Development and validation of opioid ligand-receptor interaction models: the structural basis of mu vs delta selectivity.

Opioid receptor binding conformations for two structurally related, conformationally constrained tetrapeptides, JOM-6 ( micro receptor selective) and JOM-13 (delta receptor selective), were deduced using conformational analysis of these ligands and analogs with additional conformational restrictions. Docking of these ligands in their binding conformations to opioid receptor structural models, based upon the published rhodopsin X-ray structure, implicates specific structural features of the micro and delta receptor ligand binding sites as forming the basis for the micro selectivity of JOM-6 and the delta selectivity of JOM-13. In particular, the presence of E229 in the micro receptor (in place of the corresponding D210 of the delta receptor) causes an adverse electrostatic interaction with C-terminal carboxylate-containing ligands, resulting in the observed preference of ligands with an uncharged C-terminus for the micro receptor. In addition, the requirement that the Phe3 side chain of JOM-13 assume a gauche orientation for optimal delta binding, whereas the Phe3 side chain of JOM-6 must be in a trans orientation for high-affinity micro binding can be largely attributed to the steric effect of replacement of L300 of the delta receptor by W318 of the micro receptor. Testing this hypothesis by examining the binding of JOM-6 and several of its key analogs with specific micro receptor mutants is described. Our initial results are consistent with the proposed ligand-receptor interaction models.     

Development of a model for the δ-opioid receptor pharmacophore. 4. Residue 3 dehydrophenylalanine analogues of Tyr-c[D-Cys-Phe-D-Pen]OH (JOM-13) confirm required gauche orientation of aromatic side chain

We have previously proposed a model for the δ-opioid receptor binding conformation of the high affinity tetrapeptide Tyr-c[D -Cys-Phe-D -Pen] OH (JOM-13) based on experimental and theoretical conformational analysis of this peptide and a correlation of conformational preferences of further conformationally restricted analogues of this tetrapeptide with their receptor binding affinities. A key element of this model is the requirement that the Phe³ side chain exist in the x¹ = −60° conformation. Conformational calculations on the residue 3 dehydrophenylalanine analogues of JOM-13 suggest that while the dehydro(Z) phenylalanine analogue can be superimposed easily with the proposed binding conformer of JOM-13, the dehydro(E)phenylalanine analogue cannot. These results lead to the prediction that the dehydro(Z)-phenylalanine analogue should display similar δ-receptor binding affinity as JOM-13 while the dehydro(E)phenylalanine analogue is expected to bind less avidly. Synthesis and subsequent opioid receptor binding analysis of the dehydrophenylalanine analogues of JOM-13 confirm these predictions, lending support to the δ-pharmacophore model. © 1996 John Wiley & Sons, Inc.     

Development of a model for the δ-opioid receptor pharmacophore: 3. Comparison of the cyclic tetrapeptide Tyr-c[D-Cys-Phe-D-Pen] OH with other conformationally constrained δ-receptor selective ligands

We have previously proposed a model of the δ-opioid receptor bound conformation for the cyclic tetrapeptide, Tyr-c[D -Cys-Phe-D -Pen]OH (JOM-13) based on its conformational analysis and from conformation-affinity relationships observed for its analogues with modified first and third residues. To further verify the model, it is compared here with results of conformational and structure-activity studies for other known conformationally constrained δ-selective ligands: the cyclic pentapeptide agonist, Tyr-c[D -Pen-Gly-Phe-D -Phe]OH (DPDPE); the peptide antagonist, Tyr-Tic-Phe-PheOH (TIPP); the alkaloid agonist, 7-spiroindanyloxymorphone (SIOM); and the related alkaloid antagonist, oxymorphindole (OMI). A candidate δ-bound conformer is identified for DPDPE that provides spatial overlap of the functionally important N-terminal N⁺₃ and C-terminal COO⁻ groups and the aromatic rings of the Tyr and Phe residues in both cyclic peptides. It is shown that all δ-selective ligands considered have similar arrangements of their pharmacophoric elements, i.e., the tyramine moiety and a second aromatic ring (i.e., the rings of Phe³, Phe⁴, and Tic² residues in JOM-13, DPDPE, and TIPP, respectively; the indole ring system in OMI, and the indanyl ring system in SIOM). The second aromatic rings, while occupying similar regions of space throughout the analogues considered, have different orientations in agonists and antagonists, but identical orientations in peptide and alkaloid ligands with the same agonistic or antagonistic properties. These results agree with the previously proposed binding model for JOM-13, are consistent with the view that δ-opioid agonists and antagonists share the same binding site, and support the hypothesis of a similar mode of binding for opioid peptides and alkaloids. © 1996 John Wiley & Sons, Inc.     

Modification of the Phe3 aromatic moiety in delta receptor-selective dermorphin/deltorphin-related tetrapeptides. Effects on opioid receptor binding.

The previously described cyclic delta opioid receptor-selective tetrapeptide H-Tyr-D-Cys-Phe-D-Pen-OH (JOM-13) was modified at residue 3 by incorporation of both natural and unnatural amino acids with varying steric, electronic, and lipophilic properties. Effects on mu and delta opioid receptor binding affinities were evaluated by testing the compounds for displacement of radiolabeled receptor-selective ligands in a guinea pig brain receptor binding assay. Results obtained with the bulky aromatic 1-Nal3 and 2-Nal3 substitutions suggest that the shape of the receptor subsite with which the side chain of the internal aromatic residue interacts differs for delta and mu receptors. This subsite of either receptor can accommodate the transverse steric bulk of the 1-Nal3 side chain but only the delta receptor can readily accept the more elongated 2-Nal3 side chain. Several analogs with pi-excessive heteroaromatic side chains in residue 3 were examined. In general, these analogs display diminished binding to mu and delta receptors, consistent with previous findings for analogs with residue 3 substitutions of modified electronic character. Several analogs with alkyl side chains in residue 3 were also examined. While delta receptor binding affinity is severely diminished with Val3, Ile3, and Leu3 substitutions, Cha3 substitution is very well tolerated, indicating that, contrary to the widely held belief, an aromatic side chain in this portion of the ligand is not required for delta receptor binding. Where possible, comparison of results in this delta-selective tetrapeptide series with those reported for analogous modification in the cyclic delta-selective pentapeptide [D-Pen2, D-Pen5]enkephalin (DPDPE) and linear pentapeptide enkephalins reveals similar trends.     

Application of the message-address concept to the docking of naltrexone and selective naltrexone-derived opioid antagonists into opioid receptor models

A binding site model for the opioid family of G-protein coupled receptors (GPCRs) is proposed based on the message-address concept of ligand recognition. Using ligand docking studies of the universal opioid antagonist, naltrexone, the structural basis for ‘message’ recognition is explored across all three receptor types, μ, δ, and κ. The binding mode proposed and basis for selectivity are also rationalized using the naltrexone-derived ligands, naltrindole (NTI) and norbinaltorphimine (nor BNI). These ligands are docked to the receptor according to the common naltrexone core or message. The resulting orientation places key ‘address’ elements in close proximity to amino acid residues critical to selectivity among receptor types. Selectivity is explained by sequence differences in the μ, δ, and κ receptors at these recognition points. Support for the model is derived from site directed mutagenesis studies and ligand binding data for the opioid receptors and other related GPCRs. Special issue dedicated to Dr. Eric J. Simon     

Opioid and melanocortin receptors: do they have overlapping pharmacophores?

We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the delta opioid receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the delta opioid receptor (Ki = 0.38 nM in binding assay, EC(50) = 0.48 nM in GTP-gamma-S binding assay, IC(50) = 74 nM in MVD) as an agonist instead of an antagonist and showed selective binding affinity (IC(50) = 2.3 muM) at the MC-3 receptor rather than at the MC-5 receptor. A study of the structure-activity relationships demonstrated that the residues in positions 2, 3, and the C-terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opioid receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors.     

Conformation-activity relationships of opioid peptides with selective activities at opioid receptors

The discovery of endogenous opioid peptides 25 years ago opened up a new chapter in efforts to understand the origins and control of pain, its relationships to other biological functions, including inflammatory and other immune responses, and the relationships of opioid peptides and their receptors to a variety of undesirable or toxic side effects often associated with the nonpeptide opiates such as morphine including addiction, constipation, a variety of neural toxicities, tolerance, and respiratory depression. For these investigations the need for potent and highly receptor selective agonists and antagonists has been crucial since they in principle allow one to distinguish unequivocally the roles of the different opioid receptors (mu, delta, and kappa) in the various biological and pathological roles of the opioid peptides and their receptors. Conformational and topographical constraint of the linear natural endogenous opioid peptides has played a major role in developing peptide ligands with high selectivity for mu, delta, and kappa receptors, and in understanding the conformational, topographical, and stereoelectronic structural requirements of the opioid peptides for their interactions with opioid receptors. In turn, this had led to insights into the three-dimensional pharmacophore for opioid receptors. In this article we review and discuss some of the developments that have led to potent, selective, and stable peptide and peptidomimetic ligands that are highly potent and selective, and that have delta agonist, mu antagonist, and kappa agonist biological activities (other authors in this issue will discuss the development of other types of activities and selectivities). These have led to ligands that provide unique insight into opioid pharmacophores and the critical roles opioid ligands and receptor scan play in pain, addiction, and other human maladies.     

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.     

pKa and volume of residue one influence delta/mu opioid binding: QSAR analysis of tyrosine replacement in a nonselective deltorphin analogue

[Gly(4)]deltorphin (Tyr-D-Ala-Phe-Gly-Val-Val-Gly-NH(2)) is a nonselective analogue of the opioid heptapeptides isolated from Phyllomedusa amphibian skin. Its nonselective nature allows for simultaneous characterization of the effects of sequence modification on both delta (delta) and mu (mu) receptor binding. The N-terminal regions of opioid peptides are considered to be responsible for receptor recognition, and the tyrosine at position one is relatively intolerant to alteration. In order to further investigate the role of the phenolic hydroxyl group in receptor interaction, a series of peptides was synthesized in which the position-one tyrosine residue was replaced with analogues of varying electronic, steric, and acid/base character, including ring-substituted tyrosines, para-substituted phenylalanines, and other nonaromatic and heterocyclic amino acids. The effects of these replacements on delta and mu receptor affinities were measured and then analyzed through quantitative structure-activity relationship (QSAR) calculations. Results support a dual hydrogen bond donor/acceptor role for the Tyr(1) hydroxyl moiety, with less acidic hydroxyl groups exhibiting stronger binding to opioid receptors. In addition, steric bulk in the Tyr(1) position independently strengthens mu and possibly delta binding, presumably by either a ligand conformational effect or enhanced van der Waals interactions with a 'loose' receptor site. The pK(a) effect is stronger on delta than on mu binding, generating an increase in delta selectivity with increasing residue-one pK(a).     

Molecular modeling and QSAR analysis of the interaction of flavone derivatives with the benzodiazepine binding site of the GABA(A) receptor complex

A large number of structurally different classes of ligands, many of them sharing the main characteristics of the benzodiazepine (BDZ) nucleus, are active in the modulation of anxiety, sedation, convulsion, myorelaxation, hypnotic and amnesic states in mammals. These compounds have high affinity for the benzodiazepine binding site (BDZ-bs) of the GABA(A) receptor complex. Since 1989 onwards our laboratories established that some natural flavonoids were ligands for the BDZ-bs which exhibit medium to high affinity in vitro and anxiolytic activity in vivo. Further research resulted in the production of synthetic flavonoid derivatives with increased biochemical and pharmacological activities. The currently accepted receptor/pharmacophore model of the BDZ-bs (Zhang, W.; Koeler, K. F.; Zhang, P.; Cook, J. M. Drug Des. Dev. 1995, 12, 193) accounts for the general requirements that should be met by this receptor for ligand recognition. In this paper we present a model pharmacophore which defines the characteristics for a ligand to be able to interact and bind to a flavone site, in the GABA(A) receptor. closely related to the BDZ-bs. A model of a flavone binding site has already been described (Dekermendjian, K.; Kahnberg, P.; Witt, M. R.; Sterner, O.; Nielsen, M.; Liljerfors, T. J. Med. Chem. 1999, 42, 4343). However, this alternative model is based only on graphic superposition techniques using as template a non-BDZ agonist. In this investigation all the natural and synthetic flavonoids found to be ligands for the BDZ-bs have been compared with the classical BDZ diazepam. A QSAR regression analysis of the parameters that describe the interaction demonstrates the relevance of the electronic effects for the ligand binding, and shows that they are associated with the negatively charged oxygen atom of the carbonyl group of the flavonoids and with the nature of the substituent in position 3'.     

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.     

Ligand recognition in μ opioid receptor: experimentally based modeling of μ opioid receptor binding sites and their testing by ligand docking

For three-dimensional understanding of the mechanisms that control potency and selectivity of the ligand binding at the atomic level, we have analysed opioid receptor-ligand interaction based on the receptor's 3D model. As a first step, we have constructed molecular models for the multiple opioid receptor subtypes using bacteriorhodopsin as a template. The S-activated dihydromorphine derivatives should serve as powerful tools in mapping the three-dimensional structure of the μ opioid receptor, including the nature of the agonist-mediated conformational change that permits G protein-coupling to ‘second messenger’ effector molecules, and in identifying specific ligand-binding contacts with the μ opioid receptor. The analyses of the interactions of some opioid ligands with the predicted ligand binding sites are consistent with the results of the affinity labeling experiments.     

The LMC delta opioid recognition pharmacophore: comparison of SNC80 and oxymorphindole

A recognition pharmacophore for the delta opioid receptor was developed de novo. Through the use of the pharmacophore and a novel four-point recognition model, major differences were observed between oxymorphindole and SNC80. This work suggests that these two classes of delta selective opioids do not bind to the delta opioid receptor in the same orientation.     

Exploring the unique pharmacology of a novel opioid receptor, ZFOR1, using molecular modeling and the 'message-address' concept.

Previous studies have probed the structural basis of ligand selectivity in the mu, delta and kappa opioid receptors through the application of molecular modeling techniques in concert with the 'message-address' concept. Here, this approach was used in an attempt to rationalize the unique pharmacological profile of a recently cloned novel opioid receptor, ZFOR1 (ZebraFish Opioid Receptor 1). Specifically, a model of the transmembrane domains of ZFOR1 was constructed and used to explore the binding modes of various prototypical opioid ligands. The results show that the 'message' portion of the binding pocket of ZFOR1 is highly conserved; hence, the binding modes of non-selective opioid ligands are well preserved. In contrast, a small number of variant residues at the extracellular end of the binding pocket, particularly Lys288 (VI:26) and Trp304 (VII:03), are shown to create adverse steric interactions with all delta and kappa selective ligands examined, thereby disrupting their binding modes. These results are consistent with, and serve as an explanation for, the observed pharmacology of this receptor, lending support to both the validity of the 'message-address' concept itself and to the use of molecular modeling approaches in its application.     

Ligands act as pharmacological chaperones and increase the efficiency of delta opioid receptor maturation

The endoplasmic reticulum (ER) is recognized as an important site for regulating cell surface expression of membrane proteins. We recently reported that only a fraction of newly synthesized delta opioid receptors could leave the ER and reach the cell surface, the rest being degraded by proteasomes. Here, we demonstrate that membrane-permeable opioid ligands facilitate maturation and ER export of the receptor, thus acting as pharmacological chaperones. We propose that these ligands stabilize the newly synthesized receptor in the native or intermediate state of its folding pathway, possibly by inducing stabilizing conformational constrains within the hydrophobic core of the protein. The receptor precursors that are retained in the ER thus represent fully competent folding intermediates that can be targets for pharmacological intervention aimed at regulating receptor expression and cellular responsiveness. The pharmacological chaperone action is independent of the intrinsic signaling efficacy of the ligand, since both agonists and antagonists were found to promote receptor maturation. This novel property of G protein-coupled receptor ligands may have important implications when considering their effects on cellular responsiveness during therapeutic treatments.     

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.     

Modifications of the cyclic mu receptor selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et): effects on opioid receptor binding and activation.

The previously described cyclic mu opioid receptor-selective tetrapeptide Tyr-c[D-Cys-Phe-D-Pen]NH2 (Et) (JOM-6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [35S]-GTPgammaS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr1 with conformationally restricted phenolic amino acids. In the [35S]-GTPgammaS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range 0.4-9nM. However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC50 value of less than 100nM. Lastly, we have identified a peptide, D-Hat-c[D-Cys-Phe-D-Pen]NH2 (Et), with high potency and > 1,000-fold functional selectivity for the mu over delta opioid receptor as measured by the [35S]-GTPgammaS assay.     

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.     

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.