Endogenous heptapeptide Met-enkephalin-Gly-Tyr binds differentially to duplicate delta opioid receptors from zebrafish

Met-enkephalin-Gly-Tyr (MEGY) is an endogenous peptide that binds to opioid sites in zebrafish and in rat brain homogenates. The aim of this work is to characterize the binding profile of this opioid ligand on two duplicate delta receptors from zebrafish, ZFOR1 and ZFOR4. Our results show that, while ZFOR1 presents one single binding site for [³H]-MEGY (K_D = 4.0 ± 0.4 nM), the experimental data from ZFOR4 fit better to the two-site binding model (K_D1 = 0.8 ± 0.2 nM and K_D2 = 30.2 ± 10.2 nM). Two other MEGY synthetic analogues, (D-Ala²)-MEGY and (D-Ala², Val⁵)-MEGY were also prepared and tested, together with the original peptide MEGY and other opioid ligands, in competition binding assays. While these peptides presented K_i values on the nanomolar range when using [³H]-MEGY as radioligand, these parameters were two orders higher in competition binding assays with the antagonist [³H]-diprenorphine. Functional [³⁵S]GTPγS stimulation analysis has revealed that these two receptors can be activated by several opioid agonists. Our results prove that although the MEGY peptide acts as an agonist on ZFOR1 and ZFOR4, there are subtle pharmacological differences between these two delta opioid receptors from zebrafish.     

Opioid receptor three-dimensional structures from distance geometry calculations with hydrogen bonding constraints.

Three-dimensional structures of the transmembrane, seven alpha-helical domains and extracellular loops of delta, mu, and kappa opioid receptors, were calculated using the distance geometry algorithm, with hydrogen bonding constraints based on the previously developed general model of the transmembrane alpha-bundle for rhodopsin-like G-protein coupled receptors (Biophys. J. 1997. 70:1963). Each calculated opioid receptor structure has an extensive network of interhelical hydrogen bonds and a ligand-binding crevice that is partially covered by a beta-hairpin formed by the second extracellular loop. The binding cavities consist of an inner "conserved region" composed of 18 residues that are identical in delta, mu, and kappa opioid receptors, and a peripheral "variable region," composed of 19 residues that are different in delta, mu, and kappa subtypes and are responsible for the subtype specificity of various ligands. Sixteen delta-, mu-, or kappa-selective, conformationally constrained peptide and nonpeptide opioid agonists and antagonists and affinity labels were fit into the binding pockets of the opioid receptors. All ligands considered have a similar spatial arrangement in the receptors, with the tyramine moiety of alkaloids or Tyr1 of opioid peptides interacting with conserved residues in the bottom of the pocket and the tyramine N+ and OH groups forming ionic interactions or H-bonds with a conserved aspartate from helix III and a conserved histidine from helix VI, respectively. The central, conformationally constrained fragments of the opioids (the disulfide-bridged cycles of the peptides and various ring structures in the nonpeptide ligands) are oriented approximately perpendicular to the tyramine and directed toward the extracellular surface. The results obtained are qualitatively consistent with ligand affinities, cross-linking studies, and mutagenesis data.     

Conformation and orientation of regulatory peptides on lipid membranes. Key to the molecular mechanism of receptor selection

The reaction of regulatory peptides with their membrane-bound receptors often occurs via a membrane-associated state of the peptide. From infrared studies on thin lipid films, we have shown that several ligands of the opioid kappa receptor and the neurokinin NK-1 receptor insert their message segments as an alpha-helix, more or less perpendicularly, into the membrane. The binding parameters for these membrane-associated states were determined from the capacitance minimization potential of lipid bilayers. A theory has been developed to account for the observed binding constants and the preferred conformation and orientation of these peptides. In contrast to the kappa and NK-1 receptors, ligands of the opioid mu and delta, and the neurokinin NK-2 and NK-3 receptors, are predicted not to form the inserted alpha-helical structure. A selection between the mu and delta (or NK-2 and NK-3) receptors appears to be made on the basis of an electrostatic gradient near the membrane surface. The molecular mechanism of receptor selection thus appears to be based to a large extent on the membrane-induced compartmentalization of ligands for the different receptors.     

Use of hydrophilic diamines for bridging of two opioid peptide pharmacophores. Synthesis and receptor binding of two new analogues.

The bivalent ligand approach, which assumes that two pharmacophores are connected by a spacer, was used to design receptor type-selective ligands for opioid receptors. The first two opioid peptide bivalent ligands with different spacer lengths containing different numbers of hydroxyl groups, (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-)2 (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-CHOH-)2, were synthesized and their binding to mu, delta, and kappa opioid receptors was characterized. Both analogues were found to possess high opioid in vitro activities. The length of the hydrophilic spacer does not affect the affinity for delta receptors, whereas shorter spacer length increases affinity for mu and even more so for kappa receptors. Thus receptor type-selective peptides for opioid receptors can be designed using the bivalent approach.     

Synthesis of [D-Ala2,4'-125I-Phe3,Glu4]deltorphin and characterization of its delta opioid receptor binding properties.

Both [D-Ala2,Glu4]Deltorphin and [D-Ala2,4'-I-Phe3,Glu4]Deltorphin are highly selective ligands for delta, relative to mu, opioid receptors. Radiolabeled [D-Ala2, 4'-125I-Phe3,Glu4]Deltorphin ([125I]Deltorphin) was prepared with a specific activity of 2200 Ci/mmol from [D-Ala2, 4'-NH2-Phe3, Glu4]Deltorphin through a diazonium salt intermediate. The inhibition of [125I]Deltorphin binding to rat brain membranes by ligands selective for mu, delta, and kappa opioid receptors is consistent with binding by the radioligand to a single site having the properties of a delta opioid receptor. The results of these studies are in good agreement with those obtained by structurally different delta opioid receptor ligands. The similarity between the delta receptor site labeled by [125I]Deltorphin and those labeled by other delta receptor agonists, in contrast to differences seen by in vivo studies of their analgesic effects, is discussed.     

A very high affinity opioid binding site in rat brain: demonstration by computer modeling

We present substantial new evidence for at least four distinct types of opioid receptors in rat brain, using quantitative ligand binding studies and mathematical modeling. Three of these binding sites are consistent with the well established "mu", "delta" and "kappa" receptors. The fourth has two distinctive features: 1) extremely high affinity (dissociation constant less than 1 nM); 2) almost complete lack of specificity for the classical "delta" or "mu" selective ligands. These properties are consistent with the putative "mu1" receptor described by Pasternak and coworkers.     

Molecular determinants of non-specific recognition of delta, mu, and kappa opioid receptors

Identification of the molecular determinants of recognition common to all three opioid receptors embedded in a single three-dimensional (3D) non-specific recognition pharmacophore has been carried out. The working hypothesis that underlies the computational study reported here is that ligands that bind with significant affinity to all three cloned opioid receptors, delta, mu, and kappa, but with different combinations of activation and inhibition properties at these receptors, could be promising behaviorally selective analgesics with diminished side effects. The study presented here represents the first step towards the rational design of such therapeutic agents. The common 3D pharmacophore developed for recognition of delta, mu, and kappa opioid receptors was based on the receptor affinities determined for 23 different opioid ligands that display no specificity for any of the receptor subtypes. The pharmacophore centers identified are a protonated amine, two hydrophobic groups, and the centroid of an aromatic group in a geometric arrangement common to all 23, non-specific, opioid ligands studied. Using this three-dimensional pharmacophore as a query for searching 3D structural databases, novel compounds potentially involved in non-specific recognition of delta, mu, and kappa opioid receptors were retrieved. These compounds can be valuable candidates for novel behaviorally selective analgesics with diminished or no side effects, and thus with potential therapeutic usefulness.     

3D modeling, ligand binding and activation studies of the cloned mouse delta, mu; and kappa opioid receptors

Refined 3D models of the transmembrane domains of the cloned delta, mu and kappa opioid receptors belonging to the superfamily of G-protein coupled receptors (GPCRs) were constructed from a multiple sequence alignment using the alpha carbon template of rhodopsin recently reported. Other key steps in the procedure were relaxation of the 3D helix bundle by unconstrained energy optimization and assessment of the stability of the structure by performing unconstrained molecular dynamics simulations of the energy optimized structure. The results were stable ligand-free models of the TM domains of the three opioid receptors. The ligand-free delta receptor was then used to develop a systematic and reliable procedure to identify and assess putative binding sites that would be suitable for similar investigation of the other two receptors and GPCRs in general. To this end, a non-selective, 'universal' antagonist, naltrexone, and agonist, etorphine, were used as probes. These ligands were first docked in all sites of the model delta opioid receptor which were sterically accessible and to which the protonated amine of the ligands could be anchored to a complementary proton-accepting residue. Using these criteria, nine ligand-receptor complexes with different binding pockets were identified and refined by energy minimization. The properties of all these possible ligand-substrate complexes were then examined for consistency with known experimental results of mutations in both opioid and other GPCRs. Using this procedure, the lowest energy agonist-receptor and antagonist-receptor complexes consistent with these experimental results were identified. These complexes were then used to probe the mechanism of receptor activation by identifying differences in receptor conformation between the agonist and the antagonist complex during unconstrained dynamics simulation. The results lent support to a possible activation mechanism of the mouse delta opioid receptor similar to that recently proposed for several other GPCRs. They also allowed the selection of candidate sites for future mutagenesis experiments.     

Comparison of the amino acid residues in the sixth transmembrane domains accessible in the binding-site crevices of mu, delta, and kappa opioid receptors.

We have mapped the residues in the sixth transmembrane domains (TMs 6) of the mu, delta, and kappa opioid receptors that are accessible in the binding-site crevices by the substituted cysteine accessibility method (SCAM). We previously showed that ligand binding to the C7.38S mutants of the mu and kappa receptors and the wild-type delta receptor was relatively insensitive to methanethiosulfonate ethylammonium (MTSEA), a positively charged sulfhydryl-specific reagent. These MTSEA-insensitive constructs were used as the templates, and 22 consecutive residues in TM6 (excluding C6.47) of each receptor were mutated to cysteine, 1 at a time. Most mutants retained binding affinities for [3H]diprenorphine, a nonselective opioid antagonist, similar to that of the template receptors. Treatment with MTSEA significantly inhibited [3H]diprenorphine binding to 11 of 22 mutants of the rat mu receptor and 9 of 22 mutants of the human delta receptor and 10 of 22 mutants of the human kappa receptor. Naloxone or diprenorphine protected all sensitive mutants, except the A6.42(287)C mu mutant. Thus, V6.40, F6.44, W6.48, I6.51, Y6.54, V6.55, I6.56, I6.57, K6.58, and A6.59 of the mu receptor; F6.44, I6.51, F6.54, V6.55, I6.56, V6.57, W6.58, T6.59, and L6.60 of the delta receptor; and F6.44, W6.48, I6.51, F6.54, I6.55, L6.56, V6.57, E6.58, A6.59, and L6.60 of the kappa receptor are on the water-accessible surface of the binding-site crevices. The accessibility patterns of residues in the TMs 6 of the mu, delta, and kappa opioid receptors are consistent with the notion that the TMs 6 are in alpha-helical conformations with a narrow strip of accessibility on the intracellular side of 6.54 and a wider area of accessibility on the extracellular side of 6.54, likely due to a proline kink at 6.50 that bends the helix in toward the binding pocket and enables considerable motion in this region. The wider exposure of residues 6.55-6.60 to the binding-site crevice, combined with the divergent amino acid sequences, is consistent with the inferred role of residues in this region in determining ligand binding selectivity. The conservation of the accessibility pattern on the cytoplasmic side of 6.54 suggests that this region may be important for receptor activation. This accessibility pattern is similar to that of the D2 dopamine receptor, the only other GPCR in which TM6 has been mapped by SCAM. That opioid receptors and the remotely related D2 dopamine receptor have similar accessibility patterns in TM6 suggest that these segments of GPCRs in the rhodopsin-like subfamily not only share secondary structure but also are packed similarly into the transmembrane bundle and thus have similar tertiary structure.     

High-affinity carbamate analogues of morphinan at opioid receptors

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.     

Endogenous ligands for sigma opioid receptors in the brain ("sigmaphin"): evidence from binding assays

Two endogenous ligands which interact preferentially with the sigma opioid receptors were identified from the guinea-pig brain extract in a Sephadex G-50 fractionation. These two ligands inhibited more potently the binding of [3H]SKF-10047 to sigma opioid receptors than [3H]naloxone to mu opioid receptors, [3H]ethylketocyclazocine to kappa opioid receptors and [3H]DADLE to delta opioid receptors. In the phencyclidine receptor assay, these two ligands were almost inactive. Incubation of these ligands with trypsin destroyed at least 50% of the activities in the sigma opioid receptor assay. Both ligands inhibited the sigma binding in a dose-dependent manner. The inhibition could be eliminated when the two ligands were removed from incubation media by extensive washings. It is therefore concluded that sigma opioid receptors are not phencyclidine receptors and that endogenous ligands for sigma opioid receptors may exist in the brain.     

Complementarity of delta opioid ligand pharmacophore and receptor models

The elaboration of a pharmacophore model for the delta opioid receptor selective ligand JOM-13 (Tyr-c[D-Cys-Phe-D-Pen]OH) and the parallel, independent development of a structural model of the delta receptor are summarized. Although the backbone conformation of JOM-13's tripeptide cycle is well defined, considerable conformational lability is evident in the Tyr(1) residue and in the Phe(3) side chain, key pharmacophore elements of the ligand. Replacement of these flexible features of the ligand by more conformationally restricted analogues and subsequent correlation of receptor binding and conformational properties allowed the number of possible binding conformations of JOM-13 to be reduced to two. Of these, one was chosen as more likely, based on its better superposition with other conformationally constrained delta receptor ligands. Our model of the delta opioid receptor, constructed using a general approach that we have developed for all rhodopsin-like G protein-coupled receptors, contains a large cavity within the transmembrane domain that displays excellent complementarity in both shape and polarity to JOM-13 and other delta ligands. This binding pocket, however, cannot accommodate the conformer of JOM-13 preferred from analysis of ligands, alone. Rather, only the "alternate" allowed conformer, identified from analysis of the ligands but "disfavored" because it does not permit simultaneous superposition of all pharmacophore elements of JOM-13 with other delta ligands, fits the binding site. These results argue against a simple view of a single, common fit to a receptor binding site and suggest, instead, that at least some binding site interactions of different ligands may differ.     

Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin

We reported recently that the ubiquitin-proteasome pathway is involved in agonist-induced down regulation of mu and delta opioid receptors [J. Biol. Chem. 276 (2001) 12345]. While evaluating the effects of various protease inhibitors on agonist-induced opioid receptor down regulation, we observed that while the peptide aldehyde, leupeptin (acetyl-L-Leucyl-L-Leucyl-L-Arginal), did not affect agonist-induced down regulation, leupeptin at submillimolar concentrations directly inhibited radioligand binding to opioid receptors. In this study, the inhibitory activity of leupeptin on radioligand binding was characterized utilizing human embryonic kidney (HEK) 293 cell lines expressing transfected mu, delta, or kappa opioid receptors. The rank order of potency for leupeptin inhibition of [3H]bremazocine binding to opioid receptors was mu > delta > kappa. In contrast to the effect of leupeptin, the peptide aldehyde proteasome inhibitor, MG 132 (carbobenzoxy-L-Leucyl-L-Leucyl-L-Leucinal), had significantly less effect on bremazocine binding to mu, delta, or kappa opioid receptors. We propose that leupeptin inhibits ligand binding by reacting reversibly with essential sulfhydryl groups that are necessary for high-affinity ligand/receptor interactions.     

Sturgeon orphanin, a molecular "fossil" that bridges the gap between the opioids and orphanin FQ/nociceptin

The elucidation of the cDNA sequence for sturgeon proorphanin provides a unique window for interpreting the evolutionary history of the opioid/orphanin gene family. The molecular "fossil" status of this precursor can be seen in several ancestral sequence characteristics that point to its origin as a duplication of either a prodynorphin- or proenkephalin-like gene. The sturgeon proorphanin cDNA encodes a precursor protein of 194 residues, and the orphanin heptadecapeptide itself binds not only the opioid receptor-like 1 (ORL1) receptor but also the classical (mu, kappa, and delta) opioid receptors with near equal affinity. Allowing for this broad receptor specificity are several amino acid substitutions at key positions in the heptadecapeptide sequence, relative to its mammalian orthologs, that have been linked by amino acid scans and site-directed mutagenic studies to the exclusion of mammalian orphanin FQ/nociceptin from classic opioid ligands (i.e. F1Y and L14W). The unique receptor binding profile of sturgeon orphanin not only provides insight into the evolutionary history of the opioid and opioid-related peptides but also provides an ideal context in which to investigate the underlying mechanisms by which novel and often divergent physiological functions arise in receptor-ligand systems.     

(4-Carboxamido)phenylalanine is a surrogate for tyrosine in opioid receptor peptide ligands

(S)-4-(Carboxamido)phenylalanine (Cpa) is examined as a bioisosteric replacement for the terminal tyrosine (Tyr) residue in a variety of known peptide ligands for the mu, delta and kappa opioid receptors. The Cpa-containing peptides, assayed against cloned human opioid receptors, display comparable binding affinity (Ki), and agonist potency (EC50) to the parent ligands at the three receptors. Cpa analogs of delta selective peptides show an increase in delta selectivity relative to the mu receptor. Cpa is the first example of an amino acid that acts as a surrogate for Tyr in opioid peptide ligands, challenging the long-standing belief that a phenolic residue is required for high affinity binding.     

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.     

The occurrence of different kappa opioid receptors (K1 and K2) in frog (Rana esculenta) brain membranes

Opioid receptors of the frog (Rana esculenta) brain are characterized mainly by their relatively high ethylketocyclazocine (EKC) binding properties and by their low affinity to mu and delta ligands when D-Ala2-(Me)Phe4-Gly5-ol enkephalin (DAGO) and D-Ala2-Leu5-enkephalin (DALE) is used. In competition experiments it has been established that EKC and N-cyclopropylmethyl-norazidomorphine (CAM), which are non-selective kappa-ligands, have relatively high affinity to frog brain as well as the kappa 2 (which is DALE sensitive subpopulation of the kappa receptor) ligands etorphine and Metenkephalin-Arg6-Phe7 (1.). The kappa 2 subtype in frog brain resembles more to the mu subtype than to the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and DAGO.     

In-vitro investigation of oxazol and urea analogues of morphinan at opioid receptors

A series of 2-amino-oxazole (7 and 8) analogs and 2-one-oxazole analogs (9 and 10) were synthesized from cyclorphan (1) or butorphan (2) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors and compared with their 2-aminothiozole analogs 5 and 6. Ligands 7-10 showed decreased affinities at kappa and mu receptors. Urea analogs (11-14) were also prepared from 2-aminocyclorphan (3) or 2-aminobutorphan (4) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors. The urea derived opioids retained their affinities at mu receptors while showing increased affinities at delta receptors and decreased affinities at kappa receptors. Functional activities of these compounds were measured in the [35S]GTPgammaS binding assay, illustrating that all of these ligands were kappa agonists. At the mu receptor, compounds 11 and 12 were mu agonist/antagonists.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

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.