Comparative thermodynamics of opioid receptor ligand interaction in the bovine adrenal medulla membranes—Evidence of opioid site heterogeneity

1. A marked dependence on temperature of agonist binding δ, μ and κ₁₋₃, opioid sites in the bovine adrenal medulla was observed, at the range of 0 to 37°C. These changes concern kinetic (k₁) and equilibrium constants (K_d), but not binding capacities (B_max).2. These dependences are different for each ligand and each opioid receptor, suggesting their molecular heterogeneity.3. The comparative thermodynamics indicates that the interaction of opioid agonists with their receptor is exergonic (ΔG° < 0) and entropy driven (ΔS° > 0).4. The comparison of Van't Hoff and Arrhenius plots indicates a discrete mechanism in the binding of each opioid receptor.     

X-ray structures of the δ opioid antagonist TIPP and a protected derivative of the δ opioid antagonist ICI 174,864

Summary The solid state structures of two synthetic opioid peptides have been determined by X-ray single crystal analysis. The first X-ray structure is that of N,N-diallyl-(O-t-butyl)-Tyr-Aib-Aib-Phe-Leu-OMe (RTI02), a protected derivative of the -receptor selective antagonist ICI 174,864 (N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH. ICI 174,864 is one of a series of rationally designed Aib-substituted enkephalin analogs which have shown site-specific antagonist properties. The second compound, the tetrapeptide Tyr-Tic-Phe-Phe-OH (TIPP), is one of a family of linear peptides containing the conformationally restricted Tic residue (tetrahydroisoquinoline-3-carboxylic acid). TIPP exhibits high affinity, selectivity and antagonism for the -receptor. Crystals of both peptides were obtained by slow evaporation and found to be monoclinic in space group P2₁. Unit cell dimensions for RTI02 were: a=13.619(4) , b=12.467(3) , c=13.750(4) , =96.03(4)^o and V=2322(1) ³. The asymmetric unit contained one molecule of RTI02 and one molecule of methanol, giving a calculated density of 1.156 g cm^-3. Unit cell dimensions for TIPP were: a=8.879(5) , b=20.146(8) , c=12.710(6) , =107.89(2)^o and V=2164(2) ³. The asymmetric unit contained one molecule of TIPP and three molecules of acetic acid, giving a calculated density of 1.251 g cm^-3. The RTI02 backbone has a double -bend, stabilized by two intramolecular hydrogen bonds. The TIPP backbone is also folded, but with only a single bend, stabilized by one intramolecular hydrogen bond and several hydrogen bonds to solvent molecules. Both compounds contain aromatic rings in close vicinity (4–6 ).     

Synthesis of pyrrolomorphinan derivatives as κ opioid agonists

We synthesized pyrrolomorphinan derivatives 6, 7, and 9 to examine whether the pyrrole ring would be an accessory site in the κ opioid receptor selective antagonist, nor-binaltorphimine. Derivative 6 had an α,β-unsaturated ketone substituent that strongly bound to the κ receptor. The compound with the highest κ receptor selectivity, 6e, produced a dose-dependent antinociceptive effect in the mouse acetic acid writhing test. However, derivatives 7 and 9, which did not have α,β-unsaturated ketone substituents, showed less κ receptor selectivity than compound 6. Based on structure–activity relationships, we proposed that these compounds adopted active structures for κ selective agonist activity. The pyrrole ring would not function as an accessory site, but the ability of the side chain on the pyrrole ring to localize above the C-ring appeared to confer κ selective agonist activity. These results will promote the design of novel κ agonists.     

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.     

[Dmt1]DALDA analogues with enhanced μ opioid agonist potency and with a mixed μ/κ opioid activity profile

Analogues of [Dmt¹]DALDA (H-Dmt-d-Arg-Phe-Lys-NH₂; Dmt = 2′,6′-dimethyltyrosine), a potent μ opioid agonist peptide with mitochondria-targeted antioxidant activity, were prepared by replacing Phe³ with various 2′,6′-dialkylated Phe analogues, including 2′,6′-dimethylphenylalanine (Dmp), 2′,4′,6′-trimethylphenylalanine (Tmp), 2′-isopropyl-6′-methylphenylalanine (Imp) and 2′-ethyl-6′-methylphenylalanine (Emp), or with the bulky amino acids 3′-(1-naphthyl)alanine (1-Nal), 3′-(2-naphthyl)alanine (2-Nal) or Trp. Several compounds showed significantly increased μ agonist potency, retained μ receptor selectivity and are of interest as drug candidates for neuropathic pain treatment. Surprisingly, the Dmp³-, Imp³-, Emp³- and 1-Nal³-containing analogues showed much increased κ receptor binding affinity and had mixed μ/κ properties. In these cases, molecular dynamics studies indicated conformational preorganization of the unbound peptide ligands due to rotational restriction around the C^βC^γ bond of the Xxx³ residue, in correlation with the observed κ receptor binding enhancement. Compounds with a mixed μ/κ opioid activity profile are known to have therapeutic potential for treatment of cocaine abuse.     

Are mu-opioid receptor polymorphisms important for clinical opioid therapy?

Mutations in the mu-opioid receptor--the primary site of action of opioid analgesics--are candidates for the variability of clinical opioid effects. This has been substantiated by recent advances in genetic research. A common mu-opioid receptor polymorphism was associated with higher demands for alfentanil or morphine for pain relief. It also decreased the potency of morphine for pupil constriction and experimental analgesia, but its molecular mechanisms are unclear. Another opioid receptor mutation greatly impaired receptor signalling in vitro, but is very rare. The accumulated evidence provides a solid basis for continuing research that should address the underlying molecular mechanisms and the role and benefits of OPRM1 genotyping for clinical pain therapy.     

Molecular mechanisms of allosteric probe dependence in μ opioid receptor

Allostery is one of the most important features of proteins. It greatly contributes to the complexity of life, since it enables possibility of precise tuning of protein function, as well as performing more than one function per protein. Probe dependence is one of the unique features of allostery. It allows a protein to respond differently to the same allosteric modulator when different drugs or transmitters are bound. Unfortunately, allosteric mechanisms are difficult to investigate experimentally. Instead, they can be reproduced artificially in simulations. We simulated in silico a native-like cell membrane fragment with an active-state human μ opioid receptor (MOR) in order to investigate diverse effects of a receptor’s positive allosteric modulator on various agonists. Particular emphasis on native-likeness of the environment was put. We managed to reproduce the experimentally observed effects, which allowed us to take deeper insight into their underlying mechanisms. We found an allosteric pathway in the receptor, leading from the ligand binding site to the intracellular, effector site. We observed that the modulator affected the pathway, inducing different resultant responses for full and partial agonists.     

Computer modeling of δ opioid receptor and interaction with ligands

Summary Human σ opioid receptor (σOR), a G-protein-coupled receptor, has been modeled using the helix axes as revealed by the crystallographic structure of bacteriorhodopsin and ligand binding profiles of single-point mutants of σOR. The model revealed feasibility of existence of a second disulfide bridge between the transmembrane helices (TMHs) 6 and 7, Cys²⁷³-Cys³⁰³. A common binding site has been suggested for high-affinity selective agonists DPDPE, DPLPE, DTLET, BW373U86 and antagonist Naltrindole. Docking calculations have shown that the amino group of the ligands forms a hydrogen bond with the imidazole ring of His³⁰¹ (TMH7) rather than with Asp¹²⁸ (TMH3) and is not a cation counterpart of this highly conserved aspartyl residue. All the findings and the model shed light on the putative structure and functioning of opioid receptors and can be used for designing further mutagenesis experiments.     

Four-component pharmacophore model for endomorphins toward μ opioid receptor subtypes

In the present work, a series of simulation tools were used to determine structure-activity relationships for the endomorphins (EMs) and derive μ-pharmacophore models for these peptides. Potential lowest energy conformations were determined in vacuo by systematically varying the torsional angles of the Tyr¹-Pro² (ω₁) and Pro²-Trp³/Phe³ (ω₂) as tuning parameters in AM1 calculations. These initial models were then exposed to aqueous conditions via molecular dynamics simulations. In aqueous solution, the simulations suggest that endomorphin conformers strongly favor the trans/trans pair of the ω₁/ω₂ amide bonds. From two-dimensional probability distributions of the ring-to-ring distances with respect to the pharmacophoric angles for EMs, a selectivity range of μ₁ is ca. 8.3 ~ 10.5 Å for endomorphin-2 and selectivity range of μ₂ is ca. 10.5 ~ 13.0 Å for endomorphin-1 were determined. Four-component μ-pharmacophore models are proposed for EMs and are compared to the previously published δ- and κ-pharmacophore models.

Angle NAB/C vs distance

     

Synthesis of quinolinomorphinan-4-ol derivatives as δ opioid receptor agonists

The previously reported morphinan derivative SN-28 showed high selectivity and agonist activity for the δ opioid receptor. In the course of examining the structure-activity relationship of SN-28 derivatives, the derivatives with the 4-hydroxy group (SN-24, 26, 27) showed higher selectivities for the δ receptor over the μ receptor than the corresponding SN-28 derivatives with the 3-hydroxy group (SN-11, 23, 28). Derivatives with the 4-hydroxy group showed potent agonist activities for the δ receptor in the [(35)S]GTPγS binding assay. Although the 17-cyclopropylmethyl derivative (SN-11) with a 3-hydroxy group showed the lowest selectivity for the δ receptor among the morphinan derivatives, the agonist activity toward the δ receptor was the most potent for candidates with the 3-hydroxy group.     

Synthesis of quinolinomorphinan derivatives as highly selective δ opioid receptor ligands

We have reported previously the novel δ opioid agonist KNT-127 which showed high affinity and selectivity for the δ receptor. Moreover, the analgesic effect of subcutaneously administered KNT-127 was more potent than that of a prototypical δ agonist (?)-TAN-67 in the acetic acid writhing test. This study of the structure–activity relationship of KNT-127 derivatives focused on the introduction of substituents onto the 5′-, 6′-, 7′- or 8′-position of the quinoline ring and revealed that many derivatives with 5′- or 8′-substituents showed high affinities and selectivities for the δ receptor. Especially, SYK-153 with an 8′-OH group showed the highest affinity and the most balanced and highest selectivity for the δ receptor among the synthesized compounds.     

Antisense oligodeoxynucleotide to δ opioid receptors attenuates morphine dependence in mice

The effect of intracerebroventricular (i.c.v.) treatment with antisense oligodeoxynucleotide (A-oligo) to δ opioid receptor mRNA on the morphine-induced place preference and naloxone-precipitated jumping was examined in morphine-dependent mice. Morphine (5 mg/kg, s.c.) produced a significant place preference. I.c.v. pretreatment with A-oligo (0.01–1 μg/mouse) dose-dependently attenuated this morphine (5 mg/kg, s.c.)-induced place preference, while mismatched oligodeoxynucleotide (M-oligo; 1 μg/mouse, i.c.v.) was ineffective. Naloxone (3 mg/kg, s.c.) precipitated jumping in morphine-dependent mice. I.c.v. pretreatment with A-oligo (1 μg/mouse) attenuated this naloxone (3 mg/kg, s.c.)-precipitated jumping in morphine-dependent mice, while M-oligo (1 μg/mouse, i.c.v.) was ineffective. These data demonstrate that the selective reduction in supraspinal δ opioid receptor function caused by pretreatment with A-oligo attenuated the morphine-induced place preference and naloxone-precipitated jumping in morphine-dependent mice, suggesting that the rewarding effect of and physical dependence on morphine may be modulated by central δ opioid receptors.     

Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands

In this letter, we describe a series of 4-substituted piperidine and piperazine compounds based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR). We have shown that by changing the length and flexibility profile of the side chain in this position, binding affinity is improved at both receptors by a significant degree. Furthermore, several of the compounds described herein display good efficacy at MOR, while simultaneously displaying DOR antagonism. The MOR agonist/DOR antagonist has shown promise in the reduction of negative side effects displayed by selective MOR agonists, namely the development of dependence and tolerance.     

Differential maturation of μ and δ opioid receptors in the chick embryonic brain

The developmental profiles of the binding of and opiate receptors agonists was investigated using the chick embryo brain. Binding of opioids was performed at embryonic days 5, 6, 15, 18, and 20 in the developing chick embryo brain. [³H]dihyromorphine was used as a ligand and with 5×10^–7 M levorphanol for non-specific binding, and [³H](d-Ala²-d-Leu⁵)-enkephalin was used as a with 5×10^–7 M (d-Ser-Gly-Phe-Leu-Thr)-enkephalin for non-specific binding. Crude membranes were prepared from whole brain at days, 5, 6 and cerebral hemispheres at days 15, 18, and 20 of embryonic age. Both and opiate receptors were present during early embryogenesis and as early as day 5. Analysis of binding sites revealed high and low affinity sites during early embryogenesis but only one site. By 18 days of embryonic age, only one site remained. This developmental change is interpreted as a transitory state of the receptor to the adult pattern. The presence of only one site is constant throughout embryonic age; it is high during early embryogenesis reaching a lower level by 18 days. The presence of a dual binding site pattern for the receptor in early embryogenesis is implicated to have a functional significance in the pluripotential role of the endogenous opioids in early development.     

Novel TIPP (H-Tyr-Tic-Phe-Phe-OH) analogues displaying a wide range of efficacies at the δ opioid receptor. Discovery of two highly potent and selective δ opioid agonists

Analogues of the δ opioid antagonist peptide TIPP (H-Tyr-Tic-Phe-Phe-OH; Tic=1,2,3,4-tetrahydroisoquinoline3-carboxylic acid) containing various 4'-[N-(alkyl or aralkyl)carboxamido]phenylalanine analogues in place of Tyr(1) were synthesized. The compounds showed subnanomolar or low nanomolar δ opioid receptor binding affinity and various efficacy at the δ receptor (antagonism, partial agonism, full agonism) in the [(35)S]GTPγS binding assay. Two analogues, [1-Ncp(1)]TIPP (1-Ncp=4'-[N-(2-(naphthalene-1-yl)ethyl)carboxamido]phenylalanine) and [2-Ncp(1)]TIPP (2-Ncp=4'-[N-(2-(naphthalene-2-yl)ethyl)carboxamido]phenylalanine), were identified as potent and selective δ opioid agonists.     

Prerequisite for His4 in deltorphin A for highδ opioid receptor selectivity

Summary Analysis of deltorphin A position 4 analogues included: backbone constrained N MeHis, spinacine (Spi), N MePhe and the tetrahydroisoquinoline-3-carboxylic acid (Tic); spatially confined side-chain (Phg); and imidazole alkylation ofl- andd-His⁴ enantiomers. High selectivity was lost with the following replacements: N MeHis⁴, N MePhe⁴ and Phg⁴ reduced binding and the constrained residues also increasedµ binding; ring closure between the side-chain and amino group to yield Spi⁴ or Tic⁴ increasedµ affinity. Imidazole methylation of His⁴ marginally affected opioid binding and doubled selectivity; alkylatedd-His⁴-derivatives generally maintained selectivity in spite of decreased affinities. Thus, His⁴ imidazole preserves selectivity by facilitating high binding and by repulsion at theµ receptor. Several low energy conformers of deltorphin A indicated that the His⁴ imidazole preferred a spatial orientation parallel to the phenolic side-chain of Tyr¹ suggestive that this conformation might contribute to high affinity and selectivity.     

Highly selective and potent μ opioid ligands by unexpected substituent on morphine skeleton

Unexpected substituent on the well-known morphine skeleton is described to be account for highly selective and potent μ opioid ligands, which is strongly connected to substituted aromatic groups on this omitted 8α-position.