Differential binding properties of oripavines at cloned μ- and δ-opioid receptors

doi:10.1016/S0014-2999(99)00460-4

European Journal of Pharmacology

Volume 378, Issue 3, 13 August 1999, Pages 323-330

https://doi.org/10.1016/S0014-2999(99)00460-4 Get rights and content

Abstract

This study examines the possibility that oripavine opioid receptor agonists bind equally to both high and low affinity states of the μ-opioid receptor. Studies were performed in C6 cells expressing μ- or δ-opioid receptors; high and low agonist affinity states of the receptors were defined by the absence and presence, respectively of Na⁺ ions and the GTP analog Gpp(NH)p. At the μ-opioid receptor dihydroetorphine and etorphine were full agonists, buprenorphine had moderate efficacy while diprenorphine was an antagonist. At the δ-opioid receptor, dihydroetorphine, etorphine, and diprenorphine had moderate efficacy while buprenorphine was an antagonist. The binding affinities of the oripavines at the μ-opioid receptor decreased only one to 2-fold in the presence of NaCl and Gpp(NH)p. In contrast, decreases in oripavine affinity at the δ-opioid receptor correlated with δ-opioid receptor efficacy. The ability of oripavine agonists to bind with high affinity to the low agonist affinity state of the ν-opioid receptor may explain the high potencies of these compounds in vivo.

Introduction

Opioid receptors are members of the seven transmembrane domain receptor superfamily that are coupled to pertussis toxin sensitive G-proteins (e.g., Dhawan et al., 1996). Evidence from numerous studies shows that the presence of sodium ions and guanine nucleotides reduces agonist binding to opioid receptors while the binding of antagonists is unaffected (Childers and Snyder, 1980; Puttfarcken et al., 1986; Werling et al., 1986; Emmerson et al., 1996). The shift in affinity caused by sodium ions and guanine nucleotides correlates with agonist efficacy (e.g., Childers and Snyder, 1980). These findings support a conformational selection model of agonist action whereby agonist binding to the high affinity state of the receptor shifts the equilibrium in favor of this state, and away from the low affinity form, thereby invoking a response (e.g., Kenakin, 1997).

Buprenorphine, an oripavine derived from thebaine, is a moderate efficacy, μ-opioid agonist with analgesic effects that are 25-times more potent than morphine (Cowan et al., 1977a, Cowan et al., 1977b). From a receptor interaction viewpoint, buprenorphine is of interest because, unlike other opioid agonists, its binding affinity is not affected by the presence of sodium ions (Villiger, 1984; Rothman et al., 1995). However, these conclusions were made from studies performed using rat brain membranes that contain a heterogeneous population of opioid receptors. Consequently it is unclear whether the binding properties of buprenorphine are indicative of its binding only to the μ-opioid receptor. For example, buprenorphine acts as an antagonist at the κ-opioid receptor (Leander, 1987; Negus and Dykstra, 1988; Toll et al., 1998) and has no agonist action at the δ-opioid receptor (Toll et al., 1998). Therefore, antagonist binding of buprenorphine to non-μ-opioid receptors could explain the lack of a Na⁺ and guanine nucleotide induced shift.

In order to elucidate the binding properties of buprenorphine to specific opioid receptors, cloned μ- and δ-opioid receptors expressed in C6 glioma cells (C6 mu) which do not endogenously express any opioid receptors (Klee and Nirenberg, 1974) were used. During the course of these experiments, it was established that other oripavines with opioid agonist properties also bind to the μ-opioid receptor in a Na⁺-insensitive fashion. The oripavines examined in addition to buprenorphine were the agonists, dihydroetorphine (Kamei et al., 1995) and etorphine (Blane et al., 1967; Walker et al., 1998), and the antagonist, diprenorphine (Dewey and Harris, 1971; Traynor et al., 1987). The degree of relative agonist efficacy of the compounds was compared in the same cell systems using the [ $^{35} S$ ]GTPγS binding assay. (Traynor and Nahorski, 1995; Emmerson et al., 1996; Clark et al., 1997; Alt et al., 1998).

The findings of this study show that opioid receptor agonist oripavines bind to the cloned μ-opioid receptor in a fashion that is insensitive to the presence of the non-hydrolysable GTP analog, Gpp(NH)p, while sodium ions produce a slight shift in dihydroetorphine and etorphine binding affinities. This insensitivity may be specific to the μ-opioid receptor since at the cloned δ-opioid receptor expressed in C6 cells (C6 delta) oripavine binding is sensitive to Na⁺ and guanine nucleotides.

Section snippets

Chemicals

[ $^{3} H$ ]Diprenorphine (45 Ci/mmol and 58 Ci/mmol), [ $^{3} H$ ]naloxone (54.6 Ci/mmol), [ $^{3} H$ ]naltrindole (33 Ci/mmol) and [ $^{35} S$ ]GTPγS, (guanosine-5′-O-(3-thio)triphosphate, 1250 Ci/mmol) were purchased from DuPont NEN (Boston, MA). DAMGO ([Tyr-d-Ala², N-Me-Phe⁴, Gly-ol⁵]enkephalin), pertussis toxin, Gpp(NH)p (5′-guanylylimido-diphosphate) and GDP (guanosine diphosphate) were purchased from Sigma (St. Louis, MO). DPDPE ([d-Pen²-d-Pen⁵]enkephalin) and SNC-80 ((+)-4-((α-R)-α-[(2S,5R

[ $^{3} H$ ]Diprenorphine binding

The C6 mu cells used in this study expressed μ receptors at a level of 435±75 fmols mg⁻¹ protein as determined with [ $^{3} H$ ]diprenorphine, with a K_d for [ $^{3} H$ ]diprenorphine of 0.13±0.01 nM. The number of receptors in C6 delta cells defined with [ $^{3} H$ ]naltrindole was 955±54 fmols mg⁻¹ protein, with a K_d for [ $^{3} H$ ]naltrindole of 0.01±0.003 nM. Wild-type C6 cells demonstrated no specific binding of [ $^{3} H$ ]diprenorphine at radioligand concentrations of 0.001 to 5 nM, at a protein level which afforded 80%

Discussion

The oripavines dihydroetorphine and etorphine stimulated [ $^{35} S$ ]GTPγS binding to membranes from C6 mu cells to a higher degree than the highly efficacious agonists DAMGO, fentanyl and morphine. The relative efficacy of moderately efficacious agonists at this receptor was in the order buprenorphine>pentazocine>butorphanol>nalbuphine, while diprenorphine and naloxone were antagonists in these cells confirming and extending previous findings (Emmerson et al., 1996).

Previous studies have shown that

Acknowledgements

This work was supported by National Institute of Drug Abuse Grants DA00254 and DA 02265 and NIGMS (GM07767).

References (42)

L Birnbaumer et al.
Receptor-effector coupling by G proteins
Biochim. Biophys. Acta
(1990)
Y Ishizuka et al.
Regulation of opioid antagonist and mu, kappa or delta agonist binding by guanine nucleotide and sodium
Jpn. J. Pharmacol.
(1984)
Ph Jauzac et al.
Physical separation of the agonist and antagonist forms of a mu opiate receptor?
Life Sciences
(1983)
J Kamei et al.
Antinociceptive effect of dihydroetorphine in diabetic mice
Eur. J. Pharmacol.
(1995)
H Kong et al.
A single residue, aspartic acid 95, in the δ opioid receptor specifies selective high affinity agonist binding
J. Biol. Chem.
(1993)
H Kurose et al.
Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via α-adrenergic, cholinergic, and opiate receptors in neuroblastoma×glioma hybrid cells
J. Biol. Chem.
(1983)
H.W Kosterlitz et al.
Rate of onset and offset of action of narcotic analgesics in isolated preparations
Eur. J. Pharmacol.
(1975)
M Kurowski et al.
[ $^{3} H$ ]etorphine and [ $^{3} H$ ]diprenorphine receptor binding in vitro and in vivo: differential effect of Na⁺ and guanylyl imidodiphosphate
Brain Res.
(1982)
J.D Leander
Buprenorphine has potent kappa opioid receptor antagonist activity
Neuropharmacology
(1987)
O.H Lowry et al.
Protein measurement with the folin phenol reagent
J. Biol. Chem.
(1951)

F Medzihradsky et al.

Lipophilicity of opioids determined by a novel micromethod

J. Pharmacol. Toxicol.

(1992)

S.S Negus et al.

Kappa antagonist properties of buprenorphine in the shock titration procedure

Eur. J. Pharmacol.

(1988)

M.L Richards et al.

In vivo receptor binding of oripavines to μ, δ, and κ-sites in rat brain as determined by an ex-vivo radiolabeling method

Eur. J. Pharmacol.

(1985)

J.R Traynor et al.

Diprenorphine has agonist activity at opioid kappa-receptors in the myenteric plexus of the guinea pig ileum

Eur. J. Pharmacol.

(1987)

J.W Villiger

Binding of buprenorphine to opiate receptors

Neuropharmacology

(1984)

M Wuster et al.

Sodium regulation of opioid agonist binding is potentiated by pertussis toxin

Biochem. Biophys. Res. Commun.

(1984)

A Alt et al.

Stimulation of guanosine-5′-0-(3-[ $^{35} S$ ]thio) triphosphate binding by endogenous opioids acting at a cloned mu receptor

J. Pharmacol. Exp. Ther.

(1998)

G.F Blane et al.

Actions of etorphine hydrochloride, (M99): a potent morphine-like agent

Br. J. Pharmacol.

(1967)

G Bot et al.

Mutagenesis of a single amino acid in the rat μ-opioid receptor discriminates ligand binding

J. Neurochem.

(1998)

J.A Carroll et al.

The physiological relevance of low agonist affinity binding at opioid μ-receptors

Br. J. Pharmacol.

(1995)

Y.C Cheng et al.

Relationship between the inhibition constant (K_i) and the concentration of inhibitor which causes 50 percent inhibition (I₅₀) of an enzymatic reaction

Biochem. Pharmacol.

(1973)

Cited by (102)

Thienorphine induces antinociception without dependence through activation of κ- and δ-, and partial activation of μ- opioid receptor
2020, Brain Research
Citation Excerpt :
In contrast to the G protein-mediated pathway, buprenorphine increased pERK1/2 through activation of the β-arrestin-mediated pathway in CHO– κ-PKAcatEGFP cells [Fig. 6B]. At the δ receptor, buprenorphine shows no agonistic effects (Toll et al., 1998; Lee et al., 1999; Romero et al., 1999). In the present study, buprenorphine exhibited a partial agonist activity of δ receptor compared with SNC80 or thienorphine [Fig. 4E].
Thienorphine hydrochloride is a new anti-relapse drug for opioid abusers that is currently in phase II clinical trial. In the present study, the antinociception, dependence, and signal transduction induced by thienorphine were examined. Thienorphine showed a potent antinociception effect in acetic acid-induced writhing test and formalin test. In the hot plate test and tail-flick test, thienorphine presented the typical partial opioid agonist character with a ceiling dose-response curve in addition to a bell-shaped curve. The hot plate test revealed that thienorphine induced approximately 50% of antinociception in μ receptor knockout (μ-KO) mice compared to wild-type controls (P < 0.05). The κ, δ selective antagonist nor-binaltorphimine (nor-BNI), and naltrindole decreased approximately 50–60% of theinorphine antinociception in μ-KO mice, respectively. The ORL1 receptor-selective antagonist J113397 did not affect theinorphine antinociception in μ-KO mice. Chronic treatment with thienorphine (1.5 mg/kg) induced some tolerance that was lower compared to buprenorphine or morphine addition. In contrast to buprenorphine or morphine, thienorphine did not lead to psychological dependence by conditioned place preference (CPP). The maximum inhibition of thienorphine on protein kinase A (PKA) activity was about 36%, 100%, 100%, and 12% in CHO-μ/κ/δ/ORL1-PKAcatEGFP cells, respectively. Similar results were observed in cyclic adenosine monophosphate (cAMP) accumulation inhibited by thienorphine in cells. Thienorphine significantly increased pERK1/2 in CHO-κ/δ-PKAcatEGFP cells. These results indicated that thienorphine induced analgesia through activation of κ- and δ-, partial activation of μ- opioid receptor without a bias between G-protein- and β-arrestin-mediated pathways. Thienorphine might be used for antinociception with minimal adverse effects.
In vitro profiling of opioid ligands using the cAMP formation inhibition assay and the β-arrestin2 recruitment assay: No two ligands have the same profile
2020, European Journal of Pharmacology
Citation Excerpt :
Full efficacy was only achieved at 10 μM, a concentration several orders of magnitude higher than its IC50 (0.002 μM) and that is highly unlikely to be achieved in the CNS at clinically administered doses in humans (Elkader and Sproule, 2005; Smith and Houghton, 2013). This observation is consistent with its partial agonist activity at the MOP-receptor when assessed using electrophysiological methods in rat brain slices (Virk et al., 2009), or the [35S]-GTPγS assay in membranes from cultured cells (Lee et al., 1999; Selley et al., 1997; Traynor and Nahorski, 1995), or in rat thalamic membranes (Selley et al., 1997). This partial MOP-receptor agonist activity is thought to underpin the ceiling-effect for buprenorphine in humans and animals, as well as some side-effects (Lutfy et al., 2003).
Previously, we showed that no two of seven opioids administered by the intracerebroventricular route had the same potency rank order for evoking antinociception, constipation and respiratory depression in rats. To gain insight at the cellular level, this study was designed to systematically investigate the activity profiles of six commonly used opioid ligands using the forskolin-stimulated cAMP assay and a β-arrestin2 recruitment assay in cultured HEK-293 cells transfected with MOP(μ), DOP(δ) or KOP(κ) receptors(-r).
Morphine was a potent agonist at the MOP-r in the cAMP assay whereas it was a weak agonist at the KOP-r and DOP-r. Oxycodone had moderate efficacy and low potency at the MOP-r. Buprenorphine was a potent MOP-r and DOP-r agonist; its efficacy rank order was DOP > MOP > KOP. Fentanyl was a potent agonist at the MOP-r; its efficacy rank order was MOP > DOP > KOP. For DPDPE, its agonist efficacy was confined to the DOP-r, whereas for U69593, its efficacy rank order was KOP>> MOP.
For the β-arrestin2 assay, fentanyl had full efficacy at the MOP-r whereas morphine and oxycodone were weak with insignificant efficacy at DOP and KOP receptors. Buprenorphine did not recruit β-arrestin2 at all three opioid-receptors. DPDPE and U69593 had full efficacy for β-arrestin2 recruitment to the DOP-r and KOP-r respectively.
Despite the low efficacy and potency of morphine, oxycodone and buprenorphine in recruiting β-arrestin2 to the MOP-r herein, these opioids all evoked respiratory depression and constipation in rats. Together, our findings discount a key role for β-arrestin2 recruitment at the MOP-r in evoking opioid-related side-effects.
Molecular Basis of Opioid Action: From Structures to New Leads
2020, Biological Psychiatry
Since the isolation of morphine from the opium poppy over 200 years ago, the molecular basis of opioid action has remained the subject of intense inquiry. The identification of specific receptors responsible for opioid function and the discovery of many chemically diverse molecules with unique opioid-like efficacies have provided glimpses into the molecular logic of opioid action. Recent revolutions in the structural biology of transmembrane proteins have, for the first time, yielded high-resolution views into the 3-dimensional shapes of all 4 opioid receptors. These studies have begun to decode the chemical logic that enables opioids to specifically bind and activate their receptor targets. A combination of spectroscopic experiments and computational simulations has provided a view into the molecular movements of the opioid receptors, which itself gives rise to the complex opioid pharmacology observed at the cellular and behavioral levels. Further diversity in opioid receptor structure is driven by both genetic variation and receptor oligomerization. These insights have enabled computational drug discovery efforts, with some evidence of success in the design of completely novel opioids with unique efficacies. The combined progress over the past few years provides hope for new, efficacious opioids devoid of the side effects that have made them the scourge of humanity for millennia.
Preface
2018, Progress in Brain Research
Modulation of opioid receptor affinity and efficacy via N-substitution of 9β-hydroxy-5-(3-hydroxyphenyl)morphan: Synthesis and computer simulation study
2017, Bioorganic and Medicinal Chemistry
The enantiomers of a variety of N-alkyl-, N-aralkyl-, and N-cyclopropylalkyl-9β-hydroxy-5-(3-hydroxyphenyl)morphans were synthesized employing cyanogen bromide and K₂CO₃ to improve the original N-demethylation procedure. Their binding affinity to the μ-, δ-, and κ-opioid receptors (ORs) was determined and functional (GTP_γ³⁵S) assays were carried out on those with reasonable affinity. The 1R,5R,9S-enantiomers (1R,5R,9S)-(−)-5-(3-hydroxyphenyl)-2-(4-nitrophenethyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-16), (1R,5R,9S)-(−) 2-cinnamyl-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-20), and (1R,5R,9S)-(−)-5-(3-hydroxyphenyl)-2-(4-(trifluoromethyl)phenethyl)-2-azabicyclo[3.3.1]nonan-9-ol (1R,5R,9S-15), had high affinity for the μ-opioid receptor (e.g., 1R,5R,9S-16: Ki = 0.073, 0.74, and 1.99 nM, respectively). The 1R,5R,9S-16 and 1R,5R,9S-15 were full, high efficacy μ-agonists (EC₅₀ = 0.74 and 18.5 nM, respectively) and the former was found to be a partial agonist at δ-OR and an antagonist at κ-OR, while the latter was a partial agonist at δ-OR and κ-OR in the GTP_γ³⁵S assay. The enantiomer of 1R,5R,9S-16, (+)-1S,5S,9R-16 was unusual, it had good affinity for the μ-OR (Ki = 26.5 nM) and was an efficacious μ-antagonist (Ke = 29.1 nM). Molecular dynamics simulations of the μ-OR were carried out with the 1R,5R,9S-16 μ-agonist and the previously synthesized (1R,5R,9S)-(−)-5-(9-hydroxy-5-(3-hydroxyphenyl-2-phenylethyl)-2-azabicyclo[3.3.1]nonane (1R,5R,9S-(−)-NIH 11289) to provide a structural basis for the observed high affinities and efficacies. The critical roles of both the 9β-OH and the p-nitro group are elucidated, with the latter forming direct, persistent hydrogen bonds with residues deep in the binding cavity, and the former interacting with specific residues via highly structured water bridges.
Synthesis and opioid receptor binding of indium (III) and [<sup>111</sup>In]-labeled macrocyclic conjugates of diprenorphine: novel ligands designed for imaging studies of peripheral opioid receptors
2016, Tetrahedron
Radiolabeled diprenorphine (DPN) and analogs are widely used ligands for non-invasive brain imaging of opioid receptors. To develop complementary radioligands optimized for studies of the peripheral opioid receptors, we prepared a pair of hydrophilic DPN derivatives, conjugated to the macrocyclic chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), for complexation with trivalent metals. The non-radioactive indium (III) complexes, tethered to the C6-oxygen position of the DPN scaffold by 6- to 9-atom spacers, displayed high affinities for binding to μ, δ and κ opioid receptors in vitro. Use of the 9-atom linker conferred picomolar affinities equipotent to those of the parent ligand DPN. The [¹¹¹In]-labeled complexes were prepared in good yield (>70%), with high radiochemical purity (∼99%) and high specific radioactivity (>4000 mCi/μmol). Their log D_7.4 values were –2.21 to –1.66. In comparison, DPN is lipophilic, with a log D_7.4 of +2.25. Further study in vivo is warranted to assess the suitability of these [¹¹¹In]-labeled DPN-DOTA conjugates for imaging trials.

View all citing articles on Scopus

View full text

Differential binding properties of oripavines at cloned μ- and δ-opioid receptors

Abstract

Introduction

Section snippets

Chemicals

[3H]Diprenorphine binding

Discussion

Acknowledgements

Biochim. Biophys. Acta

Jpn. J. Pharmacol.

Life Sciences

Eur. J. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

Eur. J. Pharmacol.

Brain Res.

Neuropharmacology

J. Biol. Chem.

J. Pharmacol. Toxicol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Neuropharmacology

Biochem. Biophys. Res. Commun.

Stimulation of guanosine-5′-0-(3-[35S]thio) triphosphate binding by endogenous opioids acting at a cloned mu receptor

J. Pharmacol. Exp. Ther.

Actions of etorphine hydrochloride, (M99): a potent morphine-like agent

Br. J. Pharmacol.

Mutagenesis of a single amino acid in the rat μ-opioid receptor discriminates ligand binding

J. Neurochem.

The physiological relevance of low agonist affinity binding at opioid μ-receptors

Br. J. Pharmacol.

Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction

Biochem. Pharmacol.

[ $^{3} H$ ]Diprenorphine binding

Stimulation of guanosine-5′-0-(3-[ $^{35} S$ ]thio) triphosphate binding by endogenous opioids acting at a cloned mu receptor

Relationship between the inhibition constant (K_i) and the concentration of inhibitor which causes 50 percent inhibition (I₅₀) of an enzymatic reaction