Synthesis and opioid activity of 2-substituted dynorphin A-(1-13) amide analogues.

A series of 2-substituted dynorphin A-(1-13) amide (Dyn A-(1-13)NH2) analogues was prepared by solid phase peptide synthesis and evaluated for opioid receptor affinities in radioligand binding assays and for opioid activity in the guinea pig ileum (GPI) assay. Amino acid substitution at the 2 position produced marked differences in both opioid receptor affinities and potency in the GPI assay; Ki values for the analogues in the radioligand binding assays and IC50 values in the GPI assay varied over three to four orders of magnitude. The parent peptide, Dyn A-(1-13)NH2, exhibited the greatest affinity and selectivity for kappa receptors and was the most potent peptide examined in the GPI assay. The most important determinant of opioid receptor selectivity and opioid potency for the synthetic analogues was the stereochemistry of the amino acid at the 2 position. Except for [D-Lys2]Dyn A-(1-13)NH2 in the kappa receptor binding assay, the analogues containing a D-amino acid at position 2 were much more potent in all of the assays than their corresponding isomers containing an L-amino acid at this position. The L-amino acid-substituted analogues generally retained some selectivity for kappa opioid receptors. The more potent derivatives with a D-amino acid in position 2, however, preferentially interacted with mu opioid receptors. Introduction of a positively charged amino acid into the 2 position generally decreased opioid receptor affinities and potency in the GPI assay.     

Synthesis and binding characteristics of the highly delta-specific new tritiated opioid peptide, [3H]deltorphin II.

A radiolabelled form of deltorphin II was synthesized by catalytic tritiation using [p-IPhe3]-deltorphin II as a precursor. The ligand labels rat brain membranes with a Kd value of 1.9 nM, and the Bmax was found to be 92 fmol/mg protein. This new tritiated ligand exhibits high affinity for the delta opioid binding site, whereas its binding to the mu type is weak and extremely low for the kappa type. Mu/delta and kappa/delta selectivity ratios were about 900 and 10,000, respectively. The highly delta selective binding properties of this new radioligand suggest that it could serve as an excellent tool for investigating the delta opioid receptors in various species.     

Synthesis and biological evaluation of N-substituted quinolinimides, as potential ligands for in vivo imaging studies of delta-opioid receptors

We report here the syntheses of N-substituted quinolinimide derivatives displaying sufficient affinity and high selectivity for delta-opioid receptors. Among 9-subsituted derivatives, one showed much higher selectivity for the delta receptor in binding assays than the delta antagonist methylnaltrindole (6: Ki = 42 nM; micro/delta and kappa/delta > 238 on rat brain membranes) and antagonist properties. This compound was labeled with carbon-11 (t1/2 = 20.4 min) as a potential radioligand for the noninvasive assessment of delta opioid receptors in vivo with positron emission tomography (PET). A high yielding radiosynthesis of [11C]-6, based on the [11C]methyl introduction on the pyridine moiety by a Stille reaction, was described (radiochemical yield = 60 +/- 10%, specific activities = 0.8 to 1.5 Ci/micromol). The in vivo pharmacological profile in rats indicated that the radiotracer crossed the blood-brain barrier but was not stable and underwent rapid degradation in both plasma and brain. No specific binding was consequently revealed.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

Synthesis and binding characteristics of [3H] H-Tyr-Ticpsi[CH2-NH] Cha-Phe-OH, a highly specific and stable delta-opioid antagonist.

Substitution of the Phe3 aromatic ring in H-Tyr-Ticpsi[CH2-NH]Phe-Phe-OH with cyclohexylalanine (Cha) has been reported to result in a compound, H-Tyr-Ticpsi[CH2-NH]Cha-Phe-OH (TICP[psi]), showing substantially increased delta-opioid antagonist potency and high delta selectivity. TICP[psi] was radiolabeled by catalytic tritiation of its precursor Tyr(3',5'-I2)1TICP[psi]. Binding characteristics of the new tritiated pseudopeptide were determined using the radioligand binding assay in rat brain membranes. On the basis of the results of saturation binding studies performed at 25 degrees C, an equilibrium dissociation constant (Kd) of 0.35 nM and a receptor density (Bmax) of 112 fmol/mg protein were calculated. This new tritiated ligand exhibits high affinity for delta-opioid receptors, whereas its binding to mu and kappa receptors is weak. A study of [H3]TICP[psi] binding displacement by various receptor-selective opioids showed the following rank order of potency: delta > kappa = mu. These receptor binding characteristics of the ligand, together with its high specific radioactivity (41.3 Ci/mmol) and stability, makes it a useful tool for labeling delta-opioid receptors, both in vitro and in vivo.     

Structure-activity relationships of dynorphin analogs substituted in positions 2 and 3

Following up on the observation that the dynorphin analog [Pro(3)]Dyn A(1-11)-NH(2) 2 possesses high affinity and selectivity for the kappa opioid receptor, a number of related peptides were prepared and characterized by radioligand binding and [(35)S]GTPgammaS assays. While incorporation of 2-azetidine carboxylic acid in position 3 led to the equally potent analog 3, the corresponding analog containing piperidine-2-carboxylic acid showed a nearly 90-fold reduction in kappa affinity. Differential preferred bond angles phi in the three building blocks might account for these observations. Compounds 2 and 3 were kappa antagonists with IC(50) values of 380 and 350 nM, respectively. The Sar(3) analog 7 and the Sar(2) analog 8 were kappa agonists, with greater selectivity than Dyn A(1-11)-NH(2) 1. In view of their high kappa affinities (8: K(i) = 1.5 nM; 2: K(i) = 2.4 nM), the new analogs were surprisingly weak kappa agonists or antagonists, e.g., the EC(50) value for the agonist 8 was 280 nM. Different kappa receptor subtypes in binding vs functional assays can not account for these results, since both assays were performed using the same membrane preparation.     

Inhibition of mu and delta opioid receptor ligand binding by the peptide aldehyde protease inhibitor, leupeptin

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.     

Conformationally-flexible benzamide analogues as dopamine D3 and sigma 2 receptor ligands

A series of conformationally-flexible analogues was prepared and their affinities for D2-like dopamine (D2, D3 and D4) were determined using in vitro radioligand binding assays. The results of this structure-activity relationship study identified one compound, 15, that bound with high affinity (K(i) value=2nM) and moderate selectivity (30-fold) for D3 compared to D2 receptors. In addition, this series of compounds were also tested for affinity at sigma1 and sigma2 receptors. We evaluated the affinity of these dopaminergic compounds at sigma receptors because (a) several antipsychotic drugs, which are high affinity antagonists at dopamine D2-like receptors, also bind to sigma receptors and (b) sigma receptors are expressed ubiquitously and at high levels (picomoles per mg proteins). It was observed that a number of analogues displayed high affinity and excellent selectivity for sigma2 versus sigma1 receptors. Consequently, these novel compounds may be useful for characterizing the functional role of sigma2 receptors and for imaging the sigma2 receptor status of tumors in vivo with PET.     

Synthesis of potent and selective dopamine D(4) antagonists as candidate radioligands

A series of dopamine D(4) antagonists was synthesized and evaluated as potential candidates for development as positron emission tomography (PET) radioligands. All new compounds display high affinity and selectivity for the D(4) receptors and compounds 5b, 5d, and 5e were identified as candidates for radioligand development.     

p-Fluoro-hexahydro-sila-difenidol exhibits poor selectivity between M3 and M1 muscarinic receptors

We investigated the potential ability of p-fluoro-hexahydro-sila-difenidol (p-F-HHSiD) to discriminate between M1 and M3 muscarinic receptor subtypes using Chinese hamster ovary cells stably transfected with the genes encoding the two receptors. Both radioligand binding and functional assays were utilized for this purpose. In contrast to initial reports of a 14-fold selectivity of this antagonist for M3 versus M1 receptors, we have detected a qualitatively similar selectivity that was markedly smaller in magnitude.     

Delineation of structural domains involved in the subtype specificity of tachykinin receptors through chimeric formation of substance P/substance K receptors.

The mammalian tachykinin receptors belong to the family of G protein-coupled receptors and consist of the substance P, substance K and neuromedin K receptors (SPR, SKR and NKR). We constructed 14 chimeric receptors in which seven transmembrane segments were sequentially exchanged between the rat SPR and SKR and examined the subtype specificity of the chimeric receptors by radioligand binding and inositol phosphate measurements after transfection into COS cells. All chimeric receptors showed maximum responses in agonist-induced inositol phosphate stimulation. Detailed analysis of five receptors with agonist selectivity similar to SPR indicated that the selectivity is mainly determined by the region extending from transmembrane segment II to the second extracellular loop together with a minor contribution of the extracellular N-terminal portion. This conclusion was more directly confirmed by an additional chimeric formation in which the introduction of the above middle portion of SPR into the corresponding region of SKR conferred a high affinity binding to substance P. The tachykinin receptors can thus be divided into two functional domains: the region covering transmembrane segments V-VII and responsible for fundamental recognition of the common tachykinin sequence; and its preceding portion involved in evoking subtype specificity by interacting with the divergent sequences of the peptides.     

Structure-activity relationships of arodyn, a novel acetylated kappa opioid receptor antagonist.

We previously reported that the novel dynorphin A (Dyn A, Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln) analog arodyn (Ac[Phe(1,2,3),Arg(4),d-Ala(8)]Dyn A-(1-11)NH(2), Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617-5619) is a kappa opioid receptor-selective peptide [K(i)(kappa) = 10 nm, K(i) ratio (kappa/mu/delta) = 1/174/583] which exhibits antagonist activity at kappa opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure-activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d-amino acid substitution in the N-terminal 'message' sequence, NMePhe substitution individually in positions 1-3, and modifications in position 1. The results for the Ala-substituted derivatives indicated that Arg(6) and Arg(7) are the most important residues for arodyn's nanomolar binding affinity for kappa opioid receptors. Ala substitution of the other basic residues (Arg(4), Arg(9) and Lys(11)) resulted in lower decreases in affinity for kappa opioid receptors (three- to fivefold compared with arodyn). Of particular interest, while [Ala(10)]arodyn exhibits similar kappa opioid receptor binding as arodyn, it displays higher kappa vs. mu opioid receptor selectivity [K(i) ratio (kappa/mu) = 1/350] than arodyn because of a twofold loss in affinity at mu opioid receptors. Surprisingly, the Tyr(1) analog exhibits a sevenfold decrease in kappa opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr(1)]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing kappa opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe(1)]arodyn which exhibits high affinity [K(i)(kappa) = 4.56 nm] and exceptional selectivity for kappa opioid receptors [K(i) ratio (kappa/mu/delta) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A-(1-13)NH(2). Thus [NMePhe(1)]arodyn is a highly kappa opioid receptor-selective antagonist that could be a useful pharmacological tool to study kappa opioid receptor-mediated activities.     

Characterization of kappa1-opioid receptor binding in human insular cortex.

Mesolimbic dopaminergic neurotransmission is modulated by dynorphin peptides binding to kappa-opioid receptors. The interaction between dynorphin and dopamine systems makes the kappa-opioid receptor a potential drug discovery target for the development of therapeutic agents for schizophrenia and drug abuse. This study reports the specificity and parameters of [3H]U69593 binding in the insular cortex, a representative corticolimbic area of the human brain. The results demonstrate that the radioligand [3H]U69593 labels a single population of receptors in human insular cortex with an affinity in the low nanomolar range. The pharmacological profile for inhibition of [3H]U69593 binding was determined in this brain region using drugs known to bind to mu, kappa and delta opioid receptors. The results show that kappa-opioid selective agonists and antagonists inhibit binding of this ligand in human brain with comparable affinities and rank order as previously described for rat and guinea pig brain and the cloned kappa1-opioid receptor subtype.     

Covalent labeling of opioid receptors with 3H-D-Ala2-Leu5-enkephalin chloromethyl ketone. II. Binding characteristics in frog brain membranes

3H-D-Ala2-Leu5-enkephalin chloromethyl ketone (3H-DALECK) was used to label opioid receptors of frog brain membranes. We have previously shown (15) that 70% of the opioid receptors are of kappa type in this preparation. The binding of 3H-DALECK was of high affinity, half maximal binding being achieved by 0.9 nM of the radioligand. The number of sites labeled was calculated to be 108 fmol/mg protein. Opioid ligands, incubated with the membranes prior to the label, inhibited 3H-DALECK binding with the following rank order:etorphine greater than EKC greater than DAGO greater than DALECK greater than DADLE. Dissociation experiments showed that 70% of the binding is irreversible. Fluorography performed after SDS-PAGE revealed specific covalent labeling of protein subunits of 90, 58 and 20 kD molecular weights. Results will be compared to those obtained in rat brain (13). Our two studies demonstrate that 3H-DALECK is a useful probe for investigation the subunit structure of opioid receptors.     

Receptors for cholecystokinin and gastrin peptides display specific binding properties and are structurally different in guinea-pig and dog pancreas

In the light of the strong potency of gastrin-related peptides on pancreatic exocrine secretion in dog, we analyzed the binding properties of peptides related to cholecystokinin (CCK) and gastrin on dog pancreatic acini compared to guinea-pig acini. Moreover, we determined apparent molecular masses of photoaffinity labelled CCK/gastrin receptors in the two models. Using the CCK radioligand, receptor selectivity towards CCK/gastrin agonists and antagonists was found to be lower in dog acini than in guinea-pig acini. Performing the binding with CCK and gastrin radioligands in combination with N2,O2'-dibutyryl-guanosine 3',5'-monophosphate, revealed that in dog acini there exist two different sub-classes of CCK/gastrin receptors having high and low selectivity, the latter ones being able to bind gastrin with high affinity (Kd = 2.1 nM). SDS-PAGE analysis of covalently cross-linked receptors using several photosensitive CCK and gastrin probes of different peptide chain lengths demonstrated that in guinea-pig, CCK peptides bound to a 84-kDa component whereas in dog pancreas, CCK and gastrin peptides bound to three distinct molecular species (Mr approximately equal to 78,000, 45,000, 28,000). Performing cross-linking in the presence of 1 microM CCK indicated that a 45-kDa protein is the putative CCK/gastrin receptor in dog pancreas. Our results support the concept of heterogeneity of CCK/gastrin receptors.     

In vivo rat brain opioid receptor binding of LY255582 assessed with a novel method using LC/MS/MS and the administration of three tracers simultaneously

LY255582 is a pan opioid selective receptor antagonist that has been shown to have high affinity for mu, delta, and kappa receptors in vitro. In order to better understand the in vivo opioid receptor selectivity of LY255582, we developed in vivo receptor occupancy assays in the rat for the opioid mu, kappa and delta receptors using the occupancy tracers naltrexone, GR103545 and naltriben respectively. Individual assays for each target were established and then a "triple tracer" assay was created where all three tracers were injected simultaneously, taking advantage of LC/MS/MS technology to selectively monitor brain tracer levels. This is the first report of a technique to concurrently measure receptor specific occupancy at three opioid receptors in the same animal. The opioid subtype selective antagonists cyprodime, JDTic and naltrindole were used to validate selectivity of the assay. Examination of LY255582 in dose-occupancy experiments demonstrated a relative order of potency of mu>kappa>delta, reproducing the previously reported order determined with in vitro binding.     

Pharmacological activities of Vitex agnus-castus extracts in vitro.

The pharmacological effects of ethanolic Vitex agnus-castus fruit-extracts (especially Ze 440) and various extract fractions of different polarities were evaluated both by radioligand binding studies and by superfusion experiments. A relative potent binding inhibition was observed for dopamine D2 and opioid (micro and kappa subtype) receptors with IC50 values of the native extract between 20 and 70 mg/mL. Binding, neither to the histamine H1, benzodiazepine and OFQ receptor, nor to the binding-site of the serotonin (5-HT) transporter, was significantly inhibited. The lipophilic fractions contained the diterpenes rotun-difuran and 6beta,7beta-diacetoxy-13-hydroxy-labda-8,14-dien . They exhibited inhibitory actions on dopamine D2 receptor binding. While binding inhibition to mu and kappa opioid receptors was most pronounced in lipophilic fractions, binding to delta opioid receptors was inhibited mainly by a aqueous fraction. Standardised Ze 440 extracts of different batches were of constant pharmacological quality according to their potential to inhibit the binding to D2 receptors. In superfusion experiments, the aqueous fraction of a methanolic extract inhibited the release of acetylcholine in a concentration-dependent manner. In addition, the potent D2 receptor antagonist spiperone antagonised the effect of the extract suggesting a dopaminergic action mediated by D2 receptor activation. Our results indicate a dopaminergic effect of Vitex agnus-castus extracts and suggest additional pharmacological actions via opioid receptors.     

Structure-activity relationships in a series of 8-substituted xanthines as A1-adenosine receptor antagonists

A series of 8-substituted xanthines were synthesized and their affinity in vitro towards A1, A2A-adenosine receptors was evaluated by radioligand receptor binding assays. All compounds showed a greater affinity and selectivity towards the A1-adenosine receptor than theophylline. The compounds in which the n-proyl group is in 1-position of the xanthine nucleus and the pyridazinone system in 8-position is linked through a chain of two or four carbon atoms, showed the highest affinity and selectivity.     

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.     

Opioid receptors in the rat spinal cord after longlasting deafferentation.

In vitro binding of specific opioid ligands to their respective sites in membrane fractions and the contribution of individual receptor classes (mu, delta, kappa) was studied in rats after longlasting (up to 22 months) section of spinal dorsal roots at the cervical (C5-8) or thoracic (Th1-4) level. This procedure leads to autotomy or scratching of the skin on the operated side. The total number of receptors in the cervical and thoracic spinal cord was more than doubled in both operated and contralateral part of the cord in comparison with intact controls of the same age. In the cervical region, this increase mainly represented a rise in the number of free receptors, whilst in the thoracic region both free and saturated receptors were increased. On the deafferented side, receptor selectivity, especially in the delta and kappa types was decreased.