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6,7-Benzomorphans have been investigated in medicinal chemistry for developing new drugs. This nucleus could be considered a versatile scaffold. The physicochemical properties of benzomorphan N-substituent are crucial in achieving a definite pharmacological profile at opioid receptors. Thus, the dual-target MOR/DOR ligands LP1 and LP2 were obtained through N-substituent modifications. Specifically, LP2, bearing as N-substituent the (2R/S)-2-methoxy-2- phenylethyl group, is a dual-target MOR/DOR agonist and is successful in animal models of inflammatory and neuropathic pain. To obtain new opioid ligands, we focused on the design and synthesis of LP2 analogs. First, the 2-methoxyl group of LP2 was replaced by an ester or acid functional group. Then, spacers of different lengths were introduced at N-substituent. In-vitro, their affinity profile versus opioid receptors has been performed through competition binding assays. Molecular modeling studies were conducted to deeply analyze the binding mode and the interactions between the new ligands and all opioid receptors., (© 2023 The Authors. Chemical Biology & Drug Design published by John Wiley & Sons Ltd.)

Human kappa opioid receptor (κ-OR), a G protein-coupled receptor (GPCR), has been identified as a drug target for treatment of such human disorders as pain perception, neuroendocrine physiology, affective behavior, and cognition. In order to find more selective and active agonists, one would like to do structure based drug design. Indeed, there is an X-ray structure for an antagonist bound to κ-OR, but structures for activated GPCRs are quite different from those for the inactive GPCRs. Here we predict the ensemble of 24 low-energy structures of human kappa opioid receptor (κ-OR), obtained by application of the GEnSeMBLE (GPCR Ensemble of Structures in Membrane Bilayer Environment) complete sampling method, which evaluates 13 trillion combinations of tilt and rotation angles for κ-OR to select the best 24. To validate these structures, we used the DarwinDock complete sampling method to predict the binding sites for five known agonists (ethylketocyclazocine, bremazocine, pentazocine, nalorphine, and morphine) bound to all 24 κ-OR conformations. We find that some agonists bind selectively to receptor conformations that lack the salt bridge between transmembrane domains 3 and 6 as expected for active conformations. These 3D structures for κ-OR provide a structural basis for understanding ligand binding and activation of κ-OR, which should be useful for guiding subtype specific drug design.

Several methodological approaches suggest that receptor heteromers exist in cell systems, but their presence in physiological tissue is widely contentious. We describe a novel method to determine if heterodimers exist in brain tissue sections using autoradiographic binding comparisons from single and double gene knockout mice, where tissues either have a full receptor complement and can form heterodimers, or are incapable of making heterodimers. We have tested this model, which we have named Knockout Subtraction Autoradiography, to determine if heterodimerisation of the kappa (KOP) and delta opioid (DOP) receptors occurs, as evidence from binding studies in cell systems suggest they are present in the brain. Using labeling of putative KOP receptor/DOP receptor heterodimers with either [(3)H]bremazocine or with [(3)H]naltrindole, two ligands which were used to provide evidence suggesting that these opioid receptor subtypes heterodimerize, we have applied a subtraction equation model based on the principle that receptor gene double knockout of either MOP receptor/KOP receptor (DOP receptor expression only) or MOP receptor/DOP receptor (KOP receptor expression only) produces tissue incapable of making the KOP receptor/DOP receptor heterodimer. We have shown in most brain regions that the labeling fits a simple additive model of monomer labeling, but that in a few brain regions opioid receptor heterodimerization does occur. The data does not support the conclusion that KOP receptor/DOP receptor heterodimerisation is widespread in the central nervous system, but does indicate that this novel methodology can detect heterodimerisation, when ligands with distinct binding affinities for monomer and heterodimer forms exist., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Persistent pain states, such as those caused by nerve injury or inflammation, are associated with altered sensations, allodynia and hyperalgesia, that are resistant to traditional analgesics. A contribution to development and maintenance in altered pain perception comes from nociceptive processing and descending modulation from supraspinal sites. A multitarget ligand seems to be useful for pain relief with a decreased risk of adverse events and a considerable analgesic efficacy. The multitarget MOR agonist-DOR antagonist LP1, (3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benazocin-3(2H)-yl]-N-phenylpropanamide, is a central acting antinociceptive agent with low potential to induce tolerance. LP1 was tested in models of neuropathic pain - induced by chronic constriction injury (CCI) of the left sciatic nerve - and inflammatory pain - produced by intraplantar injection of carrageenan. In CCI rats, subcutaneous (s.c.) LP1 (3 mg/kg) showed a significant antiallodynic effect, measured with von Frey filaments, and antihyperalgesic effect, evoked in response to a radiant heat stimulus with plantar test. Analogously, LP1 significantly reduced allodynic and hyperalgesic thresholds in a model of inflammatory pain induced by carrageenan. To evaluate the contribution of opioid receptor subtypes in LP1 antinociceptive effects, the multitarget LP1 profile was assessed using selective opioid antagonists. Moreover, functional electrophysiological in vitro assays, using primary cortical and spinal cord networks, allowed to define the "pharmacological fingerprint" of LP1. The EC₅₀ values in this functional screening seem to confirm LP1 as a potent opioid ligand (EC₅₀ = 0.35 fM and EC₅₀ = 44 pM in spinal cord and frontal cortex, respectively). Using a NeuroProof data-base of well characterised reference compounds, a similarity profile of LP1 to opioid and non-opioid drugs involved in pain modulation was detected. Our studies seem to support that multitarget ligand approach should be useful for persistent pain conditions in which mechanical allodynia and thermal hyperalgesia are significant components of the nociceptive response., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Aims: Opioid drugs are the principal treatment option for moderate to severe pain and exert their biological effects through interactions with opioid receptors that are widely distributed throughout the CNS and peripheral tissues. Ligands capable of simultaneously targeting different receptors could be successful candidates for the treatment of chronic pain. Enhanced antinociception coupled with a low incidence of side effects has been demonstrated for ligands possessing mixed mu-opioid receptor (MOR) and delta-opioid receptor (DOR) activity. We previously reported that 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) acted as a MOR-DOR ligand in in vitro functional assays and moreover this drug produced a valid antinociception that was longer lasting than that of morphine. The aim of this work was to determine whether the antinociceptive effect produced by LP1 was central or peripheral and to assess which opioid receptor subtypes are involved in its effects., Main Methods: We explored the effects of naloxone methiodide (NX-M), a quaternary opioid antagonist, administered either intracerebroventricularly (i.c.v.) or subcutaneously (s.c.), on LP1-mediated antinociception in male Sprague-Dawley rats. In addition, we administered s.c. selective antagonists for MOR, DOR and kappa-opioid receptor (KOR) to investigate the effects of LP1. To characterise this drug's DOR profile better, we also investigated the effects of LP1 on DPDPE, a selective DOR agonist., Key Findings: Data obtained by tail flick test showed that LP1 induced predominantly MOR-mediated supraspinal antinociception and was able to counteract DPDPE analgesia., Significance: LP1, a multitarget opioid ligand, is a supraspinal acting antinociceptive agent that is useful for the treatment of chronic pain., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Aims: Powerful analgesics relieve pain primarily through activating mu opioid receptor (MOR), but the long-term use of MOR agonists, such as morphine, is limited by the rapid development of tolerance. Recently, it has been observed that simultaneous stimulation of the delta opioid receptor (DOR) and MOR limits the incidence of tolerance induced by MOR agonists. 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2H)-yl]-N-phenylpropanamide (LP1) is a centrally acting agent with antinociceptive activity comparable to morphine and is able to bind and activate MOR and DOR. The aim of this work was to evaluate and compare the induction of tolerance to antinociceptive effects from treatment with LP1 and morphine., Main Methods: Here, we evaluated the pharmacological effects of LP1 administered at a dose of 4 mg/kg subcutaneously (s.c.) twice per day for 9 days to male Sprague-Dawley rats. In addition, the LP1 mechanism of action was assessed by measurement of LP1-induced [(35)S]GTPγS binding to the MOR and DOR., Key Findings: Data obtained from the radiant heat tail flick test showed that LP1 maintained its antinociceptive profile until the ninth day, while tolerance to morphine (10mg/kg s.c. twice per day) was observed on day 3. Moreover, LP1 significantly enhanced [(35)S]GTPγS binding in the membranes of HEK293 cells expressing either the MOR or the DOR., Significance: LP1 is a novel analgesic agent for chronic pain treatment, and its low tolerance-inducing capability may be correlated with its ability to bind both the MOR and DOR., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Using Chinese Hamster Ovary cell membranes that stably expressed the human kappa-opioid receptor, we investigated the hypothesis that kappa(1)- and kappa(2)-opioid receptors, historically defined by their pharmacological selectivity for either arylacetamides or benzomorphans are, in fact, different affinity states or binding sites on the same kappa-opioid receptors. Receptor binding studies showed that GTP gamma S potently inhibited [3H](5 alpha,7 alpha,8 beta)-(+)-N-methyl-N-(7-[1-pyrrolidinyl]-1-oxaspiro [4.5]dec-8-yl)-benzeneacetamide (U69,593) binding, compared to virtually no inhibition of [3H]bremazocine binding. Saturation binding experiments showed a three-fold decrease in [3H]U69,593 affinity in the presence of GTP gamma S, but GTP gamma S had no effect on [3H]bremazocine affinity. The kappa-opioid receptor antagonist nor-binaltorphimine had a four-fold higher affinity for [3H]U69,593-labeled receptors than for [3H]bremazocine-labeled receptors. Functional selectivity studies, measuring the stimulation of [35S]GTP gamma S agonist-induced binding, showed a significantly higher U69,593-induced G protein-receptor activation in comparison to the stimulation observed with bremazocine. These results suggest that pharmacologically defined 1 kappa-opioid receptor subtypes may be different affinity states of the same receptor.

A mouse mu opioid receptor was engineered to contain a FLAG epitope at the amino-terminus and a hexahistidine tag at the carboxyl-terminus to facilitate purification. Selection of transfected human embryonic kidney (HEK) 293 cells yielded a cell line that expressed the receptor with a B(max) of 10 pmol/mg protein. 3[H]Bremazocine exhibited high affinity binding to the epitope-tagged mu opioid receptor with a KD of 1.0 nM. The agonists [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO), morphine and [D-Ala(2),D-Leu(5)]enkephalin (DADL) competitively inhibited bremazocine binding to the tagged mu receptor with KI's of 3.5, 17 and 70 nM, respectively. Chronic treatment of cells expressing the epitope-tagged mu receptor with DAMGO resulted in down-regulation of the receptor, indicating that the tagged receptor retained the capacity to mediate signal transduction. The mu receptor was solubilized from HEK 293 cell membranes with n-dodecyl-beta-D-maltoside in an active form that maintained high affinity bremazocine binding. Sequential use of wheat germ agglutinin (WGA)-agarose chromatography, Sephacryl S300 gel filtration chromatography, immobilized metal affinity chromatography, immunoaffinity chromatography, and sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) permitted purification of the receptor. The purified mu opioid receptor was a glycoprotein that migrated on SDS/PAGE with an apparent molecular mass of 80 kDa. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry was used to identify and characterize peptides derived from the mu opioid receptor following in-gel digestion with trypsin or chymotrypsin, and precursor-derived tandem mass spectrometry (ms/ms) confirmed the identity of several peptides derived from enzymatic digestion of the mu opioid receptor.

Using quantitative receptor autoradiography we have determined if deletion of the delta-opioid receptor gene (Oprd1) results in compensatory changes in the expression of other opioid receptors. Gene targeting was used to delete exon 1 of the mouse delta-opioid receptor gene and autoradiography was carried out on brains from wild-type, heterozygous and homozygous knockout mice. Delta-opioid receptors were labeled with [(3)H]deltorphin I (7 nM), mu- with [(3)H]DAMGO (4 nM), and kappa- with [(3)H]CI-977 (2.5 nM) or [(3)H]bremazocine (2 nM in the presence of DPDPE and DAMGO) and non-specific binding determined with naloxone. [(3)H]Deltorphin I binding was reduced by approximately 50% in heterozygous animals. In homozygous animals specific binding could only be detected after long-term film exposure (12 weeks). Regions exhibiting this residual [(3)H]deltorphin I binding correlated significantly with those demonstrating high levels of the mu-receptor and were abolished in the presence of the mu-agonist DAMGO. Autoradiographic mapping showed significant overall reductions in [(3)H]DAMGO and [(3)H]CI-977 binding throughout the brain following loss of both copies of the Oprd1 gene. In contrast, overall levels of [(3)H]bremazocine binding were higher in brains from -/- than +/+ mice. Our findings suggest that residual [(3)H]deltorphin I binding in the brain of delta-receptor gene knockout mice is the result of cross-reactivity with mu-sites and that there are no delta-receptor subtypes derived from a different gene. Changes in mu- and kappa-receptor labeling suggest compensatory changes in these subtypes in response to the absence of the delta-receptor. The differences in [(3)H]CI-977 and [(3)H]bremazocine binding indicate these ligands show differential recognition of the kappa-receptor., (Copyright 2002 Elsevier Science B.V.)

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.

Despite ample pharmacological evidence for the existence of multiple mu-, delta- and kappa-opioid receptor subtypes, only three genes encoding mu-(MOR), delta-(DOR) and kappa-(KOR) opioid receptor have been cloned. The KOR gene encodes kappa(1)-sites, which specifically bind arylacetamide compounds, and the possible existence of kappa-opioid receptor subtypes derived from another kappa-opioid-receptor gene, yet to be characterized, remains a very contentious issue. kappa(2)-Opioid receptors are described as binding sites typically labelled by the non-selective benzomorphan ligand [3H]bremazocine in the presence of mu-, delta- and kappa(1)-opioid receptor blocking ligands. To investigate the genetic origin of kappa(2)-opioid receptors, we have carried out homogenate binding experiments with [3H]bremazocine in brains of single MOR-, DOR-, KOR- and double MOR/DOR-deficient mice. Scatchard analysis showed that 68+/-12% of the binding sites arise from the MOR gene, 27+/-1% from the DOR gene and 14.5+/-0.2% from the KOR gene, indicating that the three known genes account for total [3H]bremazocine binding. Experiments in the presence of mu-, delta- and kappa(1)-opioid receptor suppressor ligands further showed that non-kappa(1)-opioid receptor labelling can be accounted for by binding to both the mu- and delta-opioid receptors. Finally, [3H]bremazocine binding experiments performed on brain membranes from the triple MOR/DOR/KOR-deficient mice revealed a complete absence of binding sites, confirming definitively that no additional gene is required to explain the total population of [3H]bremazocine binding sites. Altogether the data show that the putative kappa(2)-opioid receptors are in fact a mixed population of KOR, DOR and predominantly MOR gene products.

Two of the synthesized (-)-(1R,5R,9R)-N-homologues (N-but-3-enyl- and N-but-3-ynyl-5,9-dimethyl-2'-hydroxy-6,7-benzomorphan (9, 13)) were found to be about 20 times more potent than morphine in the mouse tail-flick assay (ED(50) = 0.05 mg/kg), and (-)-(1R,5R, 9R)-N-but-2-ynyl-5,9-dimethyl-2'-hydroxy-6,7-benzomorphan ((-)-(1R, 5R,9R)-N-but-2-ynylnormetazocine, 12) was about as potent as the opioid antagonist N-allylnormetazocine (AD(50) in the tail-flick vs morphine assay = 0.3 mg/kg). All of the homologues examined had higher affinity for the kappa-opioid receptor than the mu-receptor except (-)-N-but-2-ynyl-normetazocine (12), which had a kappa/mu ratio = 7.8 and a delta/mu ratio = 118. The (-)-N-2-cyanoethyl (3), -allyl (8), and -but-3-ynyl (13) analogues had good affinity (<10 nM) for delta-opioid receptors. Two homologues in the (+)-(1S,5S,9S)-normetazocine series, N-pent-4-enyl (24) and N-hex-5-enyl (25), were high-affinity and selective sigma(1)-ligands (K(i) = 2 nM, sigma(2)/sigma(1) = 1250, and 1 nM, sigma(2)/sigma(1) = 750, respectively); in contrast, N-allylnormetazocine (22) had relatively poor affinity at sigma(1), and its sigma(1)/sigma(2) ratio was <100.

Binding pockets of the opioid receptors are presumably formed among the transmembrane domains (TMDs) and are accessible from the extracellular medium. In this study, we determined the sensitivity of binding of [(3)H]diprenorphine, an antagonist, to mu, delta, and kappa opioid receptors to charged methanethiosulfonate (MTS) derivatives and identified the cysteine residues within the TMDs that conferred the sensitivity. Incubation of the mu opioid receptor expressed in HEK293 cells with MTS ethylammonium (MTSEA), MTS ethyltrimethylammonium (MTSET), or MTS ethylsulfonate (MTSES) inhibited [(3)H]diprenorphine binding with the potency order of MTSEA > MTSET > MTSES. Pretreatment of mu, delta, and kappa opioid receptors with MTSEA dose-dependently inhibited [(3)H]diprenorphine binding with MTSEA sensitivity in the order of kappa > mu >> delta. The effects of MTSEA occurred rapidly, reaching the maximal inhibition in 10 min. (-)-Naloxone, but not (+)-naloxone, prevented the MTSEA effect, demonstrating that the reaction occurs within or in the vicinity of the binding pockets. Each cysteine residue in the TMDs of the three receptors was mutated singly, and the effects of MTSEA treatment were examined. The mutants had similar affinities for [(3)H]diprenorphine, and C7. 38(321)S, C7.38(303)S, and C7.38(315)S mutations rendered mu, delta, and kappa opioid receptors less sensitive to the effect of MTSEA, respectively. These results indicate that the conserved Cys7.38 is differentially accessible in the binding-site crevice of these receptors. The second extracellular loop of the kappa receptor, which contains several acidic residues, appears to play a role, albeit small, in its higher sensitivity to MTSEA, whereas the negative charge of Glu6.58(297) did not. To the best of our knowledge, this is the first report to show that a conserved residue among highly homologous G protein-coupled receptors is differentially accessible in the binding-site crevice. In addition, this represents the first successful generation of MTSEA-insensitive mutants of mu, delta, and kappa opioid receptors, which will allow determination of residues accessible in the binding-site crevices of these receptors by the substituted cysteine accessibility method.

Several non-peptidic opioids have been synthesized recently as part of a program to develop selective delta receptor agonists. In this study, the affinities of a set of compounds for cloned delta and mu opioid receptors expressed in HEK 293 cell lines were determined by competition analysis of [3H]bremazocine binding to membrane preparations. All compounds studied exhibited high affinity and selectivity, with apparent dissociation constants in the range of 0.6-1.7 nM for the delta opioid receptor and 240-1165 nM for the mu opioid receptor. We next sought to determine which domain of the delta receptor was critical for mediating the highly selective binding by analysis of ligand affinities for mu/delta receptor chimeras. Receptor binding profiles suggested that a critical site of receptor/ligand interaction was located between transmembrane domain 5 (TM5) and TM7 of the delta receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the mu opioid receptor, led to a spectrum of effects on affinities, depending on the ligand tested. Affinities of SB 219825 and SB 222941 were particularly sensitive to the substitution, displaying a 50-fold and 70-fold decrease in affinity, respectively. Activities of the delta receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing delta opioid receptors with selective ligands induced phosphorylation of MAP kinase, although the non-peptidic ligands were less efficacious than the enkephalin derivative DADL (Tyr-D-Ala-Gly-Phe-D-Leu). Similarly, chronic exposure of HEK 293 cells expressing delta opioid receptors with selective, non-peptidic ligands, with the exception of SB 206848, caused receptor down-regulation, however, the SB compounds were less efficacious than DADL.

We hypothesized that the selectivity profile of the rat mu-opioid receptor for opioid receptor-selective ligands is determined by the nature of the amino acid residues at highly divergent sites in the ligand-binding pocket. To determine which characteristics of these residues contribute to opioid receptor ligand selectivity, we made various mutant receptors that replaced the Lys(303) and Trp(318) residues near the extracellular interface of transmembrane domains VI and VII, respectively. Ligand binding determinations using transiently transfected monkey kidney epithelial (COS-1) cells show that Lys(303) mutations cause little change in the receptor binding profile, whereas the Trp(318) mutant receptors have considerably lower affinity for micro-opioid receptor-selective ligands and greatly increased affinity for delta-opioid receptor-selective ligands. The nature of these mutations show that this effect is not due to sterics or charge alone. [35S]guanosine-5'-O-(3-thio)-triphosphate ([35S]GTPgammaS) activity assays show that these residues may influence functional, as well as binding selection. We conclude that a primary role for Trp(318) is to form a basis for ligand selectivity.

Metal ions affect ligand binding to G-protein-coupled receptors by as yet unknown mechanisms. In particular, Na(+) increases the affinity for antagonists but decreases it for agonists. We had modeled the mu-opioid receptor (muR) based on the low-resolution structure of rhodopsin by G. F. X. Schertler, C. Villa, and R. Henderson (1993, Nature 362, 770-772) and proposed that metal ions may be directly involved in the binding of ligands and receptor activation (B. S. Zhorov and V. S. Ananthanarayanan, 1998, J. Biomol. Struct. Dyn. 15, 631-637). Developing this concept further, we present here homology models of muR using as templates the structure of rhodopsin elaborated by I. D. Pogozheva, A. L. Lomize, and H. I. Mosberg (1997, Biophys. J. 70, 1963-1985) and J. M. Baldwin, G. F. X. Schertler, and V. M. Unger (1997, J. Mol. Biol., 272, 144-164). Using the Monte Carlo minimization (MCM) method, we docked the Na(+)-bound forms of muR ligands: naloxone, bremazocine, and carfentanyl. The resultant low-energy complexes showed that the two positive charges in the protonated metal-bound ligands interact with the two negative charges at Asp(3.32) and Asp(2.50) (for notations, see J. A. Ballesteros and H. Weinstein, 1995, Methods Neurosci. 25, 366-426). MCM computation on morphine docked inside the model of muR by I. D. Pogozheva, A. L. Lomize, and H. I. Mosberg (1998, Biophys. J. 75, 612-634) yielded two binding modes with the ligand's ammonium group salt-bridged either to Asp(3.32) (generally regarded as the ligand recognition site) or to Asp(2.50). The latter is the presumed site for Na(+) ion, which is known to modulate ligand binding. Assuming that in the low-dielectric transmembrane region of muR, organic and inorganic cations would compete for Asp(3.32) and Asp(2.50), we propose that ligand binding, as visualized in the above models, would first displace Na(+) from Asp(3.32). A subsequent progress of the ligand toward Asp(2.50) would result in either the retention of Na(+) at Asp(2.50) in the case of antagonists or the displacement of Na(+) from Asp(2.50) in the case of agonists. The displaced Na(+) would move toward the salt-bridged Asp(3.49)-Arg(3.50) and disengage the salt bridge. This, in turn, would result in conformational changes at the cytoplasmic face of the receptor that facilitate the interaction with the G-protein., (Copyright 2000 Academic Press.)

This report concerns the synthesis and preliminary pharmacological evaluation of a novel series of kappa agonists related to the morphinan (-)-cyclorphan (3a) and the benzomorphan (-)-cyclazocine (2) as potential agents for the pharmacotherapy of cocaine abuse. Recent evidence suggests that agonists acting at kappa opioid receptors may modulate the activity of dopaminergic neurons and alter the neurochemical and behavioral effects of cocaine. We describe the synthesis and chemical characterization of a series of morphinans 3a-c, structural analogues of cyclorphan [(-)-3-hydroxy-N-cyclopropylmethylmorphinan S(+)-mandelate, 3a], the 10-ketomorphinans 4a,b, and the 8-ketobenzomorphan 1b. Binding experiments demonstrated that the cyclobutyl analogue 3b [(-)-3-hydroxy-N-cyclobutylmethylmorphinan S(+)-mandelate, 3b, MCL-101] of cyclorphan (3a) had a high affinity for mu, delta, and kappa opioid receptors in guinea pig brain membranes. Both 3a,b were approximately 2-fold more selective for the kappa receptor than for the mu receptor. However 3b (the cyclobutyl analogue) was 18-fold more selective for the kappa receptor in comparison to the delta receptor, while cyclorphan (3a) had only 4-fold greater affinity for the kappa receptor in comparison to the delta receptor. These findings were confirmed in the antinociceptive tests (tail-flick and acetic acid writhing) in mice, which demonstrated that cyclorphan (3a) produced antinociception that was mediated by the delta receptor while 3b did not produce agonist or antagonist effects at the delta receptor. Both 3a,b had comparable kappa agonist properties. 3a,b had opposing effects at the mu receptor: 3b was a mu agonist whereas 3a was a mu antagonist.

A convenient enantioselective synthesis of (1R,5R,9R)- and (1S,5S, 9S)-9-alkyl-2-benzyl-6,7-benzomorphans (2a-c) which starts with naphthaldehyde is described. These compounds were designed to gain additional information on the structure-sigma binding relationship of the 6,7-benzomorphan class of sigma ligands. In contrast to pentazocine and most 6,7-benzomorphans, the (1R,5R,9R)-isomers of 2a-c showed greater affinity for the sigma(1) receptor than the (1S, 5S,9S)-isomers. Despite reversal of enantioselectivity at the sigma(1) sites, moderate affinity and enantioselectivity at the sigma(2) sites [greater affinity for (1R,5R,9R)-isomers than (1S,5S, 9S)-isomers] were maintained. A comparison of the binding affinities of 2a-c to the more conformationally flexible trans-2-alkyl-1-benzaminoethyl-1,2-dihydronaphthalenes (10a-c) suggested that the relatively rigid structure of 2a-c played an important part in their sigma(1) binding properties. These compounds, particularly (1R,5R,9R)-2-benzyl-9-methyl-6,7-benzomorphan [(-)-2a], which has a K(i) value of 0.96 nM, will be useful in further characterization of the sigma(1) receptor.

Desglycinyl remacemide (DGR), a biologically active metabolite of remacemide, was radiolabeled in an attempt to develop a ligand binding assay to identify its site of action. Incubation of the radioligand with membranes obtained from P2 fractions of whole rat brain revealed a single population of specific [3H]-DGR binding sites having a Kd of 290 nM and a Bmax of 1.3 pmole/mg protein. The specific binding of [3H]-DGR is most enriched in the P2 subcellular fraction and is heterogeneously distributed throughout the brain. The binding of [3H]-DGR to rat brain membranes was inhibited most potently by MK-801 and SKF-10,047. In contrast, haloperidol, and other sigma receptor-active agents, were relatively inactive at this site. These data suggest that DGR interacts with a channel blocking site on the NMDA receptor., (Copyright 1999 Elsevier Science B.V.)

In addition to effects in the periphery through inhibition of prostaglandin synthesis, several lines of evidence suggest that nonsteroidal anti-inflammatory drugs (NSAIDs) act in the central nervous system. The possibility that the central action of NSAIDs involves regulation of opioid receptors was investigated by quantitative autoradiography of mu, delta, and kappa sites in rat brain slices. Increased (p < 0.05) labeling of mu receptors was observed in thalamic nuclei, gyrus dentate, and layers of the parietal cortex of rats treated for 10 days with lysine clonixinate. Labeling of delta receptors was lower in the lateral septum, and kappa sites decreased in thalamic nuclei. These effects were not mediated through direct interaction with opioid-binding sites, since receptor-binding assays using rat brain membranes confirmed that clonixinate up to 1 x 10(-4) mol/l does not inhibit mu, delta, and kappa receptor specific binding. Central effects of NSAIDs might, therefore, involve interaction with the opioid receptor system through indirect mechanisms.

We have investigated the role of cysteine residues in a highly purified mu opioid receptor protein (muORP) by examining the effect of -SH reagents on the binding of opioid ligands. Treatment of muORP, which is devoid of additional proteins, eliminates complications that arise from reaction of -SH reagents with other components, such as G proteins. Reagents tested include N-ethylmaleimide, 5,5'-dithiobis(2-nitrobenzoic) acid, and two derivatives of methanethiosulfonate. Specific opioid binding was inactivated by micromolar concentrations of all -SH reagents tested. Agonist binding ([3H]DAMGO) was much more sensitive to inactivation than antagonist binding ([3H]bremazocine). Prebinding muORP with 100 nM naloxone protected antagonist and agonist binding from inactivation by -SH reagents. The results of these experiments strongly suggest that at least one, and possibly more, reactive cysteine residue(s) is present on the mu opioid receptor protein molecule, positioned near the ligand binding site and accessible to -SH reagents.

We investigated the molecular basis of the discrimination between nociceptin/orphanin FQ receptor (NociR) and opioid receptors (OPRs) by bremazocine, a non-type-selective opioid ligand. Construction of several chimeric receptors between NociR and kappa-opioid receptor (KOPR) and mutant NociRs followed by binding experiments with [3H]bremazocine showed that the mutation of only four amino acid residues of NociR, Ala216, Val279, Gln280 and Val281, to the amino acid residues located at the corresponding position of KOPR, Lys227, Ile290, His291 and Ile292, made it possible for the resultant mutant NociR to bind bremazocine with high affinity. Considering that these four amino acid residues are conserved among mu-, delta- and kappa-OPRs, the present result suggests that bremazocine recognizes the difference in these four amino acid residues to discriminate between NociR and OPRs.

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.

A series of (+)-cis-N-normetazocine derivatives has been described, and their affinities for sigma1, sigma2, and phencyclidine (PCP) sites and opioid, muscarinic (M2), dopamine (D2), and serotonin (5-HT2) receptors were evaluated. The effect of the N-substitution with a substituted ethylamino spacer was investigated. Compounds 8c-11c displayed high affinities for sigma1 sites and for opioid receptors. Substitution of the second basic nitrogen either with alkyl or cycloalkyl substituents give compounds (1a-6a) with high affinity and selectivity for sigma1 binding sites. Compounds 1a-5a were further characterized in vivo, and their agonist/antagonist activity was evaluated. In mouse, compound 1a and 2a as well as haloperidol suppressed in a dose-related manner the stereotyped behavior induced by (+)-SKF 10,047. Compounds 3a-5a and (+)-pentazocine do not affect the stereotyped behavior induced by ip injection of (+)-SKF 10,047. Therefore, from this series of compounds we identified potent and selective sigma1 ligands which might prove useful to unveil the functional role of sigma1 sites.

Receptor binding studies and electrophysiological studies demonstrated the existence of at least two kappa opioid receptors, which have been designated kappa-1 and kappa-2. Several agonists and antagonists are selective for the kappa-1 receptor whereas no known ligands are selective for the kappa-2 receptor. In this study, the kappa opioid GR89,696 was tested in the guinea pig hippocampal slice preparation for kappa-1 versus kappa-2 activity. The perforant path-evoked population spike in the dentate was use to evaluate activity at the kappa-1 receptor, and the Schaffer collateral-evoked N-methyl-D-aspartate (NMDA) receptor-mediated synaptic current in CA3 pyramidal cells was used to measure kappa-2 receptor activation. GR89,696 had no effect on the perforant path-evoked dentate population spike; however, it did reverse the effects of the selective kappa-1 agonist U69,593 when co-perfused over the slices. In the CA3, GR89,696 inhibited the NMDA receptor-mediated synaptic current. The inhibition was antagonized by naloxone. The EC50 for GR89,696 on the NMDA current was 41.7 nM (95% CL, 7.0-248 nM). These findings indicate that GR89,696 is an agonist for kappa-2 opioid receptors and an antagonist at kappa-1 receptors in the guinea pig hippocampus.

Cocaine is thought to be addictive because chronic use leads to molecular adaptations within the mesolimbic dopamine (DA) circuitry that affect motivated behavior and emotion. Although the reinforcing effects of cocaine are mediated primarily by blocking DA reuptake into the presynaptic nerve terminal, reciprocal signaling between DA and endogenous opioids has important implications for cocaine dependence. The present study used the opioid antagonist 6 beta-[125iodo]-3,14-dihydroxy-17-cyclopropylmethyl-4,5 alpha-epoxymorphinan ([125I]IOXY) after pretreatment with the site-directed acylating agents 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5-isothiocyanatobenzimid iazole -HCl (mu-selective) and N-phenyl-N-[1-(2-(4-isothiocyanato)-phenethyl)-4-piperidinyl]-p ropana mide-HCl (delta-selective) to examine the effect of cocaine exposure on the distribution and density of kappa2 receptors in autopsy studies of human cocaine fatalities. The selective labeling of the kappa2 receptor subtype was demonstrated by competition binding studies, which gave a pharmacological signature (IOXY >/= (+)-bremazocine >> U50,488 >/= U69,593) distinct from either the kappa1 or kappa3 receptor subtypes. Visualization of [125I]IOXY labeling revealed that kappa2 receptors localize to mesocortical and subcortical limbic areas, including the cingulate, entorhinal, insular, and orbitofrontal cortices and the nucleus accumbens and amygdala. The number of kappa2 receptors in the nucleus accumbens and other limbic brain regions from cocaine fatalities was increased twofold as compared with age-matched and drug-free control subjects. Cocaine overdose victims, who experienced paranoia and marked agitation before death, also had elevated densities of kappa2 receptors in the amygdala. These findings demonstrate for the first time that kappa2 receptor numbers are upregulated by cocaine exposure. The molecular adaptation of kappa2 receptor numbers may play a role in the motivational incentive associated with episodes of binge cocaine use and in the dysphoria that follows abrupt cocaine withdrawal.

A novel "restoration of function" mutagenesis strategy was developed to identify amino acid sequence combinations necessary to restore the ability to bind delta-selective ligands to an inactive delta/mu receptor chimera in which 10 amino acids of the third extracellular loop of the delta receptor were replaced by the corresponding amino acids from the mu receptor (delta/mu291-300). This chimera binds a nonselective opioid ligand but is devoid of affinity for delta-selective ligands. A library of mutants was generated in which some of the 10 amino acids of the mu sequence of delta/mu291-300 were randomly reverted to the corresponding delta amino acid. Using a ligand binding assay, we screened this library to select mutants with high affinity for delta-selective ligands. Sequence analysis of these revertants revealed that a leucine at position 300, a hydrophobic region (amino acids 295-300), and an arginine at position 291 of the human delta-opioid receptor were present in all revertants. Single and double point mutations were then introduced in delta/mu291-300 to evaluate the contribution of the leucine 300 and arginine 291 residues for the binding of delta-selective ligands. An increased affinity for delta-selective ligands was observed when the tryptophan 300 (mu residue) of delta/mu291-300 was reverted to a leucine (delta residue). Further site-directed mutagenesis experiments suggested that the presence of a tryptophan at position 300 may block the access of delta-selective ligands to their docking site.

The E-isomer of 7-benzylidenenaltrexone (BNTX, la) was reported by Portoghese as a highly selective delta-opioid antagonist. The corresponding Z-isomer 1b was not readily available through direct aldol condensation of naltrexone (6) with benzaldehyde. Using the photochemical methods employed by Lewis to isomerize cinnamamides, we have obtained Z-isomer 1b in good yield from E-isomer 1a. A series of (E)- and (Z)-7-arylidenenaltrexone derivatives was prepared to study the effect of larger arylidene groups on opioid receptor affinity in this series. By aldol condensation of naltrexone (6) with benzaldehyde, 1-naphthaldehyde, 2-naphthaldehyde, 4-phenylbenzaldehyde, and 9-anthracaldehyde, the (E)-arylidenes were readily obtained. Photochemical isomerization afforded the corresponding Z-isomers. These compounds were evaluated via opioid receptor radioligand displacement assays. In these assays, the Z-isomers generally had higher affinity and were more delta-selective than the corresponding E-isomers. The (Z)-7-(1-naphthylidene)naltrexone (3b) showed the greatest selectivity (delta:mu ratio of 15) and highest affinity delta-binding (Ki = 0.7 nM). PM3 semiempirical geometry optimizations suggest a significant role for the orientation of the arylidene substituent in the binding affinity and delta-receptor selectivity. This work demonstrates that larger groups may be incorporated into the arylidene portion of the molecule with opioid receptor affinity being retained.

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

A series of N-substituted maleimides was shown to effectively inactivate bremazocine binding to delta opioid receptors. Apparent second order rate constants for inactivation increased with increasing size of the N-substituent: N-methyl < N-ethyl < N-butyl < N-phenylmaleimide. It is suggested that the positive chain length effect is attributed to nonpolar interactions with the receptor in the vicinity of the reactive group. Binding to mu and delta opioid receptors was equally sensitive to inactivation by (2-aminoethyl)methanethiosulfonate; the [2-(trimethylammonium)ethyl] and (2-sulfonatoethyl) derivatives were less active. Site-directed mutagenesis of the mu opioid receptor indicated that Cys159, Cys190, Cys235, Cys292, or Cys321, residing in transmembrane domain 3, 4, 5, 6, and 7, respectively, were not the site of modification.

Quantitative autoradiographic assessment of cerebral cortical laminar distribution of mu, delta and kappa opioid receptors was carried out in coronal sections of five post-mortem human brains obtained at autopsy. The cortical areas studied were: cingulate, frontal, insular, parietal, parahippocampal, temporal, occipitotemporal, occipital and striate area. In general, the laminar patterns of distribution for the three types of receptors are distinctive. Peak levels of delta opioid binding are in laminae I, II, and IIIa. mu-Receptors are located in lamina III followed by I and II in cingulate, frontal, insular and parietal cortices and lamina IV in temporal and occiptotemporal cortices. kappa-Receptors are found concentrated in laminae V and VI. The patterns of opioid binding in cortical laminae showed remarkable consistency in all five brains examined. In contrast to other cortical areas, the parahippocampal gyrus, at the level of the amygdaloid formation, demonstrated peak kappa receptor density in laminae I, II and III. mu-Opioid binding was undetectable in the lateral occipital cortex and in the striate area.

Diabetic rats display changes in opioid pharmacology and brain regional levels of opioid peptides and prodynorphin mRNA. Previous investigations of opioid receptor binding, carried out in whole-brain homogenates, have, however, failed to detect changes. In the present study, quantitative autoradiography was used to measure mu and kappa opioid receptor binding in discrete brain regions of streptozotocin-treated diabetic rats. Measurement was limited to regions that previously displayed opioid binding changes in chronically food-restricted rats, since our primary aim is to identify brain mechanisms that mediate adaptive responses to persistent metabolic need and adipose depletion. Diabetics displayed strong trends or statistically significant changes which matched seven of the thirteen binding changes observed in food-restricted rats. In no case did diabetics display changes in the opposite direction. The two statistically significant changes common to food-restricted and diabetic rats are increased kappa binding in the medial preoptic area and decreased mu binding in the lateral habenula. The possible functional significance of these changes is discussed.

In behavioral experiments, the cinnamoylaminomorphinone clocinnamox (CCAM) has been shown to act as an insurmountable antagonist of mu, but not delta or kappa opioid agonists. In contrast, CCAM displayed only moderate mu selectivity (29:6:1 for mu:delta:kappa) in radioligand displacement experiments using mouse brain membranes. In the present study, the apparent discrepancy between the high mu selectivity of the insurmountable functional antagonism of CCAM and its only moderate mu selectivity in in vitro binding experiments was resolved by in vitro washout experiments and ex vivo binding experiments involving all three opioid receptor types. In the ex vitro washout experiments, CCAM-mediated mu receptor binding inhibition could not be reversed even after allowing for 8 hr hr dissociation, whereas the CCAM inhibition of delta and kappa receptor binding was time-dependently reversed. In the ex vivo binding experiments, 1 hr pretreatment of mice with 10 mg/kg of CCAM i.p. decreased ex vivo [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin ([3H]DAMGO) binding (tested at > or = 5 * Kd) by 90%, paralleled by an 88% decrease in mu receptor density in equilibrium saturation binding assays. Ex vivo [3H]DAMGO binding returned to control levels with a T1/2 of 2.7 to 4.2 days (independent of the CCAM dose), the effect being predominantly due to a recovery of mu receptor density. The CCAM inhibition of ex vivo [3H]DAMGO binding was dose-dependent and could be prevented in part by simultaneous administration of the protein synthesis inhibitor cylcoheximide. In contrast to the ex vivo binding of [3H]DAMGO, ex vivo binding of p-[3H]CI-[D-Pen2, D-Pen5]enkephalin or (-)-[3H]bremazocine (in the presence of 1 microM DAMGO and 1 microM [D-Pen6, D-Pen5]enkephalin) was not affected by CCAM pretreatment. Finally, ex vivo [3H]DAMGO binding inhibition data correlated well with mu receptor population changes estimated by Black and Leff analysis of behavioral (antinociception) experiments (T1/2 of receptor reappearance, 3.2 days). Thus, although CCAM reversibly interacted with mu, delta and kappa opioid receptors, only binding to mu receptors was wash-resistant. Binding of methoclocinnamox, a codeinone CCAM precursor with mu agonistic activity, seemed to be at least partially reversible, even at mu receptors.

In order to determine the opioid receptor binding in the rat cardiac sarcolemma, direct and displacement binding assays of tritiated bremazocine, a nonselective opioid, agonist and U69593, a specific kappa-receptor agonist, were performed in a sarcolemmal membrane preparation. Saturation studies revealed that there were substantial binding sites for both radioligands. The [3H]bremazocine had a binding capacity of 100 fmol/mg protein with a Kd of 3.4 nM while the binding of [3H]U69593 showed a Kd of 7.4 nM and a Bmax of 139 fmol/mg protein. Displacement binding assay showed that [D-Ala2, MePhe4, Gly-(ol)5]enkephalin (DAGO), a specific mu-agonist, exhibited no inhibitory effect on the binding of either [3H]bremazocine or [3H]U69593. [D-Ala2,D-Leu5]enkephalin (DADLE), a specific delta-agonist, had no inhibition on the binding of [3H]U69593, but displaced the binding of [3H]bremazocine at higher concentrations (10(-5)-10(-6)M). U50488, a specific kappa-agonist, completed with the binding of [3H]U69593 23 times more potently than with the binding of [3H]bremazocine. A residual binding sites of [3H]bremazocine was found with a Kd of 6.4 nM and a Bmax of 76 fmol/mg protein under conditions in which mu, delta and kappa 1 opioid bindings were suppressed by DAGO and DADLE at 200 nM and U50488 at 1 microM. DADLE at 5 microM, a concentration known to block kappa 2 binding sites, abolished the residual U50488-insensitive specific binding of [3H]bremazocine. Met5-Enkephalin-Arg6-Phe7, a specific kappa 2 ligand, displaced the binding of [3H]bremazocine much more readily than the binding of [3H]U69593. The present study provided evidence for the presence of two kappa-receptor subtypes in the cardiac sarcolemma: one U50488-sensitive and DADLE-insensitive, and the other U50488-insensitive and DADLE-sensitive. These two kappa-receptor binding subtypes may correspond to kappa 1 and kappa 2 receptor subtypes. Computer assisted non-linear regression analysis in the competition experiments of U50488 with [3H]U69593 afforded a best fit of the inhibition curves for two displacing components of kappa 1 binding sites: one with high affinity and low Bmax, another with low affinity and high Bmax.

Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding.

We determined the ability of a new benzomorphan derivative [2R-[2 alpha, 3(R*),6 alpha]]-1,2,3,4,5,6-hexahydro-3-(2-methoxypropyl)- 6,11,11-trimethyl-2,6-methano-3-benzazocin-9-ol hydrochloride (BIII 277 CL) to inhibit the N-methyl-D-aspartic acid (NMDA) receptor-channel complex in vitro and in vivo. BIII 277 CL potently displaced [3H]MK-801 binding from the NMDA receptor-channel complex in synaptosomal membrane preparations from rat brain cortex (Ki = 4.49 nmol/l). It was much less effective at displacing [3H]dihydromorphine, [3H]naloxone and [3H]ditolyguanidine binding in similar membrane preparations: the Ki values were 3323, 8031 and 1017 nmol/l, respectively. BIII 277 CL did not exhibit any marked affinities for a variety of other central neurotransmitter receptors. BIII 277 CL antagonized NMDA-induced [3H]noradrenaline release (EC50 = 1.7 mumol/l) and NMDA-induced inhibition of protein synthesis in rat hippocampal slices (EC50 = 3.0 mumol/l). In mice, BIII 277 CL prevented NMDA-induced lethality (ID50 = 0.54 mg/kg s.c.) and, as expected, also caused disturbances in motor coordination in the same dose range (ED50 = 0.47 mg/kg s.c.). The duration of BIII 277 CL was much shorter than than of (+)MK-801 in both tests. Finally, BIII 277 CL (0.3 mg/kg s.c. 5 times over 24 h) reduced the cortical infarct area in mice that had been subjected previously to focal cerebral ischemia by unilateral occlusion of the middle cerebral artery. In summary, these results indicate that BIII 277 CL is a potent and specific ion-channel blocker of the NMDA receptor-channel complex which could be used for the treatment of acute thromboembolic stroke in humans.

Whether there is an endogenous ligand for sigma receptors remains unknown. A total lipid fraction was isolated from the porcine liver, which was then subjected to chromatographic separation, yielding nine chromatographic fractions eluated at various chloroform/methanol ratios: each fraction was tested for its capacity of inhibiting the binding of (+)-[3H]SKF 10047 to hepatic sigma-receptors. The chromatographic fraction 7 + 7A (eluated at chloroform/methanol ratios of 60:40-10:90) which consisted mainly of phospholipids was found to have the highest activity. However, a complex analysis of this fraction revealed no relationship of its sigma-inhibiting activity to phospholipids. It is suggested that the non-competitive inhibition of (+)-[3H]SKF 10047 binding by the isolated active fraction is due to its presence of an endogenous ligand which, interacting with sigma-2 receptor, allosterically inhibits the binding of the labelled (+)-benzomorphan.

The synthesis and sigma 1 and sigma 2 binding properties of several (+)- and (-)-2-benzyl- and 2-dimethylallyl-2'-substituted-5,9 alpha-dimethyl-6,7-benzomorphans (3 and 4) are presented. In agreement with previously reported binding data for 2-substituted 5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans (N-substituted-N-normetazocine), all (1S,5S,9S)-(+)-isomers showed higher affinity for the sigma 1 site than the corresponding (1R,5R,9R)-(-)-isomers. Replacement of the 2'-hydroxy group of (+)-2-benzyl-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan [(+)-1f] with a 2'-NH2 and 2'-N(CH3)2 [(+)-3b and (+)-3c, respectively] had only a small effect on the sigma 1 Ki values. Changing the 2'-hydroxy group of (+)-1f to an H, F, Cl, Br, I, NHAc, or NHSO2CH3 resulted in a 5-fold or greater loss in potency. In contrast, replacement of the 2'-hydroxy group of (+)-2-(dimethylallyl)-5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphan [(+)-1b, (+)-pentazocine] with a 2'-H or 2'-F group resulted in a 2-fold increase in potency. Conversion of (+)-1f to its 2'-desoxy analogue (+)-2d resulted in a 27.5-fold loss in affinity. This suggests that (+)-1f and other N-substituted benzomorphan analogues may be binding to single sigma 1 receptors in a different way or to different sigma 1 receptors. (-)-Pentazocine [(-)-1b] and its 2'-fluoro analogue, (-)-2-(dimethylallyl)-5,9 alpha-dimethyl-2'-fluoro-6,7-benzomorphan [(-)-4a] showed the highest potency for the sigma 2 binding site.

Following the cloning of the opioid receptors mu, kappa, and delta, we conducted a search for related receptors. Using oligonucleotides based on the opioid and also the structurally related somatostatin receptors, we amplified genomic DNA using the polymerase chain reaction and isolated fragments of novel G protein-coupled receptor genes. Two of these gene fragments designated clones 12 and 11 were used to isolate the full-length genes. The intronless coding sequences of these genes, named GPR7 and GPR8, shared 70% identity with each other, and each shared significant similarity with the sequences encoding transmembrane regions of the opioid and somatostatin receptors. GPR7 was mapped to chromosome 10q11.2-q21.1 and GPR8 to chromosome 20q13.3. Northern blot analysis using human mRNA demonstrated expression of GPR7 mainly in cerebellum and frontal cortex, while GPR8 was located mainly in the frontal cortex. In situ hybridization revealed expression of GPR7 in the human pituitary. A partial sequence of the mouse orthologue of GPR7 was obtained, and in situ hybridization demonstrated expression in discrete nuclei of brain, namely suprachiasmatic, arcuate, and ventromedial nuclei of hypothalamus. A stable cell line expressing the GPR7 gene was created, but expression levels of the receptor were low. The available pharmacology indicated binding to several opioid drugs such as bremazocine, levorphanol, and beta-FNA, but not to the opioid receptor subtype-selective mu, delta, or kappa agonists.

The mouse thymoma R1.1 cell line was shown previously to express a single high-affinity kappa 1 opioid receptor that is negatively coupled through a pertussis toxin-sensitive G-protein to adenylyl cyclase. This study compared opioid receptor binding and inhibition of adenylyl cyclase activity in three unique derivatives of the R1.1 cell line. Membranes from the R1.G1 and R1E/TL8x.1.G1.OUAr.1 (R1EGO) cell lines bound both [3H]U69,593 and [3H](-)-bremazocine with similar affinities compared with R1.1 membranes, whereas membranes from the R1E/TL8x.1 (R1E) cell line did not possess any opioid binding sites, detected by radioreceptor binding. The Bmax values for [3H]U69,593 and [3H]-(-)-bremazocine binding to R1.G1 and R1EGO cell membranes were, respectively, 3- and 6-fold greater than those obtained with the parent R1.1 cell line. GTP and its nonhydrolyzable analog, Gpp(NH)p, inhibited [3H]U69,593 binding to all three cell lines. Stimulation of low-Km GTPase activity by the kappa-selective agonist (-)U50,488 was greatest in R1.G1 membranes, followed by R1EGO and R1.1. The maximal inhibition of forskolin-stimulated adenylyl cyclase activity by (-)U50,488 was 66 +/- 2% in R1.G1 and 49 +/- 2% in R1EGO, compared with 37 +/- 1% in R1.1 membranes. Whereas maximal inhibition of adenylyl cyclase activity did not correlate with receptor number among cell lines, the inhibition of cyclic AMP production did correlate with stimulation of low-Km GTPase activity. The R1.1 cell line and its derivatives, R1.G1 and R1EGO, express a similar type of kappa opioid receptor, which exhibits differences in coupling to G-proteins and to adenylyl cyclase among cell lines. These cell lines provide an excellent model system for studying the regulation of opioid receptor-adenylyl cyclase coupling efficiency.

One of the functions of glial receptors is to regulate synthesis and release of a variety of neuropeptides and growth factor peptides, which in turn act on neurons or other glia. Because of the potential importance of these interactions in injured brain, we have examined the role of two different receptors in the regulation of astrocyte neuropeptide synthesis. Stimulation of beta-adrenergic receptors on type 1 astrocytes resulted in increased mRNA and protein for the proenkephalin (PE) and somatostatin genes. This receptor also increased expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The potential role of opiate receptors was examined in several ways. Treatment of newborn rats for 7 days with the opiate antagonist naltrexone, prior to preparation of astrocytes, had no effect on PE mRNA or met-enkephalin content but resulted in a significant increase in NGF content. However, treatment of astrocytes in culture with met-enkephalin, morphine, or naltrexone had no effect on any of these parameters. No opiate binding could be detected, using either etorphine or bremazocine, to membranes of astrocytes prepared from cortex, cerebellum, striatum, or hippocampus of 1-day, 7-day, or 14-day postnatal rats. Thus we conclude that type 1 astrocytes do not express opiate receptors and that the in vivo effects of naltrexone are mediated indirectly via some other cell type/receptor.

Three opioid receptor types have been identified in the CNS and periphery that are referred to as mu, delta, and kappa. The present study examines the mRNA distribution of the kappa 1 receptor in the rat brain and compares it to the distribution of kappa receptor-binding sites and prodynorphin mRNA using a combination of in situ hybridization and receptor autoradiographic techniques. kappa 1 receptor mRNA was localized with a cRNA probe generated with a BamHI-HindIII cDNA fragment of the rat kappa 1 receptor and corresponds to the last 45 bp of the protein coding region and 728 nucleotides of the 3' untranslated region. Prodynorphin mRNA was localized with a cRNA probe corresponding to a 733-bp BamHI-HincII fragment of prodynorphin. kappa receptor-binding sites were labeled in one of two ways: [3H]U69,593 or [3H]bremazocine in the presence of a 300-fold excess of DAMGO and DPDPE. A high degree of correspondence between the kappa 1 receptor mRNA and kappa receptor binding was observed in several brain regions, including the endopiriform nucleus, claustrum, nucleus accumbens, olfactory tubercle, bed nucleus of the stria terminalis, medial preoptic area, paraventricular, supraoptic, suprachiasmatic, dorsomedial and ventromedial hypothalamic nuclei, basolateral, medial and cortical amygdaloid nuclei, midline thalamic nuclei, periaqueductal grey, parabrachial nucleus, locus coeruleus, and the nucleus of the solitary tract. Differences in the localization of kappa 1 receptor mRNA and binding and the relationship between the distribution of kappa 1 receptor and prodynorphin mRNAs are discussed.

Studies were directed at determining whether the kappa-opioid receptor expressed on the mouse R1.1 thymoma cell line contained either a disulfide bond or a sulfhydryl group at the opioid binding site. The binding of the kappa-opioid receptor agonist [3H](-)-bremazocine to R1.1 cell membranes was unchanged following treatment with the disulfide bond-reducing reagent dithiothreitol at concentrations up to 130 mM. However, treatment of membranes with the sulfhydryl-alkylating reagent N-ethylmaleimide, followed by extensive washing, reduced [3H](-)-bremazocine binding by as much as 90%. Inhibition of [3H](-)-bremazocine binding by N-ethylmaleimide was concentration- and time-dependent. When R1.1 cell membranes were treated with 1 mM N-ethylmaleimide for 10 min at 24 degrees C, the Bmax value for [3H](-)-bremazocine binding decreased by 50%, with no change in receptor affinity. N-Ethylmaleimide-induced reduction of [3H](-)-bremazocine binding was attenuated by pretreatment of membranes with the kappa-selective opioids U50,488 and U69,593. The results indicate that a sulfhydryl group is present at or near the binding site on the kappa-opioid receptor expressed by the R1.1 thymoma cell line.

The presence of kappa-opioid receptor subtypes has been clearly established in guinea pig brain. Using [3H]bremazocine in the presence of reversible blockers of mu, delta and kappa 1 receptors, two additional binding sites can be determined in guinea pig brain membranes. The site with higher affinity for the opioid ligands represents kappa 2, while the other site has low affinity and is poorly characterized. The kappa 2 site has high affinity for ethylketocyclazocine and other benzomorphans, as well as for the dynorphin gene products tested. The dynorphin analogs have no appreciable affinity for the low affinity site, so this site should not be called a kappa receptor. With an appropriate membrane preparation, kappa 2 binding can also be demonstrated in the guinea pig ileum. Binding affinities for selected ligands at kappa 2 in guinea pig ileum membranes are very similar to affinities found in brain membranes.

Three novel benzomorphans, (+)-N-benzylnormetazocine, (-)-deoxy-N-benzylnormetazocine, and (-)-deoxypentazocine were tested for their ability to produce circling behavior in rats following intranigral microinjections. Dose studies revealed the following rank order of potency: (-)-deoxypentazocine > (-)-deoxy-N-benzylnormetazocine > (+)-N-benzylnormetazocine. This rank order approximates that for affinities for sigma 2 receptors but not sigma 1 receptors. It is very unlikely that the effects of the (-)-deoxybenzomorphans were mediated by opiate receptors for the following reasons: (1) consistent with the known requirement for the phenolic hydroxyl group for opiate activity, both (-)-deoxy compounds showed very low affinity for opiate receptors; (2) naloxone (4 micrograms) co-administered with (-)-deoxy-N-benzylnormetazocine failed to reduce its efficacy; (3) both (-)-deoxy compounds failed to produce marked analgesic effects in the tail flick test following systemic injections of 20 mg/kg s.c. These finding suggest that sigma 2 receptors mediate the motor effects of sigma ligands in rats.

The present study describes the characterization of an opioid binding site on membranes prepared from the R1.1 cell line, a murine thymoma. Specific (-)[3H]bremazocine binding was saturable, stereoselective, and limited to a single high affinity binding site with a Kd value of 15.2 +/- 1.6 pM and a Bmax value of 54.8 +/- 6.0 fmol/mg of protein. The kappa-selective alkaloids and dynorphin peptides inhibited (-)[3H]bremazocine binding with Ki values of less than 1 nM, in contrast to mu- and delta-selective ligands. The high affinity of this site for alpha-neo-endorphin and U50,488 suggests that this kappa opioid binding site resembles the kappa 1b subtype. NaCl, as well as other mono- and divalent cations, inhibited (-)[3H]bremazocine binding. In the presence of NaCl, the nucleotides GTP, GDP, and the nonhydrolyzable analog guanylyl-5'-imidodiphosphate (Gpp(NH)p) also decreased (-)[3H]bremazocine binding, suggesting that this kappa opioid binding site is coupled to a G-protein. In summary, R1.1 cells possess a single high affinity kappa opioid receptor that shares many properties with brain kappa 1b opioid receptors.

Opioid receptors are currently classified as mu, delta, and kappa types, but various subtypes have also been proposed. We have investigated whether subtypes exist by using [3H]bremazocine. [3H]Bremazocine binds to twice as many naloxone-sensitive sites as other nonselective opioid agonists, as shown in four membrane types that have very different ratios of mu, delta, and kappa receptor types. [3H]Bremazocine binding is completely inhibited by an excess (in unlabeled form) of other opioid ligands, with Hill coefficients of 0.8-0.95. These paradoxes can be explained if there are high- and low-affinity states of the mu, delta, and kappa receptors and bremazocine binds with similar affinities to both states. We propose that these states are the guanine nucleotide-binding protein (G-protein)-coupled form and the uncoupled form of each receptor. As evidence for this proposal, the [3H]bremazocine binding suffered little or no loss with G-protein-uncoupling treatments, whereas binding of other opioid agonists was fully sensitive. We conclude that [3H]bremazocine offers a tool for the measurement of the total pools of coupled and uncoupled opioid receptors and that much of the previous characterization of opioid receptor subtypes reflects, instead, a significant pool of G-protein-uncoupled opioid receptors.

Previous experiments resolved four kappa binding sites in guinea pig brain termed kappa 1a, kappa 1b, and kappa 2b. The present study was undertaken to examine the occurrence of kappa receptor subtypes in rat and human brain. [3H]U69,593 and [3H]bremazocine were used to label kappa 1 and kappa 2 binding sites, respectively, present in brain membranes depleted of mu and delta binding sites by pretreatment with the irreversible ligands, BIT and FIT. Low levels of [3H]U69,593 binding precluded a detailed quantitative study of kappa 1 binding sites in these species. Quantitative examination of [3H]bremazocine binding resolved two kappa 2 binding sites in both rat and human brain whose ligand selectivity patterns differed from that of the guinea pig. These observations suggest that there may be considerable variation in the ligand recognition site of kappa receptor subtypes among mammalian species.

Unlabeled fluoro- and iodo-substituted ligands exhibiting very high affinity and selectivity for sigma receptors were synthesized based on three different structural classes of sigma receptor ligands. These compounds were evaluated for sigma receptor affinity and specificity in order to assess their potential as PET/SPECT imaging agents. Thus, (+)- and (-)-N-(5-fluoro-1-pentyl)normetazocines [(+)- and (-)-4] based on the (+)-benzomorphan class of sigma ligands were synthesized via N-alkylation of optically pure (+)- and (-)-normetazocine with 5-[(methylsulfonyl)oxy]-1-pentyl fluoride (11). (+)- and (-)-4 displaced [3H](+)-3-PPP with Ki values of 0.29 and 73.6 nM and [3H](+)-pentazocine with Ki values of 10.5 and 38.9 nM, respectively. The second class of PET/SPECT ligands was based upon the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class of sigma ligands; N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-(3-fluoro-1-propyl)-2-(1- pyrrolidinyl)ethylamine (5) was obtained via N-alkylation of N-[2-(3,4-dichlorophenyl)-1-ethyl]-2-(1-pyrrolidinyl)ethylamine (14) with 3-fluoropropyl p-toluenesulfonate. 5 exhibited Ki values of 4.22 and 5.07 nM for displacement of [3H](+)-3-PPP and [3H](+)-pentazocine, respectively, comparable with the parent N-propyl compound. Attempts to synthesize N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-[3- [(methylsulfonyl)oxy]-1-propyl]-2-(1-pyrrolidinyl)ethylamine (26), a precursor to 5 that could conceivably be converted to [18F]-5 by treatment with 18F-, proved unsuccessful. The sequence of regioselective nitration, catalytic hydrogenation, and diazotization followed by NaI quench of N-[2-(3,4-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (2) afforded the iodinated ethylenediamine N-[2-(2-iodo-4,5-dichlorophenyl)-1-ethyl]-N-methyl-2-(1- pyrrolidinyl)ethylamine (8), a potential SPECT ligand for sigma receptors. This compound showed an affinity of 0.54 nM ([3H](+)-3-PPP) comparable with the parent compound 2 (Ki = 0.34 nM, [3H](+)-3-PPP). Ligand 8 exhibited a similar potency against [3H](+)-pentazocine. The third class of high-affinity sigma receptor ligands was rationalized based on rearrangement of the bonds in ethylenediamine 2 to give 1-[2-(3,4-dichlorophenyl)-1-ethyl]-4-(1-propyl)piperazine (3). This compound exhibited very high affinity (Ki = 0.31 nM, [3H](+)-3-PPP) and selectivity for sigma receptors.(ABSTRACT TRUNCATED AT 400 WORDS)