Cyclic somatostatin octapeptide analogues with high affinity and selectivity toward mu opioid receptors

A series of cyclic conformationally restricted penicillamine containing somatostatin octapeptide analogues have been prepared by standard solid phase synthetic techniques and tested for their ability to inhibit specific [125I]CGP 23,996 (des-Ala1-,Gly2-[desamino-Cys3Tyr11]-dicarba3, 14-somatostatin), [3H]naloxone or [3H]DPDPE ([D-Pen2-D-Pen5]enkephalin) binding in rat brain membrane preparations. We now report structure-activity relationship studies with the synthesis of our most potent and selective mu opioid receptor compound D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2, which we refer to as Cys2Tyr3Orn5Pen7-amide. While this octapeptide exhibited high affinity (IC50 = 2.80 nM) for an apparently single population of binding sites (nH = 0.89 +/- 0.1) and exceptional selectivity for mu opioid receptors with an IC50(DPDPE)/IC50 (naloxone) ratio of 4,829, it also displayed very low affinity for somatostatin receptors (IC50 = 22,700 nM). Thus, Cys2Tyr3Orn5Pen7-amide may be the ligand of choice for further characterization of mu opioid receptors and for examining the physiological role of this class of receptors.     

125I][D-Ala2]deltorphin-I: a high affinity, delta-selective opioid receptor ligand.

The selective delta opioid agonist [D-Ala2]deltorphin-I was radioiodinated and the product purified using reverse phase HPLC. The binding characteristics and distribution profile of [125I][D-Ala2]deltorphin-I were assessed in mouse brain using homogenate binding techniques and quantitative autoradiography. [125I][D-Ala2]deltorphin-I bound with high affinity to a single class of sites (KD = 0.5 nM) in brain membrane preparations and striatal sections. Competition studies indicated that [125I][D-Ala2]deltorphin-I was selectively labeling delta opioid receptors as shown by the ratio of apparent affinities for mu and delta receptors (KI mu/KI delta = 1388). The autoradiographical distribution profile of [125I][D-Ala2]deltorphin-I binding sites was also consistent with that of other delta-selective radioligands. The data indicate that [125I][D-Ala2]deltorphin-I binds to delta opioid receptors with high affinity and selectivity. Because of its very high specific activity, it can be detected rapidly with high sensitivity by autoradiographic emulsion.     

Complementarity of delta opioid ligand pharmacophore and receptor models

The elaboration of a pharmacophore model for the delta opioid receptor selective ligand JOM-13 (Tyr-c[D-Cys-Phe-D-Pen]OH) and the parallel, independent development of a structural model of the delta receptor are summarized. Although the backbone conformation of JOM-13's tripeptide cycle is well defined, considerable conformational lability is evident in the Tyr(1) residue and in the Phe(3) side chain, key pharmacophore elements of the ligand. Replacement of these flexible features of the ligand by more conformationally restricted analogues and subsequent correlation of receptor binding and conformational properties allowed the number of possible binding conformations of JOM-13 to be reduced to two. Of these, one was chosen as more likely, based on its better superposition with other conformationally constrained delta receptor ligands. Our model of the delta opioid receptor, constructed using a general approach that we have developed for all rhodopsin-like G protein-coupled receptors, contains a large cavity within the transmembrane domain that displays excellent complementarity in both shape and polarity to JOM-13 and other delta ligands. This binding pocket, however, cannot accommodate the conformer of JOM-13 preferred from analysis of ligands, alone. Rather, only the "alternate" allowed conformer, identified from analysis of the ligands but "disfavored" because it does not permit simultaneous superposition of all pharmacophore elements of JOM-13 with other delta ligands, fits the binding site. These results argue against a simple view of a single, common fit to a receptor binding site and suggest, instead, that at least some binding site interactions of different ligands may differ.     

Piperazinyl benzamidines: synthesis and affinity for the delta opioid receptor.

Piperazinyl benzamidines were prepared and found to bind to the rat delta (delta) opioid receptor. The most active compounds had a N,N-diethylcarboxamido group and a N-benzyl piperazine. The most potent among these was N,N-diethyl-4-[4-(phenylmethyl)-1-piperazinyl][2-(trifluoromethyl)phenyl]iminomethyl]benzamide (27) with a 1.22nM K(i) for the rat delta opioid receptor and ca. 1000 x selectivity relative to the mu opioid subtype.     

Conformationally constrained cyclic enkephalin analogs with pronounced delta opioid receptor agonist selectivity

The enkephalin analogs, [D-Pen2,L-Cys5]- and [D-Pen2,D-Cys5]-enkephalin are cyclic compounds, conformationally constrained by virtue of their 14-membered, disulfide containing rings and by the rigidizing effect of the beta, beta dimethyl substituents of the penicillamine side chain. The analogs exhibit profound delta receptor specificity as assessed by their relative potencies in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assays, exhibiting, respectively, 666 and 215 times higher potency in the latter assay system. By contrast, the receptor selectivities measured in rat brain binding assays in the absence of sodium were much more modest, the cyclic analogs being, respectively, 15.2 and 6.0 times more effective at displacing [3H] [D-Ala2,D-Leu5]enkephalin than [3H]naloxone. However, for binding assays performed in the presence of a sodium concentration equivalent to that used in the GPI and MVD assays, these binding selectivities increased to 167 and 49, respectively.     

Aladan3]TIPP: a fluorescent delta-opioid antagonist with high delta-receptor binding affinity and delta selectivity

Fluorescent analogues of the potent and highly selective delta-opioid antagonist TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) containing the exceptionally environmentally sensitive fluorescent amino acid beta-(6'-dimethylamino-2'-naphthoyl)alanine (Aladan [Ald]) in place of Phe3 were synthesized. The Ald3- and D-Ald3 analogues of TIPP and TIP all retained delta-opioid antagonist properties. The most potent analogue, [Ald3]TIPP, showed a K(e) value of 2.03 nM in the mouse vas deferens assay and five times higher delta vs. mu selectivity (K(i)mu/K(i)delta = 7930) than the TIPP parent peptide in the opioid receptor binding assays. Theoretical conformational analyses of [Ald3]TIPP and [Ald3]TIP using molecular mechanics calculations resulted in a number of low-energy conformers, including some showing various patterns of aromatic ring stacking and others with the Ald side chain and a carbonyl group (fluorescence quencher) in close proximity. These ensembles of low-energy conformers are in agreement with the results of steady-state fluorescence experiments (fluorescence emission maxima and quantum yields) and fluorescence decay measurements (fluorescence lifetime components), which indicated that the fluorophore was either engaged in intramolecular hydrophobic interactions or in proximity of a fluorescence quencher (e.g., a carbonyl group). These fluorescent TIP(P) delta-opioid antagonists represent valuable pharmacological tools for various applications, including studies on membrane interactions, binding to receptors, cellular uptake and intracellular distribution, and tissue distribution.     

A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2, pCl-Phe4, d-Pen5]enkephalin

Binding characteristics of a new, conformationally constrained, halogenated enkephalin analogue, [3H]-[D-penicillamine2, pCl-Phe4, D-penicillamine5]enkephalin ([3H]pCl-DPDPE), were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C determined a dissociation constant (Kd) of 328 +/- 27.pM and a receptor density (Bmax) of 87.2 +/- 4.2 fmol/mg protein. Kinetic studies demonstrated biphasic association for [3H]pCl-DPDPE, with association rate constants of 5.05 x 10(8) +/- 2.5 x 10(8) and 0.147 +/- 10(8) +/- 0.014 x 10(8) M-1 min-1. Dissociation was monophasic with a dissociation rate constant of 2.96 x 10(-3) +/- 0.25 x 10(-3) min-1. The average Kd values determined by these kinetic studies were 8.4 +/- 2.7 pM and 201 +/- 4 pM. Competitive inhibition studies demonstrated that [3H]pCl-DPDPE has excellent selectively for the delta opioid receptor. [3H]pCl-DPDPE binding was inhibited by low concentrations of ligands selective for delta opioid receptor relative to the concentrations required by ligands selective for mu and kappa sites. These data show that [3H]pCl-DPDPE is a highly selective, high affinity ligand which should be useful in characterizing the delta opioid receptor.     

Derivatives of 17-(2-methylallyl)-substituted noroxymorphone: variation of the delta address and its effects on affinity and selectivity for the delta opioid receptor

In an effort to establish the importance of the N-(2-methylallyl) substituent in the noroxymorphone series, several derivatives have been synthesized, retaining that N-substituent and modifying the delta address moiety. A few compounds showed moderate binding affinity and selectivity for the delta receptor; none displayed a pharmacological profile as exceptional as N-(2-methylallyl)noroxymorphindole. A second study showed that 3-O-methylation of all derivatives decreases binding affinity. The present results indicate that only a combination of the N-(2-methylallyl) group and an indole delta address provided high selectivity for the delta receptor.     

3-Ethoxy-beta-carboline: a high affinity benzodiazepine receptor ligand with partial inverse agonist properties

3-Ethoxy-beta-carboline binds with high affinity to benzodiazepine receptors in the central nervous system (Ki approximately equal to 10.1, 15.3, and 25.3 nM in rat cerebellum, cerebral cortex, and hippocampus, respectively). This compound has pharmacological actions reminiscent of benzodiazepine receptor partial inverse agonists such as FG 7142 and 3-carboethoxy-beta-carboline. Thus, while not a convulsant, 3-ethoxy-beta-carboline potentiated the convulsant actions of pentylenetetrazole in mice. Furthermore, this compound reduced both the time spent and the total entries in the open arms of an elevated plus maze and also inhibited stress-induced ulcer formation, effects that are also observed with benzodiazepine receptor inverse agonists. These findings suggest that 3-ethoxy-beta-carboline is a partial inverse agonist at benzodiazepine receptors which may prove useful for in vivo studies since it has a higher affinity for benzodiazepine receptors and better solubility than the commonly used partial inverse agonist FG 7142. Furthermore, 3-ethoxy-beta-carboline appears to be less vulnerable to metabolic degradation than ester analogs with a similar pharmacological profile such as 3-carboethoxy-beta-carboline.     

Topographical requirements for delta opioid ligands: presence of a carboxyl group in position 4 is not critical for deltorphin high delta receptor affinity and analgesic activity.

To investigate the role of the carboxyl group in deltorphin molecules, we have synthesized three new analogues in which the acidic amino acid residues in position 4 of the deltorphins were replaced by non-acidic but hydrophilic amino acids residues. The three analogues, [Ser4]-, [Gln4]-, and [Cys4]-deltorphin, all are as potent or more potent than either deltorphin I or II at delta opioid receptors and possess good delta selectivities. The excellent correlation between their in vitro delta receptor potencies and their intrathecal antinociception activity forms a strong argument for involvement of those receptors in spinal nociceptive modulation in the rats.     

Deltorphin, a naturally occurring peptide with high selectivity for delta opioid receptors, improves memory consolidation in two inbred strains of mice

Deltorphin, a heptapeptide that selectively binds to delta receptors, was intracerebroventricularly administered to mice belonging to the strains C57BL/6 and DBA/2 immediately after training in a one-trial inhibitory avoidance task. The retention performance of both strains was improved by the peptide administration, with DBA mice being more sensitive to this effect. The results show different effects of delta and mu receptor agonists on memory consolidation and of strain differences in number and/or distribution of delta opioid receptors in the brain.     

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.     

Desmethylnafoxidine aziridine: an electrophilic affinity label for the estrogen receptor with high efficiency and selectivity

Desmethylnafoxidine aziridine (Naf-Az), an affinity label for the estrogen receptor based structurally on the antiestrogen nafoxidine, has been prepared in unlabeled and in high specific activity, tritium-labeled form and has been evaluated for its apparent competitive binding, and time-dependent irreversible, covalent attachment to the estrogen receptor. Naf-Az was synthesized through a key 1,2-diaryl-3,4-dihydronaphthalene intermediate that was prepared from 6-methoxy-1-tetralone by two routes involving alternate strategies for arylation. Conversion of the diaryldihydronaphthalene to Naf-Az through a series of deprotection-activation reactions culminated in ethyleneimine displacement of a methanesulfonate. The tritium-labeled material was prepared by tritium-iodine exchange on an iodinated methanesulfonate precursor, followed by ethyleneimine displacement. Compared to our previously-prepared reagent tamoxifen aziridine (Tam-Az), Naf-Az has a higher apparent competitive binding affinity, and it reacts with the estrogen receptor in cytosol preparations and in intact MCF-7 breast cancer cells rapidly and with at least comparable efficiency and selectivity. SDS-polyacrylamide gel electrophoretic analysis confirms its selective labeling of the Mr 66,000 estrogen receptor. Naf-Az should prove to be useful in studies aimed at characterizing the properties and structure of estrogen receptors.     

Novel 1,2,3,4-tetrahydroisoquinolines with high affinity and selectivity for the dopamine D3 receptor

Using clearance and brain penetration studies as a screen, tetrahydroisoquinoline 3 was identified as a lead having low clearance in rats (CLb 20 ml/min/kg). Introduction of a 7-CF3SO2O- substituent into the tetrahydroisoquinoline, followed by replacement of the biphenylamido group of 3 by a 3-indolylpropenamido group gave 31, having high D3 receptor affinity (pKi 8.4) and 150 fold selectivity over the D2 receptor.     

Dermorphin and deltorphin heptapeptide analogues: replacement of Phe residue by Dmp greatly improves opioid receptor affinity and selectivity

The usefulness of 2,6-dimethylphenylalanine (Dmp) as a Phe surrogate in two opioid peptides, dermorphin (DM) and deltorphin II (DT), was investigated. Compared to DM, [L-Dmp(3)]DM (1) showed a 170-fold increase in mu affinity and only a 4-fold increase in delta affinity, resulting in a 40-fold improvement in mu receptor selectivity. Compared to DT, [L-Dmp(3)]DT (3) showed a 22-fold increase in delta affinity and somewhat of a loss in mu affinity, and consequently a marked (75-fold) improvement in delta receptor selectivity. The D-Dmp replacement, however, resulted in a great loss in receptor selectivity in each of the peptides. The specific receptor interactions of 1 and 3 were confirmed by in vitro bioassays. Analogues 1 and 3 seem to be useful as pharmacological tools for the study of opioid systems.     

D-Ala2]deltorphin II analogs with high affinity and selectivity for delta-opioid receptor.

Nine [D-Ala2]deltorphin II (DL-II:Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH2) analogs having various aliphatic amino acids at positions 5 and 6 were synthesized to gain more information about the role of hydrophobic Val5,6 residues for the delta-opioid receptor selectivity. Binding assays of analogs replaced by Ala demonstrated the importance of hydrophobic Val5,6 residues in DL-II for delta-affinity and selectivity, and especially critical importance of Val5 residue for higher delta-selectivity. By enhancing the hydrophobicity of residues at positions 5 and 6, we have developed analogs with very high delta-affinity and selectivity over those of DL-II, e.g., [Ile5,6], [norleucine5,6] and [gamma-methyl-leucine5,6]DL-II, which will be useful as delta-selective ligands for investigation of the physiological role of opioid receptors.     

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.     

Preliminary ligand binding data for subtypes of the delta opioid receptor in rat brain membranes

Delta opioid binding sites were assayed using [³H][D-ala²,D-leu⁵]enkephalin and rat brain membranes depleted of μ binding sites with the site-directed acylating agent, 2-(p-ethoxybenzyl)-1-diethylaminoethyl-5 -isothiocyanatobenzimidazole-HCI. [D-Pen², D-Pen⁵]enkephalin (DPDPE), [D-Pen²,L-Pen⁵]enkephalin, [D-Ala²]deltorphin-I and [D-Ala²]deltorphin-II inhibition curves were characterized by slope factors (Hill coefficients) less than 1. The low slope factor of DPDPE persisted in the presence of 50 μM 5'-guanylyimidodiphosphate in the assay. Quantitative analysis of [D-ala²,D-leu⁵]enkephalin, DPDPE and [D-Ala²]deltorphin-I binding surfaces resolved two binding sites. Whereas [D-ala²,D-leu⁵]enkephalin had equal affinity for both sites, DPDPE and [D-Ala²]deltorphin-I had high affinity for the high capacity binding site, and low affinity for the low capacity binding site. These data support pharmacological studies demonstrating δ receptor subtyes which mediate antinociception.