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

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

Exploration of universal cysteines in the binding sites of three opioid receptor subtypes by disulfide-bonding affinity labeling with chemically activated thiol-containing dynorphin A analogs.

A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of mu and delta opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys(Npys)-containing analogs of dynorphin A, namely, [D-Ala2, Cys(Npys)8]dynorphin A-(1-9) amide (1) and [D-Ala2, Cys(Npys)12]dynorphin A-(1-13) amide (2). When rat (mu and delta) or guinea pig (kappa) brain membranes were incubated with these Cys(Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (mu), deltorphin II (delta), and U-69593 (kappa), the number of receptors decreased sharply, depending upon the concentrations of these Cys(Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label mu receptors (EC50 = 27-33 nM), but also label delta receptors fairly well (160-180 nM). However, for kappa receptors they showed drastically different potencies as to affinity labeling; i.e., EC50 = 210 nM for analog 1, but 10,000 nM for analog 2. Analog 2 labeled kappa receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in mu, delta, and kappa receptors, whereas analog 2 only labels the Cys residues conserved in mu and delta receptors.     

Biotinylated human beta-endorphins as probes for the opioid receptor

The reaction of human beta-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH+), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human beta-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Tyr-1. Three HPLC fractions were isolated for receptor binding studies with monobiotinylation of Lys-9 (B1 beta and B1X beta; X = C6 spacer arm), Lys-19 (B1 gamma), and a mixture of Lys-24, Lys-28, and Lys-29 derivatives (B1 alpha, BX1 alpha). All derivatives displayed tight binding to avidin, and no dissociation from avidin was detectable over several hours at 0 degrees C for the derivatives (BX1 alpha) tested. IC50 values for binding to mu and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human beta-endorphin (IC50,mu = 1.5 nM, IC50,delta = 1.3 nM). Association with avidin decreased opioid receptor affinities for the C6 spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to mu and delta sites for derivatives biotinylated at the alpha-helical part of the molecule (Lys-19, -24, -28, and -29). Thus, when bound to avidin, the biotinylated human beta-endorphin derivatives with spacer arm (BX1 alpha), substituted near the carboxyl terminal (Lys-24, -28, and -29), displayed mu binding affinities equal to and delta binding affinities only four times lower than underivatized human beta-endorphin. Biotinylated human beta-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were considerably reduced when derivatives were bound to avidin. The ability of biotinylated human beta-endorphin to cross-link the mu and delta opioid receptors to avidin allows application of the biotin-avidin system as a molecular probe of the opioid receptor.     

Mixed kappa agonists and mu agonists/antagonists as potential pharmacotherapeutics for cocaine abuse: synthesis and opioid receptor binding affinity of N-substituted derivatives of morphinan

A series of new N-substituted derivatives of morphinan was synthesized and their binding affinity for the three opioid receptors (mu, delta, and kappa) was determined. A paradoxical effect of N-propargyl (MCL-117) and N-(3-iodoprop-(2E)-enyl) (MCL-118) substituents on the binding affinities for the mu and kappa opioid receptors was observed. All of these novel derivatives showed a preference for the mu and kappa versus delta binding.     

Pharmacological effects of naltriben as a ligand for opioid mu and kappa receptors in rat cerebral cortex

Naltriben (NTB) has been used to differentiate the subtypes of delta opioid receptors, delta1 and delta2. However, there is considerable evidence suggesting that NTB may act on other types of opioid receptors too. We examined the effects of NTB on the specific binding of radiolabeled ligands for opioid mu and kappa2 receptors, and the effects on the release of [3H]norepinephrine ([3H]NE) in rat cerebral cortex slices. NTB displaced the specific binding of [3H]DAMGO with Ki value of 19.79 +/- 1.12 nM in rat cortex membranes. Specific binding of [3H]diprenorphine ([3H]DIP) was inhibited by NTB with Ki value of 82.75 +/- 6.32 nM in the presence of DAMGO and DPDPE. High K+ (15 mM)-stimulated release of [3H]NE was attenuated by DAMGO in rat cerebral cortex slices. NTB (30 nM) shifted the dose-response curve of DAMGO to the right and attenuated the maximal effect. In the meantime, NTB inhibited high K+-stimulated [3H]NE release at concentrations above 100 nM. The inhibitory effect of NTB was not attenuated by CTAP (10 nM) and naloxone (3 nM) but by higher concentration of naloxone (30 nM), nor-BNI (300 nM) and bremazocine (3 nM). These results indicate that NTB, depending on the dosage, could acts not only as an antagonist at delta but also as a noncompetitive antagonist for mu receptors, and as an agonist for kappa2 receptors in rat cerebral cortex.     

High-affinity carbamate analogues of morphinan at opioid receptors

A series of carbamate analogues were synthesized from levorphanol (1a), cyclorphan (2a) or butorphan (3a) and evaluated in vitro for their binding affinity at mu, delta, and kappa opioid receptors. Functional activities of these compounds were measured in the [(35)S]GTPgammaS binding assay. Phenyl carbamate derivatives 2d and 3d showed the highest binding affinity for kappa receptor (K(i)=0.046 and 0.051 nM) and for mu receptor (K(i)=0.11 and 0.12 nM). Compound 1c showed the highest mu selectivity. The preliminary assay for agonist and antagonist properties of these ligands in stimulating [(35)S]GTPgammaS binding mediated by the kappa opioid receptor illustrated that all of these ligands were kappa agonists. At the mu receptor, compounds 1b, 1c, 2b, and 3b were agonists, while compounds 2c-e and 3c-e were mu agonists/antagonists.     

Understanding the structural requirements of 4-anilidopiperidine analogues for biological activities at mu and delta opioid receptors

New 4-anilidopiperidine analogues in which the phenethyl group of fentanyl was replaced by several aromatic ring-contained amino acids (or acids) were synthesized to study the biological effect of the substituents on mu and delta opioid receptor interactions. These analogues showed broad (47 nM-76 microM) but selective (up to 17-fold) binding affinities at the mu opioid receptor over the delta opioid receptor, as predicted from the message-address concept.     

A very high affinity opioid binding site in rat brain: demonstration by computer modeling

We present substantial new evidence for at least four distinct types of opioid receptors in rat brain, using quantitative ligand binding studies and mathematical modeling. Three of these binding sites are consistent with the well established "mu", "delta" and "kappa" receptors. The fourth has two distinctive features: 1) extremely high affinity (dissociation constant less than 1 nM); 2) almost complete lack of specificity for the classical "delta" or "mu" selective ligands. These properties are consistent with the putative "mu1" receptor described by Pasternak and coworkers.     

In-vitro investigation of oxazol and urea analogues of morphinan at opioid receptors

A series of 2-amino-oxazole (7 and 8) analogs and 2-one-oxazole analogs (9 and 10) were synthesized from cyclorphan (1) or butorphan (2) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors and compared with their 2-aminothiozole analogs 5 and 6. Ligands 7-10 showed decreased affinities at kappa and mu receptors. Urea analogs (11-14) were also prepared from 2-aminocyclorphan (3) or 2-aminobutorphan (4) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors. The urea derived opioids retained their affinities at mu receptors while showing increased affinities at delta receptors and decreased affinities at kappa receptors. Functional activities of these compounds were measured in the [35S]GTPgammaS binding assay, illustrating that all of these ligands were kappa agonists. At the mu receptor, compounds 11 and 12 were mu agonist/antagonists.     

Synthesis and biological activity of 8beta-substituted hydrocodone indole and hydromorphone indole derivatives.

The 8beta-unsubstituted and substituted analogues of hydrocodone indole and hydromorphone indole were synthesized and their binding affinities to opioid receptors were determined. Introduction of an 8beta-methyl group into the indolomorphinan nucleus increased affinity at all opioid receptors. 6,7-Dehydro-4,5alpha-epoxy-8beta-methyl-6,7,2',3'-indolomorphinan (9) was found to be a delta antagonist with subnanomolar affinity (0.7 nM) for the delta-opioid receptor, and to have good delta-selectivity (mu/delta=322).     

16-Me cyprenorphine (RX 8008M): a potent opioid antagonist with some delta selectivity

16-Me cyprenorphine (RX 8008M) has been investigated in a number of isolated tissue preparations and found to be a pure opioid antagonist with Ke values at the delta, mu and kappa receptors of 0.73, 1.77 and 59.6 nM respectively. Comparisons of the mu, kappa and delta Ke values with a number of other antagonists in the mouse vas deferens have been made and show that the 16-Me substituent results in a marked enhancement of delta activity, making RX 8008M the most selective non-peptide delta antagonist available at the present time.     

Synthesis and opiate receptor binding properties of 17-methyl-6,7-dehydro-3,14-dihydroxy-4,5alpha-epoxy-6,7:4',5'-pyrimidin omorphinans

A class of opioid receptor active derivatives of oxymorphone has been synthesized using a common enaminone intermediate. The derivatives have heterocyclic groups fused to the 6,7-positions of the morphinan system and all were synthesized in high yield. A pyrazolo derivative is an agonist for the mu and delta receptors and an antagonist for the kappa receptor.     

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.     

8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one derivatives as orphanin FQ receptor agonists.

A series of 8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan+ ++-4-one derivatives 1 was studied with respect to the binding affinity for the orphanin FQ (OFQ) and opioid (mu, kappa, delta) receptors. The influence of stereochemistry as well as the substitution pattern of the phenyl-ring in position 1 on the affinity for the orphanin FQ receptor and selectivity to opioid (mu, kappa, delta) receptors is discussed. The most interesting compound 1c was tested for its anxiolytic-like properties in vivo.     

Characterization of opioid receptors on isolated canine gallbladder smooth muscle cells

Smooth muscle cells were isolated from the fundus of the canine gallbladder and examined for the presence of opioid receptors. The cells contracted in a concentration-dependent manner in response to three opioid peptides (Met-enkephalin, dynorphin1-13 and Leu-enkephalin), which are known derivatives of opioid precursors present in myenteric neurons of the gut. The order of potency was Met-enkephalin greater than dynorphin1-13 greater than Leu-enkephalin. The contractile response to opioid agonists was selectively inhibited by opioid antagonists (naloxone and Mr2266) but not by muscarinic, CCK/gastrin or tachykinin antagonists. Equivalent responses to the three opioid peptides exhibited differential sensitivity to preferential antagonists of mu (naloxone) and kappa (Mr2266) opioid receptors consistent with the presence of the three main types of opioid receptors (mu, delta and kappa) on canine gallbladder muscle cells.     

Design and synthesis of opioidmimetics containing 2',6'-dimethyl-L-tyrosine and a pyrazinone-ring platform

Twelve 2',6'-dimethyl-L-tyrosine (Dmt) analogues linked to a pyrazinone platform were synthesized as 3- or 6-[H-Dmt-NH(CH(2))(n)],3- or 6-R-2(1H)-pyrazinone (n=1-4). 3-[H-Dmt-NH-(CH(2))(4)]-6-beta-phenethyl-5-methyl-2(1H)-pyrazinone 11 bound to mu-opioid receptors with high affinity (K(i)mu=0.13 nM; K(i)delta/K(i)mu=447) with mu-agonism (GPI IC(50)=15.9 nM) and weak delta-antagonism (MVD pA(2)=6.35). Key factors affecting opioid affinity and functional bioactivity are the length of the aminoalkyl chain linked to Dmt and the nature of the R residue. These data present a simplified method for the formation of pyrazinone opioidmimetics and new lead compounds.     

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.     

Delta opioidmimetic antagonists: prototypes for designing a new generation of ultraselective opioid peptides.

BACKGROUND: Tyr-Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) and Tyr-Tic-Ala were the first peptides with delta opioid antagonist activity lacking Phe, considered essential for opioid activity based on the N-terminal tripeptide sequence (Tyr-D-Xaa-Phe) of amphibian skin opioids. Analogs were then designed to restrain the rotational flexibility of Tyr by the substitution of 2,6-dimethyl-L-tyrosine (Dmt). MATERIALS AND METHODS: Tyr and Dmt peptides were synthesized by solid phase and solution methods using Fmoc technology or condensing Boc-Dmt-OH or Boc-Tyr(But)-OH with H-L-Tic-OBut or H-D-Tic-OBut, respectively. Peptides were purified (> 99%) by HPLC and characteristics determined by 1H-NMR, FAB-MS, melting point, TLC, and amino acid analyses. RESULTS: H-Dmt-Tic-OH had high affinity (Ki delta = 0.022 nM) and extraordinary selectivity (Ki mu/Ki delta = 150,000); H-Dmt-Tic-Ala-OH had a Ki delta = 0.29 nM and delta selectivity = 20,000. Affinity and selectivity increased 8700- and 1000-fold relative to H-Tyr-Tic-OH, respectively. H-Dmt-Tic-OH and H-Dmt-Tic-NH2 fitted one-site receptor binding models (eta = 0.939-0.987), while H-Dmt-Tic-ol, H-Dmt-Tic-Ala-OH and H-Dmt-Tic-Ala-NH2 best fitted two-site models (eta = 0.708-0.801, F 18.9-26.0, p < 0.0001). Amidation increased mu affinity by 10- to 100-fold and acted synergistically with D-Tic2 to reverse selectivity (delta-->mu). Dmt-Tic di- and tripeptides exhibited delta antagonist bioactivity (Ke = 4-66 nM) with mouse vas deferens and lacked agonist mu activity (> 10 microM) in guinea-pig ileum preparations. Dmt-Tic analogs weakly interacted with kappa receptors in the 1 to > 20 microM range. CONCLUSIONS: Dmt-Tic opioidmimetic peptides represent a highly potent class of opioid peptide antagonists with greater potency than the nonopioid delta antagonist naltrindole and have potential application as clinical and therapeutic compounds.     

A new synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) and activity in a calcium mobilization assay

A new chiral synthesis of the ORL-1 antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (2, J-113397) was developed. J-113397 has a K(e)=0.85nM in an ORL-1 calcium mobilization assay and is 89-, 887-, and 227-fold selective for the ORL-1 receptor relative to the mu, delta, and kappa opioid receptors.     

A human neuroblastoma cell line expresses mu and delta opioid receptor sites

A series of neuroblastoma cell lines were screened for the presence of opioid receptor sites with the tracers [3H]diprenorphine (mu, delta, kappa ligand) and [3H]naloxone (mu-selective ligand). One human neuroblastoma cell line, SK-N-SH, displayed avid binding for both tracers. Binding experiments with multiple tracers revealed the presence of both mu and delta sites. These sites were stereospecific, saturable, and proteinaceous in character. Saturation binding experiments provided an estimate of 50,000 mu and 10,000 delta sites/cell. NaCl (100 mM) and guanine nucleotide, guanylyl imidodiphosphate (50 microM), reduced opioid agonist but not antagonist binding to these sites. Etorphine at 1 nM inhibited prostaglandin E1-stimulated cyclic AMP production by approximately 20%, which was reversible by naloxone. The opioid-binding sites on SK-N-SH cells closely resemble the previously reported mu and delta sites in human and rodent brain. Therefore, the SK-N-SH neuroblastoma cell line represents a useful tool to study the molecular functions of opioid receptors.