Role of steric interactions in the delta opioid receptor selectivity of [D-Pen2, D-Pen5]enkephalin

In order to assess the individual effects of each of the 3-methyl groups in residue 2 of [D-Pen2, D-Pen5]enkephalin on binding affinity to mu and delta opioid receptors, (2S,3S)methylcysteine ((3S)Me-D-Cys) and (2S,3R)methylcysteine ((3R)Me-D-Cys) were synthesized and incorporated into the analogs, [(3S)Me-D-Cys2, D-Pen5] enkephalin and [(3R)Me-D-Cys2, D-Pen5]enkephalin. Of these analogs, [(3S)Me-D-Cys2, D-Pen5]enkephalin appears from 1H n.m.r. spectra to assume a conformation similar to those of [D-Pen2, D-Pen5]enkephalin and the less delta receptor-selective, but more potent, [D-Cys2, D-Pen5]enkephalin. Assessment of binding affinity to mu and delta receptors revealed that [(3S)Me-D-Cys2, D-Pen5]enkephalin exhibits delta receptor affinity intermediate between [D-Pen2, D-Pen5]enkephalin and [D-Cys2, D-Pen5]enkephalin while its mu receptor affinity is similar to that of [D-Cys2, D-Pen5]enkephalin. These results suggest that, for [D-Pen2, D-Pen5]enkephalin, adverse steric interactions between the D-Pen2 pro-R methyl group and the mu receptor binding site lead to the low mu receptor binding affinity observed for this analog. By contrast, both the pro-R and pro-S D-Pen2 methyl groups lead to minor steric interactions which contribute to the somewhat lower delta receptor affinity of this compound.     

Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.     

Dimeric pentapeptide enkephalin: a novel probe of delta opiate receptors

A dimeric pentapeptide enkephalin (DPE2) consisting of two molecules of [D-Ala 2, Leu 5] enkephalin linked at C-terminal leucine with ethylenediamine, (H-Tyr-D-Ala-Gly-Phe-Leu-NH-Ch2)2 is a bivalent ligand for the delta enkephalin receptors of rat brain and neuroblastoma-glioma hybrid (NG108-15) cells. This new enkephalin analog shows dramatically increased affinity in radioligand assays using whole brain membranes when delta but not mu specific radioligands are employed. When membranes from NG108-15 cells are used, the dimer shows greatly increased activity irrespective of the mu or delta specificity of the tracer. The dimer DPE2 shows a four-fold, "sodium shift" in its IC50 for competition with [3H]naloxone, suggestive of agonist behavior. Agonist activity was confirmed by demonstrating that DPE2 inhibits cyclic AMP production in prostaglandin E1 stimulated NG108-15 cells, and by demonstrating very high potency in the mouse vas deferens bioassay. DPE2 binds to the same delta sites as the delta-selective monomer [D-Ala2, D-Leu5] enkephalin, since the two ligands show complete crossdisplacement. Radiolabeled 3H-DPE2 shows a five-fold higher affinity constant, a 2.5-fold higher association rate constant, and a two-fold lower dissociation rate than the monomer. These results are consistent with the hypothesis that the dimeric pentapeptide enkephalin can bridge two delta receptors. This enkephalin dimer provides a valuable new probe of opiate receptors and their organization in cell membranes.     

Side chain to side chain cyclization of an enkephalin analog results in loss of opioid receptor selectivity

The cyclic enkephalin analog H-Tyr-D-Lys-Gly-Phe-Glu-NH2 (I) and the structurally related open chain analogs H-Tyr-D-Nle-Gly-Phe-Gln-NH2 (II) and H-Tyr-D-Lys(For)-Gly-Phe-Abu-NH2 (III) were tested in mu and delta opioid receptor-representative binding assays and bioassays. Whereas both linear analogs showed a pronounced preference for mu receptors over delta receptors, the conformationally restricted cyclic peptide I was found to be unselective. This finding represents the first reported example of a peptide cyclization resulting in a loss of receptor selectivity. From this and earlier studies, it was concluded that the receptor selectivity of cyclized peptide analogs relative to that of their linear correlates may depend on the size and relative rigidity of their ring structures.     

Inhibition of mu and delta but not kappa opioid binding to membranes by Fab fragments from a monoclonal antibody directed against the opioid receptor

Fab fragments from a monoclonal antibody, OR-689.2.4, directed against the opioid receptor, selectively inhibited opioid binding to rat and guinea pig neural membranes. In a titratable manner, the Fab fragments noncompetitively inhibited the binding of the mu selective peptide [D-Ala2,(Me)Phe4,Gly(OH)5][3H] enkephalin and the delta selective peptide [D-Pen2,D-Pen5] [3H]enkephalin (where Pen represents penicillamine) to neural membranes. In contrast, kappa opioid binding, as measured by the binding of [3H]bremazocine to rat neural membranes and guinea pig cerebellum in the presence of mu and delta blockers, was not significantly altered by the Fab fragments. In addition to blocking the binding of mu and delta ligands, the Fab fragments displaced bound opioids from the membranes. When mu sites were blocked with [D-Ala2,(Me)Phe4,Gly(OH)5]enkephalin, the Fab fragments suppressed the binding of [D-Pen2,D-Pen5][3H]enkephalin to the same degree as when the mu binding site was not blocked. The Fab fragments also inhibited binding to the mu site regardless of whether or not the delta site was blocked with [D-Pen2,D-Pen5]enkephalin. This monoclonal antibody is directed against a 35,000-dalton protein. Since the antibody is able to inhibit mu and delta binding but not kappa opioid binding, it appears that this 35,000-dalton protein is an integral component of mu and delta opioid receptors but not kappa receptors.     

Cystamine-enkephalin dimer. Syntheses and biological activities of enkephalin analogs containing cystamine and cysteamine

A cystamine-enkephalin dimer, containing two molecules of [D-Ala2, Leu5] enkephalin cross-linked at the COOH-terminal leucine residue with cystamine, (NH2-CH2-CH2-S-)2, has been synthesized in order to examine directly the dimerization effect of an enkephalin molecule on the opiate receptor interactions. In a comparison of potencies against [3H]-[D-Ala2,D-Leu5] enkephalin (3H-DADLE) and [3H]-[D-Ala2,MePhe4,Gly-ol5] enkephalin (3H-DAGO) as delta and mu tracers, respectively, enkephalin dimer showed a very high affinity, especially for the delta opiate receptors. Dimer was almost threefold more potent than DADLE, which is one of the most utilized delta ligand to date. When the binding affinity of cystamine-dimer was compared with that of its reduced thiol-monomer, namely [D-Ala2,Leu5,cysteamine6] enkephalin, the increment in affinity was four to fivefold for both delta and mu receptors. The results strongly indicate that the dimeric enkephalin is more potent presumably due to the simultaneous interaction with the two binding sites of the opiate receptors.     

A study of the effect of the irreversible delta receptor antagonist [D-Ala2,Leu5,Cys6]-enkephalin on delta cx and delta ncx opioid binding sites in vitro and in vivo.

Several lines of data support the existence of two classes of delta receptors: the delta cx binding site, which is the delta binding site of the mu-delta opioid receptor complex, and the delta ncx, which is the noncomplexed delta receptor. [D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is an extended analog of [Leu5]enkephalin, which has been shown to bind irreversibly to delta receptors via the terminal cysteine by formation of a disulfide bond with the receptor. In vivo studies have shown that DALCE produces short-lived antinociceptive actions, followed by long-term antagonism of delta receptor-mediated antinociception. The major goal of the present study was to examine the effect of DALCE on the delta cx and delta ncx binding sites in vitro and in vivo. Intracerebroventricular administration of 40 micrograms DALCE failed to decrease [3H][D-Ala2,D-Leu5]enkephalin binding to the delta cx and delta ncx binding sites. Pretreatment of membranes with DALCE in vitro greatly reduced the Bmax of the delta ncx binding site, without significantly altering the Bmax of the delta cx binding site. These findings suggest that when administered in vivo, DALCE fails to distribute uniformly throughout the brain, and that it therefore binds covalently to opioid receptors mostly in the periventricular regions. Viewed collectively, these data support the hypothesis that DALCE acts as a selective delta ncx antagonist, and that the delta ncx binding site, which is sensitive to DALCE, is most likely synonymous with the recently described delta 1 receptor.     

Loss of striatal mu1 opiate binding by substantia nigra lesions in the rat

Opiate receptors have been identified within the striatum and some have been localized presynaptically to nigrostriatal neurons. Using unilateral ablative lesions of the substantia nigra, we examined binding in the ipsilateral and contralateral striata. Lesions significantly lowered both 3H[D-Ala2,MePhe4,Gly(ol)5]enkephalin (DAGO) and 3H[D-Ala2,Leu5]enkephalin (DADL) binding. The inclusion of competitors in these assays revealed a decrease in both mu1 and mu2 receptors. Mu1 binding was slightly more sensitive to the lesioning than mu2 binding. Selective mu1 and mu2 binding assays supported these observations. No change in delta binding was observed in the lesioned striata. These studies raise the possibility that both mu1 and mu2, but not delta, receptors are localized presynaptically on nigrostriatal neurons.     

Cyclic enkephalin analogs containing various para-substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists.

The cyclic enkephalin analog H-Tyr-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) is a highly potent opioid agonist with IC(50)s of 35 pm and 19 pm in the guinea-pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe(1)-analog of this peptide showed 370-fold and 6790-fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr(1) hydroxyl-group in the interaction with mu and delta opioid receptors. In the present study, the effect of various substituents (-NH(2), -NO(2), -CN, -CH(3), -COOH, -COCH(3), -CONH(2)) introduced in the para-position of the Phe(1)-residue of H-Phe-c[D-Cys-Gly-Phe(pNO(2))-D-Cys]NH(2) on the in vitro opioid activity profile was examined. Most analogs showed enhanced mu and delta agonist potencies in the two bioassays, except for the Phe(pCOOH)(1)-analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH(3))(1)- and the Phe(pCONH(2))(1)-analogs. The latter compound showed subnanomolar mu and delta agonist potencies and represents the most potent enkephalin analog lacking the Tyr(1) hydroxyl-group reported to date. Taken together, these results indicate that various substituents introduced in the para-position of Phe(1) enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure-activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

Interaction of S-activated enkephalin analogs with opiate receptors

Enkephalin analogs containing a thiol activated by a thiomethyl (SCH3)*** or 3-nitro-2-pyridinesulfenyl (Npys) group were synthesized. Incubation of such S-activated enkephalin analogs as [D-Ala2, Leu(CH2S)SCH(3)5]enkephalin or [D-Ala2,Leu(CH2S)Npys5]enkephalin with guinea pig ileum (GPI) resulted in the continuous stimulation of the mu opiate receptors. This sustained GPI-activity was completely reversed with the antagonist naloxone, while subsequent washings elicited again the full enkephalin activity. When GPI showing full enkephalin activity was incubated with 1 mM dithiothreitol, about 70% of the activity was eliminated. Examination of enkephalin analogs containing Cys(Npys) at position 1, 5, or 6 suggested that no other thiols occur near the enkephalin binding site of the mu receptor. From these results, it is considered that only one thiol group exists near the binding site of the mu receptor in GPI. Similar results were also obtained for the mu receptors in mouse vas deferens.     

Synthesis and receptor binding affinity of both E- and Z-dehydrophenylalanine4 enkephalins

The dehydrophenylalanine4-enkephalin having the E-configuration (delta EPhe; phenyl and C = 0, cis) was prepared by photoisomerization of the Z-isomer with 3100 A light, followed by reversed-phase HPLC separation of the resulting mixture of the Z- and E-isomers. In the radioligand receptor binding assays, the E-isomer of [D-Ala2, delta Phe4, Leu5]enkephalin exhibited an extremely diminished affinity as compared with the Z-isomer, namely 150-260-fold loss of affinity for the delta and mu opiate receptors. The results indicate that the interrelationship of the Tyr1 and Phe4 residues in the enkephalin molecule seems to be of great importance in receptor recognition.     

Opiate receptor binding characteristics of dimeric analogues of mu-selective DAGO-enkephalin

DAGO-enkephalin ([ D-Ala2, MePhe4, Gly-ol5]enkephalin), a highly selective ligand for mu opiate receptors, was dimerized with a series of alpha,omega-alkanedioic acids (n = 2-12) at the OH-terminus. In the radioligand receptor binding assays with rat brain, most of the DAGO-enkephalin dimers with cross-linking methylene chain n (DEDn) were more potent than DAGO monomer. For delta receptors, affinity of DEDn was maximized with n = 8, which might be related to an optimal distance between two binding sites. For mu receptors, an increase in chain length resulted in a progressive loss of potency. Although all of DEDn are considerably mu-selective, with a mu/delta ratio of 15-50, DEDn exhibited fairly flat binding curves with 15-50% smaller sloped than that of DAGO, suggesting that the dimers interact more strongly with one of the possible two mu binding sites.     

Probing the opioid receptor complex with (+)-trans-superfit. I. Evidence that [D-Pen2,D-Pen5]enkephalin interacts with high affinity at the delta cx binding site.

A variety of data support the existence of an opioid receptor complex composed of distinct but interacting mu cx and delta cx binding sites, where "cx" indicates "in the complex." The ability of subantinociceptive doses of [Leu5]enkephalin and [Met5]enkephalin to potentiate and attenuate morphine-induced antinociception, respectively, is thought to be mediated via their binding to the delta cx binding site. [D-Pen2,D-Pen5]Enkephalin also modulates morphine-induced antinociception, but has very low affinity for the delta cx binding site in vitro. In the present study, membranes were depleted of their delta ncx binding sites by pretreatment with the site-directed acylating agent, (3S,4S)-(+)-trans-N-[1-[2-(4-isothiocyanato)phenyl)-ethyl]-3-methy l-4- piperidyl]-N-phenylpropaneamide hydrochloride, which permits selective labeling of the delta cx binding site with [3H][D-Ala2,D-Leu5]enkephalin. The major findings of this study are that with this preparation of rat brain membranes: a) there are striking differences between the delta cx and mu binding sites; and b) both [D-Pen2,D-Pen5]enkephalin and [D-Pen2,L-Pen5]enkephalin exhibit high affinity for the delta cx binding site.     

The actions of naloxazone on the binding and analgesic properties of morphiceptin (NH2Tyr-Pro-Phe-Pro-CONH2), a selective mu-receptor ligand

Active in both binding and biological assays, morphiceptin (NH₂ Tyr-Pro-Phe-Pro-CONH₂), a potent opioid peptide derivative of β-casamorphine, binds specifically and selectively to mu or morphine-type receptors with little affinity for delta sites. Displacement studies of a variety of ³H-labeled opiates and enkephalins show biphasic curves. Naloxazone, which blocks irreversibly and selectively high affinity opiate and enkephalin binding, abolishes morphiceptin's inhibition of binding at low concentrations, suggesting that the high affinity binding of enkephalins and opiates represents a mu or morphine-type receptor. Unlike the reversible antagonist naloxone, naloxazone treatment $\text{in}$$\text{vivo}$ inhibits for over 24 hours the analgesic activity of morphiceptin like it inhibits morphine, β-endorphin and enkephalin analgesia. Together, these studies imply that opiates and enkephalins bind with highest affinity to a mu receptor which mediates their analgesic activity. The ³H-D-ala²-D-leu⁵-enkephalin binding remaining after naloxazone treatment, representing a lower affinity site (K_D 4 nM), is quite insensitive to morphiceptin inhibition and has the characteristics of a delta receptor. However, the ³H-dihydromorphine binding present after naloxazone treatment, which also represents a lower affinity site (K_D 6 nM), is far more sensitive to both morphine and morphiceptin and may represent a second morphine-like, or mu, receptor.     

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.     

The effect of modification of the carboxyl group in short D-arginine 2-containing enkephalin analogs on their affinity and selectivity for opiate receptors

Radioreceptor binding assay using a membrane fraction from the rat brain was applied to study [D-Arg2, Leu5] enkephalin and two series of its analogues truncated at the C-terminus with a free or modified carboxyl group: tetra- and tripeptide amides and ethyl esters. The affinity to mu-specific opiate receptor subtype of the N-terminal [D-Arg2] tetrapeptide ethyl ester was 44 times as high as that of the tripeptide with a free carboxyl, and thus the ester retained up to 10% of leucine-enkephalin binding potency. However, a comparable esterification of the carboxyl group in the N-terminal [D-Arg2] tripeptide led to a 6-fold reduction in its affinity to mu-receptors. Consequently, identical modifications of the C-terminal carboxyl group in enkephalin analogues of various length can have completely different effects. Substitution of the natural glycine residue by D-arginine residue in position 2 of the enkephalin molecule truncated at the C-terminus increased the mu-receptor binding potency of the tetrapeptide, whereas its delta receptor binding potency declined by more than one order of magnitude. Simultaneous replacement of glycine2 by D-arginine2 and carboxyl amidation resulted in the short enkephalin analogue Tyr--D--Arg--Gly--Phe--NH2, whose affinity to mu receptors was four times as high as that of leucine--enkephalin, the tetrapeptide being 284 times more selective for the mu vs. delta opiate receptors.     

Characterization of [125I]AR-M100613, a high-affinity radioligand for delta opioid receptors

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.     

The nature of cation-binding groups in binding sites for [[3H]Tyr1, D-Ala2, D-Leu5]enkephalin

The influence of pH on binding of labeled stable analog of enkephalin, [3H]Tyr1, D-Ala2, D-Leu5]enkephalin, to high- and low-affinity receptors of rat brain membranes was studied. It was shown that alkali-earth metal ions combine with a deprotonated group (pKa 7,0) of the high-affinity receptor, thereby activating the latter. The effect of cations on the low-affinity enkephalin binding is independent of pH. The presence of phosphate group in the high-affinity binding site, as well as of imidazole residue in the low-affinity binding site was surmised. The latter supposition was supported by data on chemical modification of the membrane preparation with the aid of diethylpyrocarbonate.     

Patterns of product inhibition for bacterial nitrite reductase

PO +Dehydrophenylalanine (delta Phe) having the E-configuration (delta EPhe ; phenyl and C = O cis) was incorporated into [Leu5]-enkephalin in order to restrict its conformation. Compared with the Z-isomer, in the radio-ligand receptor binding assays, [D-Ala2, delta EPhe4 , Leu5] enkephalin showed drastically decreased potency for the delta and mu opiate receptors, i.e., 260- and 150-fold loss of affinity, respectively. The results strongly indicate that the opiate receptors require the Z-configuration (phenyl and C = O, trans) of the delta Phe4 residue and may require a specific interrelationship between the aromatic rings of the Tyr1 and Phe4 residues in the molecule for binding. The conformation of [Leu5]-enkephalin specific for the delta receptors was analyzed and a comparison made with its crystal structure recently elucidated.     

Multiple opioid receptors: An examination of the dissociation of [3H]leucine enkephalin from rat brain membranes

The experiments reported in this paper address the hypothesis that [3H]leucine enkephalin labels both mu and delta receptors. As reported by other workers, this peptide dissociates from rat brain membranes in a biphasic manner. This is consistent with a two site binding model which hypothesizes that the peptide labels both opioid mu and delta receptors from which it dissociates at different rates. To test this hypothesis, we determined the dissociation of bound ligand from rat brain membranes incubated to equilibrium with [3H]leucine enkephalin in the absence and presence of 100 nM morphine. The data were not significantly different. We conclude that the biphasic off-kinetics of [3H]leucine enkephalin is not evidence for a two-site binding model.