Single residue modifications of the delta opioid receptor selective peptide, [D-Pen2,D-Pen5]-enkephalin (DPDPE). Correlation of pharmacological effects with structural and conformational features

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [D-Pen2,D-Pen5]enkephalin, Tyr-D-Pen-Gly-Phe-D-PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by 1H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine1-DPDPE ([DAT1]DPDPE) and phenylglycine4-DPDPE ([Pgl4]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced mu receptor binding affinity of the carboxamide terminal DPDPE-NH2 appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the alpha-aminoisobutyric acid substituted analog [Aib3]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the delta opioid receptor.     

Delta opioid receptor-selective ligands: [D-Pen2,D-Pen5]enkephalin-dermenkephalin chimeric peptides.

A number of DPDPE-dermenkephalin chimeric peptides have been synthesized in which the putative C-terminal delta-address of dermenkephalin has been linked to the highly delta opioid selective cyclic peptide [D-Pen2,D-Pen5]enkephalin (DPDPE). Asp, Met-Asp and Leu-Met-Asp have been added to the C-terminus of DPDPE and both the carboxyl terminal and the carboxamide terminal series have been prepared. The bioassays using the mouse vas deferens and guinea pig ileum preparations have revealed a steady decrease in potency (compared to DPDPE) at delta and mu receptors as the dermenkephalin sequences were added. Some of the analogues, however, retained high delta selectivity. Similar results were obtained using radioligand binding assays. These findings suggest that the C-terminal amino acid sequence of dermenkephalin plays a role of delta-address which is specific to dermenkephalin itself, and is not additive with another delta selective ligand such as DPDPE.     

A three-dimensional model of the delta-opioid pharmacophore: comparative molecular modeling of peptide and nonpeptide ligands

A comparative molecular modeling study of delta-opioid ligands was performed under the assumption that potent peptide and nonpeptide agonists may have common three-dimensional (3D) arrangement of pharmacophore groups upon binding to the delta-receptor. Low-energy conformations of the agonists 7-spiroindanyloxymorphone (SIOM) and 2-methyl-4a-alpha-(3-hydroxyphenyl)-1,2,3,4,4a,5,12, 12a-alpha-octahydro-quinolino[2,3,3-g]isoquinoline (TAN-67), and a partial agonist oxomorphindole (OMI) were determined by high-temperature molecular dynamics (MD). A good spatial overlap was found for the pharmacophore groups of SIOM, TAN-67, and OMI, including the basic nitrogen, phenol hydroxyl, and two aromatic ring. Based on this overlap we proposed a 3D pharmacophore model for nonpeptide delta-opioid agonists with a distance of 7.0 +/- 1.3 A between the two aromatic rings and of 8.2 +/- 1.0 A between the nitrogen and phenyl ring. The potent and highly delta-opioid receptor selective agonist [(2S,3R)-TMT(1)]DPDPE, which shares global backbone constraints of the 14-membered disulfide cycle and a strong preference for the trans rotamer of the TMT(1) side chain, was chosen as a peptide template of the delta-opioid pharmacophore. Extensive MD simulations at 300 K with the AMBER force field were performed for [(2S,3R)-TMT(1)]DPDPE and the less potent [(2S, 3S)-TMT(1)]DPDPE analogue. Multiple MD trajectories were collected for each peptide starting from the x-ray structures of DPDPE and [L-Ala(3)]DPDPE and from models proposed in the literature. Low-energy MD conformations were filtered by the nonpeptide pharmacophore query and then directly superimposed with SIOM, OMI, and TAN-67. Two conformers of [(2S,3R)-TMT(1)]DPDPE that showed the best overlap with the nonpeptide pharmacophore (rms deviation 相似文献   

Cyclic, disulfide- and dithioether-containing opioid tetrapeptides: development of a ligand with high delta opioid receptor selectivity and affinity

Tetrapeptides of primary sequence Tyr-X-Phe-YNH2, where X is D-Cys or D-Pen (penicillamine) and where Y is D-Pen or L-Pen, were prepared and were cyclized via the side chain sulfurs of residues 2 and 4 to disulfide or dithioether-containing analogs. These peptides are related to previously reported penicillamine-containing pentapeptide enkephalin analogs but lack the central glycine residue of the latter and were designed to assess the effect of decreased ring size on opioid activity. Binding affinities of the tetrapeptides were determined to both mu and delta opioid receptors. Binding affinity and selectivity in the tetrapeptide series were observed to be highly dependent on primary sequence. For example, L-Pen4 analogs displayed low affinity and were nonselective, while the corresponding D-Pen4 diastereomers were of variable affinity and higher selectivity. Among the latter compounds were examples of potent analogs in which selectivity shifted from delta selective to mu selective as the ring size was increased. The relatively high binding affinity and delta receptor selectivity observed with one of the carboxamide terminal disulfide analogs led to the synthesis of the corresponding carboxylic acid terminal, Tyr-D-Cys-Phe-D-PenOH. This analog displayed delta receptor binding selectivity similar to that of the standard delta ligand, [D-Pen2,D-Pen5]enkephalin (DPDPE), and was found to have a 3.5-fold higher binding affinity than DPDPE. All the tetrapeptides were further evaluated in the isolated mouse vas deferens (mvd) assay and all displayed opioid agonist activity. In general, tetrapeptide potencies in the mouse vas deferens correlated well with binding affinities but were somewhat lower. Receptor selectivity in the mvd, assessed by examining the effect of opioid antagonists on the tetrapeptide concentration-effect curves, was similar to that determined in the binding studies.     

Comparative binding properties of linear and cyclic delta-selective enkephalin analogues: [3H]-[D-Thr2, Leu5] enkephalyl-Thr6 and [3H]-[D-Pen2, D-Pen5] enkephalin

The range of delta-selectivity of linear and cyclic analogues of enkephalin in rat brain was found to be: [D-Pen2, L-Pen5] enkephalin (DPLPE) greater than [D-Pen2, D-Pen5] enkephalin (DPDPE) greater than [D-Thr2, Leu5] enkephalyl-Thr6 (DTLET) greater than [D-Ser2, Leu5] enkephalyl-Thr6 (DSLET). Saturation experiments performed with [3H]DPDPE and [3H]DTLET in NG108-15 cells and rat brain showed similar binding capacities for both the ligands, but the delta-affinity of [3H]DTLET (KD approximately 1.2 nM) was much better than that of [3H]DPDPE (KD approximately 7.2 nM). The rather low delta-affinity of DPDPE induced high experimental errors cancelling the benefit of its better delta-selectivity. Binding experiments in rat or guinea-pig brains showed, in both cases, the better delta-selectivity of [3H]DTLET compared to [3H]DSLET. The former peptide remains at this time the most appropriate radioactive probe for binding studies of delta-receptor.     

Assessment of stereoselectivity of trimethylphenylalanine analogues of delta-opioid [D-Pen(2),D-Pen(5)]-enkephalin

[D-Pen(2),D-Pen(5)]-Enkephalin (DPDPE) is an enzymatically stable delta-opioid receptor-selective peptide, which was modified by the trimethylation of the Phe(4) residue to give beta-methyl-2', 6'-dimethylphenylalanine (TMP), resulting in four conformations : (2R,3S)-beta-Phe-DPDPE, (2R,3R)-beta-Phe-DPDPE, (2R, 3S)-beta-Phe-DPDPE, and (2S,3R)-beta-Phe-DPDPE. Synthesis was by solid-phase techniques using enantiomerically pure amino acids to give the four optically pure diastereoisomer peptides. The potency and selectivity (delta- versus mu-opioid receptor) were evaluated by radioreceptor binding in rat brain, with a mu/delta ratio decrease for all TMP conformations, compared with the parent compound (DPDPE). Octanol/buffer distribution analysis showed enhanced lipophilicity of all TMP forms, with a sixfold enhancement associated with (2S,3S)-TMP. In situ vascular perfusion in anesthetized rats showed a 1.6-fold (p < 0.01) increase in the ratio of brain uptake for (2S,3S)-TMP and a 1.5-fold (p < 0.01) decrease in uptake for (2R,3R)-TMP. Saturability of (2S,3S)-TMP was shown (p < 0.01) against 100 microM unlabeled DPDPE, showing a shared nondiffusionary transport system. P-glycoprotein affinity was shown in situ for the parent and (2S,3S)-TMP (p < 0.01). Protein binding capacity of the TMP compounds in rat plasma and in situ mammalian bovine serum albumin-Ringer showed (2R,3S)-TMP and (2S,3R)-TMP with the lowest degree of protein binding (p < 0.01), and (2S,3S)-TMP and (2R,3R)-TMP with comparable affinities to DPDPE. Analgesia, via intravenous administration, showed significantly reduced (p < 0.01) end effect and time course for (2R,3R)-TMP, (2R,3S)-TMP, and (2S, 3R)-TMP as compared with DPDPE. These results demonstrate that topographical modification in a conformationally restricted peptide can significantly modulate potency and receptor selectivity, binding capacity, enzymatic stability, lipophilicity, P-glycoprotein affinity, and blood-brain barrier permeability, resulting in a change of bioavailability, and thereby provides insight for future peptide drug design.     

Conformational analysis of enkephalin analogs containing a disulfide bond. Models for delta- and mu-receptor opioid agonists

Conformational analysis of the cyclic opioids H-Tyr-D-Pen-Gly-Phe-D-Pen-OH (DPDPE) and H-Tyr-D-Cys-Gly-Phe-D-Cys-OH (DCDCE) have been performed using the AMBER program. DPDPE is considerably more selective for delta-receptors than DCDCE. Using the RNGCFM program, a large number of ways were found to close the 14-membered disulfide-containing ring structure. However, intramolecular hydrogen bonds were only possible in gamma-turn and inverse gamma-turn conformations centered on the glycine residue which were associated with opposite chiralities of the disulfide bond. With the cyclic part of the molecules in either a gamma-turn or inverse gamma-turn, a systematic conformational analysis was performed on the tyrosine and phenylalanine sidechains. This showed that conformers with the tyrosine and phenylalanine phenyl rings in the vicinity of the disulfide bond were preferred due to attractive van der Waals forces. For DPDPE, however, this was only possible with a positive dihedral angle for the disulfide bond due to the presence of the beta-carbon methyls of Pen2. In contrast, these preferred conformers were possible with both chiralities of the disulfide bond in DCDCE. Conformational entropies and free energies were computed from the translational, rotational, and vibrational energy levels available to each conformer. The conformational entropies were found to vary significantly and to result in a re-ordering of the lowest energy minima. Based on these conformational differences in DPDPE and DCDCE and their differing pharmacological selectivities, tentative conformational preferences for delta- and mu-receptor opioid peptides are proposed.     

D-Pen2-[D-Pen5]enkephalin impairs acquisition and enhances retention of a one-way active avoidance response in rats.

We reported previously that D-Pen2-[D-Pen5]enkephalin (DPDE), a delta-opioid receptor selective analog of Leu-enkephalin, impairs acquisition of an automated jump-up avoidance response in rats and acquisition of a one-way active avoidance response in mice. In the present study we investigated the effects of DPDPE on one-way avoidance conditioning in rats. The rats received two escape-only trials on day 1 and eight additional training trials on day 2. DPDPE (1.16 micrograms/kg IP) administered prior to training on day 2 impaired acquisition of the avoidance response. On the other hand, DPDPE (0.332 microgram/kg IP) administered following presentation of the two escape-only trials on day 1 significantly enhanced retention, as measured by improved one-way active avoidance performance on day 2. These results indicate that activation of delta-opioid receptors by DPDPE has a modulatory effect on acquisition and retention of aversively motivated performance.     

Evidence for δ-Opioid Binding and GTP-Regulatory Proteins in 5-Day-Old Rat Brain Membranes

The availability of the bispenicillamine enkephalin [3H] [D-Pen2,D-Pen5]enkephalin ([3H]DPDPE) a highly selective ligand for delta-opioid receptors, has made possible a more definitive examination of the ontogeny of this receptor subtype. In this report, the binding characteristics of [3H]DPDPE in 5-day-old neonatal (P-5) and adult rat brain are compared. Analysis of saturation curves as well as homologous displacement data revealed no significant difference in the binding affinity of [3H]DPDPE between P-5 animals and adults. Conversely, the binding capacity increased fivefold during this period. The delta-specificity of the sites was further proven by competition experiments with mu- and delta-selective ligands. Mn2+ (0.5 mM) elevated [3H]DPDPE specific binding by lowering the Kd, whereas 50 microM 5'-guanylylimidodiphosphate inhibited it by decreasing the total number of high-affinity binding sites in both P-5 animals and adults. Pertussis toxin-catalyzed ADP ribosylation experiments revealed the presence of 40-kDa proteins, with a molecular mass corresponding to G protein subunits alpha i/alpha o, as early as 1 h after birth. There was a low, but detectable, basal low-Km GTPase activity in P-5 animals, which increased fivefold during postnatal development. The present report establishes the existence of high-affinity [3H]DPDPE binding as well as GTP-regulatory proteins 5 days after birth. Yet, heterologous competition studies and ionic effects suggest that neonatal binding sites differ from adult receptors. Whether the neonatal sites are newly synthesized, incompletely processed sites or a developmentally programmed isoform remains to be determined.     

Partial depletion of endogenous zinc level by (D-Pen2, D-Pen5)enkephalin in the rat brain.

The effects of the potent delta opioid agonist (D-Pen2, D-Pen5)enkephalin (DPDPE) were studied on the endogenous levels and regional distribution of Zn2+ in rat central nervous system by means of flame atomic absorption spectrophotometry. The olfactory bulb exhibited the highest Zn2+ level, followed by the frontal and parietal cortices, striatum and hippocampus; the lowest ion levels were found in the medulla and thoracic spinal cord. Intracerebroventricular administration of DPDPE resulted in significant, time- and dose-dependent decreases in endogenous Zn2+ contents in the parietal cortex, hippocampus and striatum. The action of DPDPE was antagonized by a 30 min naloxone pretreatment. Naloxone alone was without effect in eliciting these responses. Thus, delta opioid receptors may regulate or modulate endogenous Zn2+ levels in the rat brain.     

Examination of the conformational meaning of "delta-address" in the dermenkephalin sequence

Comprehensive energy calculations were applied to four opioid-related peptides with different receptor selectivities, namely the delta-selective dermenkephalin (Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, DRE), the mu-selective dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, DRM) and their "hybrid" peptides DRM/DRE (Tyr-D-Ala-Phe-Gly-Leu-Met-Asp-NH2) and DRE/DRM (Tyr-D-Met-Phe-His-Tyr-Pro-Ser-NH2). It was shown that the N-terminal tripeptide "mu-messages" in the delta-selective ligands DRE and DRM/DRE can possess similar low energy space arrangements of their functionally important elements (the N-terminal alpha-amino group and the aromatic moieties of Tyr and Phe), but that these are different from the space arrangement of these moieties in mu-selective DRM and DRE/DRM. These results suggest that the C-terminal tripeptide "delta-address" in DRE may influence the conformation of the "mu-message" in DRM. A refined model for the delta-receptor-bound conformation of DRE is proposed based on these calculations which is similar to that previously suggested for the cyclic delta-selective peptide [D-Pen2, D-Pen5]enkephalin (DPDPE). This model also has partial correspondence with the structure of the delta-selective alkaloid naltrindole.     

The role of progestin receptors and the mitogen-activated protein kinase pathway in delta opioid receptor facilitation of female reproductive behaviors

The present study investigated the role of the progestin receptor (PR) and the mitogen-activated protein kinase (MAPK) pathway in the facilitation of lordosis behavior by the delta opioid receptor agonist [D-Pen(2), D-Pen(5)]-enkephalin (DPDPE). Ovariectomized, estrogen-primed rats were treated with the PR antagonist RU486 or the MAPK inhibitor PD98059 prior to intraventricular (icv) infusion of DPDPE. Both RU486 and PD98059 blocked receptive and proceptive behaviors induced by DPDPE at 60 min, and RU486 continued to inhibit estrous behavior at 90 min. Because delta opioid receptors can activate the p42/44 MAPKs, extracellular signal regulated kinases (ERK), we determined the effects of DPDPE on ERK phosphorylation. Icv infusion of DPDPE increased the levels of phosphorylated ERK in the hypothalamus and preoptic area of female rats, assessed by immunoblotting. These results support the participation of the PR and the MAPK pathway in the facilitation of lordosis behavior by delta opioid receptors.     

Ligand binding profiles of delta-opioid receptor in human cerebral cortex membranes: evidence of delta-opioid receptor heterogeneity

In this study, receptor binding profiles of opioid ligands for subtypes of opioid delta-receptors were examined employing [3H]D-Pen2,D-Pen5-enkephalin ([3H]DPDPE) and [3H]Ile(5,6)-deltorphin II ([3H]Ile-Delt II) in human cerebral cortex membranes. [3H]DPDPE, a representative ligand for delta1 sites, labeled a single population of binding sites with apparent affinity constant (Kd) of 2.72 +/- 0.21 nM and maximal binding capacity (Bmax) value of 20.78 +/- 3.13 fmol/mg protein. Homologous competition curve of [3H]Ile-Delt II, a representative ligand for delta2 sites, was best fit by the one-site model (Kd = 0.82 +/- 0.07 nM). Bmax value (43.65 +/- 2.41 fmol/mg) for [3H]Ile-Delt II was significantly greater than that for [3H]DPDPE. DPDPE, [D-Ala2,D-Leu5]enkephalin (DADLE) and 7-benzylidenaltrexone (BNTX) were more potent in competing for the binding sites of [3H]DPDPE than for those of [3H]Ile-Delt II. On the other hand, deltorphin II (Delt II), [D-Ser2,Leu5,Thr6]enkephalin (DSLET), naltriben (NTB) and naltrindole (NTI) were found to be equipotent in competing for [3H]DPDPE and [3H]Ile-Delt II binding sites. These results indicate that both subtypes of opioid delta-receptors, delta1 and delta2, exist in human cerebral cortex with different ligand binding profiles.     

Selective delta-opioid receptor antagonism by ICI 174,864 in the central nervous system

The effects of the novel gamma-opioid receptor antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH: Aib = alpha-aminoisobutyric acid) have been examined in the CNS in vivo using spontaneous reflex contractions of the rat urinary bladder as an index of activity. Bladder contractions were inhibited by equipotent intracerebroventricular (ICV) doses of the selective mu-agonist DAGO [D-Ala2, MePhe4,Gly-(ol)5]enkephalin and the delta-agonist DPDPE[D-Pen2, D-Pen5]enkephalin. ICI 174,864 (1-3 micrograms) administered by the same route produce a selective and reversible antagonism of DPDPE effects. At higher doses (6-15 micrograms, ICV) ICI 174,864 exhibited marked agonistic activity, producing inhibition of bladder contractions that were resistant to ICV naloxone (1-2 micrograms). Thus ICI 174,864 was considered a selective central delta-opioid receptor antagonist but its usefulness was limited by additional agonistic properties.     

Conformational analysis of β-methyl-para-nitrophenylalanine stereoisomers of cyclo[D-Pen2, D-Pen5]enkephalin by NMR spectroscopy and conformational energy calculations

Solution conformations of β-methyl-para-nitrophenylalanine⁴ analogues of the potent δ-opioid peptide cyclo[D-Pen², D-Pen⁵]enkephalin (DPDPE) were studied by combined use of nmr and conformational energy calculations. Nuclear Overhauser effect connectivities and ³J_HNC^α_H coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of[β-Me-p-NO₂Phe⁴]DPDPE in DMSO were compared with low energy conformers obtained by energy minimization in the Empirical Conformational Energy Program for Peptides #2 force field. The conformers that satisfied all available nmr data were selected as probable solution conformations of these peptides. Side-chain rotamer populations, established using homonuclear (³J_H^α_H^β) and heteronuclear (³J_H^αC^γ) coupling constants and ¹³C chemical shifts, show that the β-methyl substituent eliminates one of the three staggered rotamers of the torsion angle x¹ for each stereoisomer of the β-Me-p-NO₂Phe⁴. Similar solution conformations were suggested for the L-Phe⁴-containing (2S, 3S)- and (2S, 3R)-stereoisomers. Despite some local differences, solution conformations of L- and D-Phe⁴-containing analogues have a common shape of the peptide backbone and allow similar orientations of the main δ-opioid pharmacophores. This type of structure differs from several models of the solution conformations of DPDPE, and from the model of biologically active conformations of DPDPE suggested earlier. The latter model is allowed for the potent (2S, 3S)- and (2S, 3R)-stereoisomers of [β-Me-p-NO₂Phe⁴] DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [β-Me-p-No₂Phe⁴] DPDPE in the δ-receptor-bound state may assume a conformation different from their favorable conformations in DMSO. © 1996 John Wiley & Sons, Inc.     

Conformational features responsible for binding of cyclic analogues of enkephalin to opioid receptors. I. Low-energy peptide backbone conformers of analogues containing Phe4

Low-energy peptide backbone conformers were found by means of energy calculation for several cyclic analogues of enkephalin in an attempt to assess models for receptor-bound conformations for opioid receptors of the mu- and delta-types. They included [D-Cys2, L-Cys5]- and [D-Cys2, D-Cys5]-enkephalinamides showing moderate preference for mu-receptors, the delta-selective compounds [D-Pen2, L-Pen5] and [D-Pen2, D-Pen5]-enkephalins and Tyr-D-Lys-Gly-Phe- analogue possessing very high affinity to receptors of the mu-type. The low-energy conformers obtained for these analogues were in good agreement with the results of calculations by other authors and with experimental evidence. All of the analogues contain a Phe residue in position 4 of the peptide chain which facilitates the eventual search for geometrical similarity between the low-energy backbone conformers of different analogues in question.     

Opioid peptide interactions with lipid bilayer membranes.

The interaction of the delta-opioid receptor selective peptides, cyclic [D-Pen2, D-Pen5]-enkephalin [DPDPE] and its acyclic analog, DPDPE(SH)2, with neutral phospholipid bilayer membranes was examined by permeability and calorimetry measurements. The permeabilities were accomplished by entrapping either peptide inside of unilamellar liposomes (composed of a mixture of a molar ratio 65:25:10 phosphatidylcholine/phosphatidylethanolamine/cholesterol) then monitoring the peptide efflux through the bilayer. The initial permeability of DPDPE (first 12 h) averaged over four experiments was (0.91 +/- 0.47).10(-12) cm s-1. In contrast the average permeability of the acylic DPDPE(SH)2 was (4.26 +/- 0.23).10(-12) cm s-1. The effect of these peptides on the phase transition, Tm, of 1,2-dipalmitoylphosphatidylcholine (DPPC) bilayers was examined by high sensitivity differential scanning calorimetry. The Tm, the calorimetric enthalpy, and the van 't Hoff enthalpy of DPPC were not significantly altered by the presence of DPDPE, whereas the calorimetric data for DPPC with DPDPE(SH)2 showed a small, yet significant, increase (0.2 degrees C) in the Tm with a 30% decrease in the cooperative unit. Both the permeability and calorimetry data reveal a stronger peptide-membrane interaction in the case of the more flexible acyclic peptide.     

Prolonged in vivo antagonism of central mu- and delta-opioid receptor activity by beta-funal trexamine

beta-Funaltrexamine (beta-FNA) was tested in the spinal cord and supraspinally against inhibition of reflex bladder contractions produced in the anesthetized rat by the opioid-receptor selective agonists [D-Ala2, MePhe4, Gly (ol)5]enkephalin (DAGO, mu-agonist) and [D-Pen2, D-Pen5] enkephalin (DPDPE, delta-agonist). All agents were microinjected either intracerebroventricularly (i.c.v.) or intrathecally (i.t.). beta-FNA (1-8 micrograms) produced long-lasting antagonism of both DAGO and DPDPE. Complete recovery from its effects was only observed some 24-32 h later. Higher doses of beta-FNA (4 and 8 micrograms i.t.) produced short-lived agonistic activity though the selectivity of this was not determined. It was concluded that beta-FNA was a potent, long-lasting antagonist at central opioid receptors in vivo but was unselective for the mu and delta opioid receptor.     

Influence of peptidase inhibitors on the apparent agonist potency of delta selective opioid peptides in vitro.

Several peptides of diverse structure, reported to possess high affinity and selectivity for the delta opioid receptor, were studied using the mouse isolated vas deferens preparation to determine the effect of peptidase inhibition on their apparent potency. The peptides evaluated included [Leu5] enkephalin, the cyclic enkephalin analogs [D-Pen2,D-Pen5]enkephalin (DPDPE) and [D-Pen2,p-F-Phe4,D-Pen5]enkephalin (F-DPDPE), the linear enkephalin analogs [D-Ala2,D-Leu5]enkephalin (DADLE) and [D-Ser2(O-tBu), Leu5,Thr6]enkephalin (DSTBULET), and the naturally occurring amphibian peptides Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (dermenkephalin), Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin I) and Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin II). Concentration-response curves were determined for each peptide in the absence and presence of a combination of the peptidase-inhibiting agents bacitracin, bestatin, and captopril. A wide range of potencies was observed, both in the control state and in the presence of peptidase inhibition. The synthetic enkephalin analogs demonstrated small increases in potency with peptidase inhibition (no increase in the case of DPDPE), whereas the naturally occurring peptides were markedly increased in potency, up to as much as 123-fold for dermenkephalin. In the presence of peptidase inhibition, deltorphin II was the most potent peptide tested (IC50 = 1.13 x 10(-10) molar), and as such is the most potent delta opioid agonist reported to date. Stability to metabolism must be considered in the design and evaluation of in vitro experiments using peptides of this type.