Ligand-induced changes in surface mu-opioid receptor number: relationship to G protein activation?

In this study, we explored the relationship between regulation of surface mu-opioid receptor number, ligand-induced G protein activation (measured by [(35)]S]guanosine-5'-O-(3-thio)triphosphate (GTPgammaS) binding) and second messenger signaling (measured by the inhibition of cAMP accumulation). Etorphine and two isomers of cis-beta-hydroxy-3-methylfentanyl (RTI-1a and RTI-1b), which were full agonists for G protein activation and signaling, caused approximately a 50% loss of surface receptors after 1 h of treatment. Fentanyl and morphine were full agonists for inhibiting cAMP accumulation and partial agonists for stimulating [(35)S]GTPgammaS binding and internalization. Although both agonists were approximately 80% as efficacious as etorphine in stimulating [(35)S]GTPgammaS binding, fentanyl induced a 35% loss of surface receptors, whereas morphine only caused a 10% loss. Additionally, both long- and short-term treatment with the opioid antagonist naloxone caused increases in surface receptors. Unexpectedly, the weak partial agonists buprenorphine and one isomer of cis-beta-hydroxy-3-methylfentanyl (RTI-1d) also were found to cause an increase in surface receptors. Treatment with pertussis toxin (PTX) diminished agonist-induced loss of surface receptors. Furthermore, the abilities of morphine and fentanyl to cause internalization were more impaired after PTX treatment than that of etorphine. PTX treatment also significantly enhanced the increase in surface receptor number caused by 18-h treatment with naloxone and buprenorphine. The results of this study suggest that disruption of G protein coupling by PTX treatment affects ligand-regulated mu-receptor trafficking and that partial agonists for signaling can vary greatly in the ability to regulate the number of surface mu-opioid receptors.

Opioid peptide receptor studies, 11: involvement of Tyr148, Trp318 and His319 of the rat mu-opioid receptor in binding of mu-selective ligands.

Previous data obtained with the cloned rat mu opioid receptor demonstrated that the "super-potent" opiates, ohmefentanyl (RTI-4614-4) and its four enantiomers, differ in binding affinity, potency, efficacy, and intrinsic efficacy. Molecular modeling (Tang et al., 1996) of fentanyl derivatives binding to the mu receptor suggests that Asp147, Tyr148, Trp318, and His319 are important residues for binding. According to this model, Asp147 interacts with the positively charged opiate agonist to form potent electrostatic and hydrogen-bonding interactions. In this study, the role of weak electrostatic and hydrogen-bonding "pi-pi" interactions of the O atom of the carbonyl group and the phenyl ring structures of RTI-4614-4 and its four enantiomers with residues Tyr148, Trp318, and His319 were explored via site-directed mutagenesis. Tyr148 (in transmembrane helix 3 {TMH3}), Trp318 (TMH7), and His319 (TMH7) were individually replaced with phenylalanine or alanine. Receptors transiently expressed in COS-7 cells were labeled with [125I]IOXY according to published procedures. Mutation of Tyr148 to phenylalanine reduced the binding affinities of some mu-selective agonists (2-7 fold) but did not alter the affinities of DAMGO, naloxone, and the non-selective opiates etorphine and buprenorphine. In contrast, this mutation significantly increased the binding affinities (decreased the Kd values) of [D-Ala2,D-Leu5]enkephalin, IOXY, and dermorphin. Mutation of Trp318 decreased opioid receptor binding to almost undetectable levels. Substitution of alanine for His319 significantly reduced binding affinities for the opioid ligands tested (1.3- to 48-fold), but did not alter the affinities of naloxone and bremazocine. These results indicate the importance of Tyrl48 and His319 for the binding of fentanyl derivatives to the mu receptor. Functional studies using the mutant receptors will provide additional insight into the mechanism of action of RTI-4614-4 and its four enantiomers.

Opioid peptide receptor studies. 7. The methylfentanyl congener RTI-4614-4 and its four enantiomers bind to different domains of the rat mu opioid receptor.

Mutational analysis of opioid receptors supports the hypothesis that dissimilar receptor domains contribute to the binding affinity of different ligands. To determine whether enantiomeric ligands can serve to distinguish between different binding pockets (which focuses the analysis on asymmetric structural factors while avoiding confounding changes in physiochemical characteristics), we analyzed the binding of the 3-methylfentanyl congeners RTI-4614-4 [(+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl)], its four stereoisomers [(2S,3R,4S)-1a, (2R,3R,4S)-1b, (2R,3S,4R)-1c, and (2S,3S,4R)-1d], and other mu agonists with cloned rat mu opioid receptors stably expressed in HEK-293 cells and mu/kappa receptor chimeras. Chimera III (kappa[aminoacids 1-141]/mu[aminoacids 151-398]), chimera IV (mu[aminoacids 1-150]/kappa[aminoacids 142-380]), and chimera XII (kappa[aminoacids 1-262]/mu[aminoacids 269-398]) bound [(125)I]IOXY (6beta-iodo-3,14-dihydoxy-17-cyclopropylmethyl-4,5alpha++ +-epoxymorphinan) with high affinities. The Ki values of 1a, 1b, 1c, and 1d at the wild-type mu receptor were 0.55 nM, 0.66 nM, 124 nM, and 59.2 nM, respectively. When the region from the N terminal to the start of the transmembrane helix 3 (TMH3) of the mu receptor was substituted by that of the kappa receptor (chimera III), the Ki value of 1b was increased (relative to the mu receptor) 590-fold compared to a 73-fold increase for 1a. When this portion of the kappa receptor was replaced by that of the mu receptor (chimera IV), the loss of affinity was not as great: 11.7-fold for 1a and 58.5-fold for 1b. Replacement of the middle of the third intracellular loop and third extracellular loop (e3) of the kappa receptor with that of the mu receptor (chimera XII) lowered (relative to their Ki values at the kappa receptor) the Ki values of [D-Ala2,D-Leu5]enkephalin and [D-Ala2-MePhe4,Gly-ol5]enkephalin to a much greater extent than the Ki values of the isomers. The kappa/chimera XII shift was greater for isomers 1c and 1d than for 1b and 1a. Viewed collectively, these data suggest that the region from the N terminal to the start of the TMH3 of the mu opioid receptor determines the binding affinity of RTI-4614-4 and its isomers and that the e3 loop also plays a major role in determining the binding affinity of mu agonist peptides. These data also show that the stereoisomers of RTI-4614-4 probably bind to different domains of the mu receptor and suggest that manipulation of stereochemistry may be a useful tool for designing domain-specific ligands.

X-ray structure of Tyr-D-Tic-Phe-Phe-NH2 (D-TIPP-NH2), a highly potent mu-receptor selective opioid agonist. Comparison with proposed model structures.

Tyr-D-Tic-Phe-Phe-NH2 (D-TIPP), a linear tetrapeptide containing the conformationally restricted Tic residue (tetrahydroisoquinoline-3-carboxylic acid), is an opioid agonist which exhibits high affinity and selectivity for the mu-receptor. Its conformational features have been studied using a combination, a solid-state (X-ray) and modeling (molecular mechanics and Monte Carlo simulations) methods. The results of the X-ray study showed two distinct conformers for D-TIPP, with the main differences lying in the orientation of the Tyr side-chain and the presence of both D-Tic(+) and D-Tic(-) conformations for the D-Tic residue. The peptide backbone is folded and stabilized by the formation of one intramolecular hydrogen bond. The modeling results also indicated a folded backbone for the peptide and both cis and trans conformers for the D-Tic residue are found in the lowest-energy structures. Comparison of the X-ray and modeling results shows many similarities especially around the D-Tic residue.

Enantiomers of diastereomeric cis-N-[1-(2-hydroxy-2-phenylethyl)- 3-methyl-4-piperidyl]-N-phenylpropanamides: synthesis, X-ray analysis, and biological activities.

(+/-)-cis-N-[1-(2-Hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide (1) is a mixture of four stereoisomers [(2S,3R,4S)-1a, (2R,3R,4S)-1b, (2R,3S,4R)-1c, and (2S,3S,4R)-1d], which together constitute two diastereoisomeric pairs of optical isomers. These four stereoisomers were prepared from optically active intermediates of known absolute configuration by procedures which had no effect on the configurations of the piperidine 3- and 4-carbons. The configuration of the phenylethyl 2-carbon in the final products was determined by X-ray analysis of (2S,3S,4R)-1d. A 1H NMR comparison of the final products to ohmefentanyl established that the racemic pair previously known as ohmefentanyl was a mixture of (2S,3R,4S)-1a and (2R,3S,4R)-1c. The individual activities of 1a, 1b, 1c, and 1d were evaluated in a variety of binding and pharmacological assays. The binding data revealed that isomers 1b and 1c had the highest affinity and selectivity for the mu site labeled with [3H]DAMGO. In contrast, the four isomers displaced [3H]etorphine in the order 1a approximately 1b > 1c approximately 1d. Evaluation of the four isomers on the mouse vas deferens (MVD) preparation revealed a potency order of 1a > 1b > 1c > 1d with concentrations of 1a and 1b in the femtomolar range causing inhibition. Experiments using the antagonists naltrexone (mu), ICI 174864 (delta), and norbinaltorphimine (kappa) demonstrated that the effects of 1a were mediated largely by the mu receptor while both delta and kappa agonist effects contributed to the actions of 1b and 1c. Isomer 1d acted as a weak mu antagonist in the MVD preparation. The same potency order was observed in a mouse analgesic assay and a rhesus monkey single dose suppression study. From the latter study the potency of 1a was estimated to be 20,000-50,000 times that of morphine, making this isomer one of the most potent opiates known. In the rhesus monkey study, isomer 1d failed to substitute for morphine and seemed to exacerbate withdrawal at doses of 0.6, 3.0, and 6.0 mg/kg. On the basis of the mouse data, isomer 1a was 21,000 times more potent than 1d, whereas isomers 1b and 1c were similar in their opiate activity in vivo. Using the optical isomers of cis-3-methylfentanyl as reference compounds, we analyzed the effects on the pharmacological activities of introducing a phenylethyl 2-hydroxyl group into the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)

Receptor reserve and affinity of mu opioid agonists in mouse antinociception: correlation with receptor binding.

In order to quantitate the extent to which opioid agonist potencies obtained in behavioral assays are determined by the apparent in vivo affinity and efficacy of the agonist, the antinociceptive effects of the mu opioid agonists morphine, fentanyl, etonitazene, and NIH 10741 were assessed before and after administration of the insurmountable mu opioid antagonist clocinnamox (CCAM) in a 55 degrees C warm-water tail withdrawal test in Swiss albino mice. Under control conditions, all four mu opioid agonists produced a full antinociceptive response with the following ED50 values (in mg/kg): morphine, 12; fentanyl, 0.47; etonitazene, 0.039; NIH 10741, 0.0051. Analysis of CCAM's effects according to Black and Leff gave the following agonist efficacy or tau values: Morphine, 4; fentanyl 15, etonitazene, 7; and NIH 10741, 59. The respective KA values were (in mg/kg): morphine, 29; fentanyl, 7.3; etonitazene, 0.22; and NIH 10741, 0.30. The major determinant of the experimentally observed ED50 values seemed to be the apparent in vivo affinity of the respective agonist and not its efficacy. KA values (expressed as mol/kg) correlated with the Ki values (in mol/l) obtained with [3H]DAMGO radioligand binding (r = 0.96 for pKA vs. pKi), although being on average 11,000-fold higher. Values for q, the available receptor fraction as determined in the behavioral experiments, correlated strongly (r = 0.96) with the q values determined by ex vivo [3H]DAMGO- and [3H]naltrexone equilibrium binding (i.e., Bmax,clocinnamox/Bmax,control), the relationship approaching unity.

Stereochemical requirements for pseudoirreversible inhibition of opioid mu receptor binding by the 3-methylfentanyl congeners, RTI-46144 and its enantiomers: evidence for different binding domains.

Fentanyl and its congeners are of interest not only because of their clinical applications, but also because certain members of this series of opioid analgesics exhibit unique properties, such as acting as pseudoirreversible inhibitors of mu receptor binding, both in vitro and in vivo. Previous studies showed that pretreatment of membranes with (+)-cis-3-methylfentanyl resulted in a lower affinity interaction of [3H]ohmefentanyl with the mu binding site, as well as an increased dissociation rate. The present study was undertaken to determine the stereochemical requirements for pseudoirreversible inhibition of mu receptor binding using the methylfentanyl congeners, (+-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl (RTI-4614-4) and its four resolved enantiomers. AR configuration of the 2-hydroxy group was essential for high affinity binding and pseudoirreversible inhibition. The two enantiomers with this configuration, 1b((2R,3R,4S)-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide oxolate) and 1c 1c ((2R,3S,4R)-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]-N- phenylpropanamide HCl), acted as pseudoirreversible inhibitors of the mu receptor as labeled with [3H][D-Ala2-MePhe4,Gly-ol5]enkaphalin, [3H]fentanyl or [3H]etorphine. RTI-4614-4, 1b, and 1c decreased the Bmax of [3H][D-Ala2-MaePhe4,Gly-ol5]enkepalin binding sites without altering the dissociation rate. These drugs had a lesser effect on steady-state [3H]fentanyl and [3H]etorphine binding but did produce statistically significant changes in the parameters of the two-component dissociation model, which accurately described the dissociation of these [3H]ligands. Viewed collectively, these data indicate that the mechanism of the pseudoirreversible inhibition appears to depend on the radioligand used to label the mu receptor. To explain these data, a pseudoallosteric model is proposed that postulates that certain mu ligands bind to different domains of the drug recognition site of the mu receptor and that the prebinding of pseudoirreversible inhibitors to the recognition site changes the domains available to a radioligand, leading to alterations in steady-state binding levels and dissociation kinetics.

Enantiomeric N-substituted N-normetazocines: a comparative study of affinities at sigma, PCP, and mu opioid receptors.

The optical antipodes of N-allyl-N-normetazocine (2; SKF 10047, NANM) were the original compounds used for the classification of the sigma receptor as distinct from other receptors such as the PCP (NMDA), opioid, and dopamine receptors. Later studies showed that (+)-N-(dimethylallyl)-N-normetazocine [(+)-4, (+)-pentazocine] was more potent and selective for the sigma receptor. In order to gain additional structure-activity relationship information, several N-substituted N-normetazocine analogs were prepared and evaluated for their sigma-1 ([3H]-(+)-3-PPP or [3H]-(+)-pentazocine), PCP ([3H]TCP), and mu opioid ([3H]DAMGO) receptor binding affinities. (+)-N-Benzyl-N-normetazocine [(+)-10)] possessed subnanomolar affinities for the sigma site, Ki = 0.67. The analog (+)-10 showed greater than 14,000- and 2400-fold selectivity, respectively, for the sigma receptor relative to the PCP and mu opioid receptors. The N-substituted N-normetazocines were enantioselective for the sigma site. The (+)-N-benzyl analog, (+)-10, showed a 55-fold selectivity relative to (-)-10. Analysis of the data also revealed that (+)-normetazocine [(+)-1] [Ki = 30 nM] possessed the highest affinity for the PCP receptor. However, (+)-metazocine [(+)-5] (Ki = 41 nM) was the most selective compound for the PCP receptor relative to the sigma (51-fold) and mu opioid (greater than 200-fold) sites.

RTI-4614-4: an analog of (+)-cis-3-methylfentanyl with a 27,000-fold binding selectivity for mu versus delta opioid binding sites.

The objective of this study was to determine the binding affinities of (+/-)-cis-N-[1-(2-hydroxy-2-phenylethyl)-3-methyl-4-piperidyl]- N-phenylpropanamide-HCI (RTI-4614-4), which is an analog of (+)-cis-3-methylfentanyl for opioid receptor subtypes. The Ki values (nM) of this agent for opioid receptor subtypes were as follows: mu (0.0055), delta (148), kappa 1 (84.8), kappa 2a (2275), and kappa 2b (22.3). The selectivity of this agent for the mu binding site was 27,000 vs. the delta binding site, 15,400 vs. the kappa 1 binding site, 413,700 vs the kappa 2a and 4,054 vs the kappa 2b binding site. In contrast, two other fentanyl analogs, N-(2-(4-methylpyridinyl))-N-(1-phenethyl-4-piperidinyl) 2-furamide and N-(2-pyrazinyl)-N-(1-phenethyl-4-piperdinyl)2-furamide had considerably higher Ki values at, and were less selective for, the mu binding site. Since RTI-4614-4 is composed of a mixture of four stereoisomers, the resolution of these isomers should permit identification of an extremely potent and selective agent for the opioid mu receptor.

Synthesis and anticonvulsant screening of 3,3-diphenyl-2-pyrrolidone derivatives.

Six derivatives of 3,3-diphenyl-2-pyrrolidone were synthesized and screened for anticonvulsant activity. The synthetic route involved a mono-N-demethylation of an intermediate N,N-dimethylaminonitrile with methyl chloroformate followed by cleavage of the carbamate group. Of the six derivatives, (+/-)-2-imino-1,5-dimethyl-3,3-diphenylpyrrolidine hydrochloride was effective in protecting mice against maximal electroshock (MES) -induced seizures at a 30-mg/kg dose level.

Phencyclidine metabolism: resolution, structure, and biological activity of the isomers of the hydroxy metabolite, 4-phenyl-4-(1-piperidinyl)cyclohexanol.

One of the major biotransformation pathways in the metabolism of phencyclidine is hydroxylation at C-4 of the cyclohexane ring to give 4-phenyl-4-(1-piperidinyl)cyclohexanol (1). Since the latter compound can exist as cis and trans isomers and the synthetic mixture has been reported to be biologically active, it was of interest to separate the isomers, test them for biological activity, and determine their ratio as metabolic products of phencyclidine. The synthetic mixture of 1 was separated by TLC and the individual isomers were characterized by 13C and 1H NMR and MS analyses. Preliminary testing of the isomers in the mouse rotarod assay indicates that the trans isomer (1b) is only slightly more active then the cis isomer (1a). Both isomers produced seizure activity and lethality at doses required to produce maximal ataxia.

Carbon-13 nuclear magnetic resonance identification of 2-hydroxy-3-O-methyl-6beta-naltrexol as a minor naltrexone metabolite.

A carbon-13 nuclear magnetic resonance comparison of synthetic and biological samples was used to identify unequivocally 2-hydroxy-3-O-methyl-6beta-naltrexol as a minor naltrexone metabolite in humans. This study points up the increasing usefulness of natural abundance carbon-13 nuclear magnetic resonance techniques in metabolism studies.

The identification and measurement of two new metabolites of naltrexone in human urine.

Two metabolites of naltrexone were identified in human and rat urine following drug administration. The metabolites, 2-hydroxy-3-0-methylnatrexone and 2-hydroxy-3-0-methyl-6 beta-naltrexol, were identified by comparison of their respective gas chromatographic retention times and methane chemical ionization spectral patterns with authentic standards. The standards were obtained by chemical synthesis from naltrexone. Urinary excretion of the two metabolites was measured by single ion recording of the pseudomolecular ions of the metabolites and an internal standard. Excretion data was collected from 5 human subjects over a period of 5 days following a single dose of naltrexone. The metabolites, 2-hydroxy-3-0-methylnatrexone and 2-hydroxy-3-0-methyl-6 beta-natrexol were found to account for an average (+/- standard error) of 0.45 +/- 0.08% and 4.6 +/- 2.1% of the administered dose, respectively. An unusually long half-life of elimination of the latter metabolite (mean of 20.2 +/- 1.8 hr) was observed. Acid-hydrolysis of the samples failed to produce a significant increase of either metabolite indicating that the major protion of these metabolites is excreted in the free form.

Carbon-13 nuclear magnetic resonance study of the alpha- and beta- isomers of methadol and acetylmethadol hydrochlorides.

The 13C-NMR spectra reported in these studies give direct evidence for the presence of two contributing conformers for alpha-methadol hydrochloride, alpha-acetylmethadol hydrochloride and beta-acetylmethadol hydrochloride. Indirect evidence is also available for the presence of more than one conformer for beta-methadol hydrochloride. However, in order to be more descriptive about the structures of the conformers, it is necessary to obtain 13C-NMR spectra that have a higher degree of resolution than is available from our present NMR system. Such systems are available, and plans are currently in progress to obtain 13C-NMR spectra or these compounds at high enough magnetic fields and low enough temperatures to give us the necessary data.