agonists produced a clear depressive action on thermal as well as mechanical noxious stimuli. The depressive action of DTLET (3 mg/kg i.v.) was lower and of shorter duration than that of DAGO (2 mg/kg i.v.). However, this effect is unambiguously related to the selective stimulation of opioid receptors since a consistent effect was also observed for a dose as low as 1.5 mg/kg i.v. of DTLET. Moreover, DTLET effect needs a high concentration of naloxone (0.5 mg/kg i.v.) to be reversed, while DAGO effect is totally reversed with 0.1 mg/kg i.v.

Asunto(s)

Encefalinas/farmacología , Oligopéptidos/farmacología , Dolor/fisiopatología , Receptores Opioides/efectos de los fármacos , Tálamo/efectos de los fármacos , Potenciales de Acción , Animales , Electrofisiología , Encefalina Ala(2)-MeFe(4)-Gli(5) , Calor , Masculino , Naloxona/farmacología , Neuronas/fisiopatología , Estimulación Física , Ratas , Ratas Endogámicas , Receptores Opioides delta , Receptores Opioides mu , Tálamo/fisiopatología

RESUMEN

The antinociceptive effects obtained in arthritic rats with morphine, the opioid mu-agonist DAGO [D-Ala2,MePhe4,Gly-ol5]enkephalin, the delta-selective agonist DTLET [D-Thr2, Leu5]enkephalyl-Thr, and the kappa-agonist U-50,488H were compared to their corresponding effects in normal animals and morphine-pretreated arthritic rats, respectively, using a paw pressure test. The effects of the mu- and kappa-agonists were increased in arthritic rats. While morphine-treated rats were cross-tolerant to the mu- and kappa-agonists, no tolerance to the delta-selective agonist was found. The possibility that the potent action of morphine in this model for chronic inflammatory pain is mediated partly through kappa-mechanisms is discussed.

Asunto(s)

Analgésicos , Artritis Experimental/metabolismo , Artritis/metabolismo , Receptores Opioides/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Enfermedad Crónica , Endorfinas/farmacología , Masculino , Morfina/farmacología , Ratas , Ratas Endogámicas , Receptores Opioides/fisiología , Receptores Opioides kappa , Receptores Opioides mu

RESUMEN

The conformational behavior of CCK7, Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2, and CCK8, Asp-Tyr-Met-Gly-Trp-Met-Asp-Phe-NH2, in their sulfated and unsulfated forms, was studied both by 1H NMR spectroscopy in dimethyl-d6 sulfoxide and water and by fluorescence-transfer measurements at pH 7. In neutral conditions, both experimental methods show that these peptides exist preferentially in folded forms with beta and gamma turns around the sequence Gly-Trp-Met-Asp and Met-Asp-Phe-NH2, respectively. The presence of stable folded conformations is supported by through-space effects during the titration of the ionizable groups and by the weak temperature dependency of some amide protons not only in dimethyl sulfoxide but also in water. The folding of the C-terminal part, already shown in CCK5, seems to be a common conformational characteristic in CCK peptides. The N-terminal part of CCK8 presents an equilibrium between beta and gamma turns, whereas this part of the peptide is more flexible in CCK7. The low quantum yield of Tyr and the large mean distance (R = 15 A) between Tyr and Trp, determined by fluorescence-transfer measurements, support the occurrence of folded conformations pushing the aromatic rings far from each other. Interestingly, the introduction of the sulfate group enhances the folding tendency even in aqueous medium. The larger amide temperature dependency and the decrease in the R distance at acidic pH suggest that an intramolecular ionic interaction involving the N-terminal amino group and the beta-carboxyl groups of Asp32 stabilize the folded forms. Metropolis calculations performed on CCK8 support the existence of stable folded conformations closely related to those deduced from experimental data.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Colecistoquinina/análogos & derivados , Sincalida/análogos & derivados , Secuencia de Aminoácidos , Transferencia de Energía , Espectroscopía de Resonancia Magnética/métodos , Fragmentos de Péptidos , Conformación Proteica , Espectrometría de Fluorescencia/métodos , Relación Estructura-Actividad