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.     

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.     

Analgesic activity and selectivity of isothiocyanate derivatives of fentanyl analogs for opioid receptors

The analgesic activity and opioid receptor binding characteristics were studied for the isothiocyanate ohmefentanyl (OMFIT), and isothiocyanate carfentanil (CarFIT), isothiocyanate 4-methoxymethylfentanyl (MethoFIT), isothiocyanate 3-methylfentanyl (superFIT) and their amide analogs. Antinociceptive activity was evaluated using the mouse hot plate test; selectivity for opioid receptor was determined in bioassay and binding assay. SuperFIT, CarFIT, OMFIT and MethoFIT exhibited an analgesic ED50 lower than those of their parent compounds without isothiocyanate (SCN) group. Furthermore these compounds exhibited potent inhibitory actions on the electrically evoked contractions of mouse vas deferens, which could be antagonized by naloxone, but their actions were weaker than those of their parent compounds without SC N-group. The inhibitory actions of these compounds on binding of [3H]OMF to mouse brain membrane was weaker than those of their parent compounds without SCN-group. CarFIT and MethoFIT showed weaker inhibitory actions on the binding of [3H] DADLE than their parent compounds without SCN-group, but SuperFIT and OMFIT stronger than their parent compounds, 3-methylfentanyl and ohmefentanyl. The selectivity of these isothiocyanate derivatives for delta opioid receptors increased. In conclusion, introducing isothiocyanato-group into 1-position of phenyl ring of ohmefentanyl and other fentanyl analogs would enhance the selectivity of these compounds for delta-opioid receptors, but decrease their analgesic activity.     

Cation dependence of opioid receptor binding supports theory on membrane-mediated receptor selectivity

A quantitative analysis of the binding of dynorphin A-(1-9)-nonapeptide to the opioid Kappa-receptors of the guinea pig cerebellum [Paterson et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6216-6220] shows that changes in electrostatic surface accumulation of the ligand fully account for the observed suppression of binding by a series of univalent and divalent salts. Binding to mu- and delta-receptors, on the other hand, is subject to additional ion-specific effects. These observations support the membrane locations for the receptor sites proposed by the "membrane compartments" theory for opioid receptor selection.     

Molecular modeling studies to predict the possible binding modes of endomorphin analogs in μ opioid receptor

The molecular docking of a series of endomorphin analog with the μ opioid receptor was performed. The successive molecular dynamics of several proposed ligand–receptor complexes inserted into the phospholipid bilayer were carried out to optimize the complex and explore the conformational changes. Meaningful differences of their binding modes were detected and the involvement of some essential residues in ligand binding was also identified. Our proposed ligand–receptor model is in good agreement with previous site-directed mutagenesis experiments.     

Cyclic enkephalin analogs that are hybrids of DPDPE-related peptides and metenkephalin-Arg-Gly-Leu: prohormone analogs that retain good potency and selectivity for delta opioid receptors.

We report here on the binding affinity and bioassay results of cyclic enkephalin analogs comprising a cyclic moiety and C-terminal fragment of MERGL, where ME denotes methionine enkephalin. MERGL (YGGFMRGL) has been suggested to be cleaved enzymatically by membrane-bound enkephalinase 24.11 to leave ME and the tripeptide RGL. In our study we have synthesized hybrids of DPDPE or DPLCE and the C-terminal tripeptide RGL in order to mimic a prohormone able to cross the blood-brain barrier. The study has shown that of the homologs presented here, analogs of DPLCE often are more potent at delta opioid receptors both in binding affinity and in bioactivity at the MVD, than DPDPE. Our hypothesis that hybrids (consisting of the drug and the spacer for the carrier) could be designed which would either have no opioid activity or, alternatively, be by themselves very active, has been verified.     

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.     

Studies on opioid receptor selectivity of beta-endorphin antagonists

Opioid receptor selectivity of several beta-endorphin (beta-EP) analogs which antagonize beta-EP-induced analgesia has been assessed using partially selective binding assays. Although the apparent affinity dissociation constant of beta-EP in these assays varies from 0.2 to 360 nm, the potency of beta-EP antagonists relative to beta-EP remains largely unchanged. It is unlikely that differences in receptor affinities can account for the antagonist properties of these analogs in vivo.     

Synthesis and opioid activity of new fentanyl analogs

Three new fentanyl analogs (compounds 3-4-5) have been synthesized and evaluated for antinociceptive properties using the writhing test. The analgesic property of the active compound, N-[1-phenylpyrazol-3-yl]-N-[1-(2-phenethyl)-4-piperidyl)] propenamide (compound 4), was tested using the hot plate test in mice. Its opioid agonistic activity was characterized using three isolated tissues: guinea pig ileum, mouse vas deferens, and rabbit vas deferens. Compound 4 was as effective as fentanyl or morphine and it showed less antinociceptive potency than fentanyl but it was more potent than morphine. The duration of the antinociception was similar to that of fentanyl. This compound inhibited the electrically evoked contractions of myenteric plexus-longitudinal muscle strips of guinea pig ileum and of mouse vas deferens but not those of rabbit vas deferens. These effects could be reversed by micro selective antagonists (naloxone and/or CTOP) but not by the delta selective antagonist naltrindole, thus indicating that the compound acted as a micro opioid agonist. Finally, the binding data confirmed that compound 4 had high affinity and selectivity for the micro-receptor.     

DAT/SERT selectivity of flexible GBR 12909 analogs modeled using 3D-QSAR methods

The dopamine reuptake inhibitor GBR 12909 (1-{2-[bis(4-fluorophenyl)methoxy]ethyl}-4-(3-phenylpropyl)piperazine, 1) and its analogs have been developed as tools to test the hypothesis that selective dopamine transporter (DAT) inhibitors will be useful therapeutics for cocaine addiction. This 3D-QSAR study focuses on the effect of substitutions in the phenylpropyl region of 1. CoMFA and CoMSIA techniques were used to determine a predictive and stable model for the DAT/serotonin transporter (SERT) selectivity (represented by pK(i) (DAT/SERT)) of a set of flexible analogs of 1, most of which have eight rotatable bonds. In the absence of a rigid analog to use as a 3D-QSAR template, six conformational families of analogs were constructed from six pairs of piperazine and piperidine template conformers identified by hierarchical clustering as representative molecular conformations. Three models stable to y-value scrambling were identified after a comprehensive CoMFA and CoMSIA survey with Region Focusing. Test set correlation validation led to an acceptable model, with q(2)=0.508, standard error of prediction=0.601, two components, r(2)=0.685, standard error of estimate=0.481, F value=39, percent steric contribution=65, and percent electrostatic contribution=35. A CoMFA contour map identified areas of the molecule that affect pK(i) (DAT/SERT). This work outlines a protocol for deriving a stable and predictive model of the biological activity of a set of very flexible molecules.     

Antimicrobial peptide-lipid binding interactions and binding selectivity

Surface pressure measurements, external reflection-Fourier transform infrared spectroscopy, and neutron reflectivity have been used to investigate the lipid-binding behavior of three antimicrobial peptides: melittin, magainin II, and cecropin P1. As expected, all three cationic peptides were shown to interact more strongly with the anionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-(phosphor-rac-(1-glycerol)) (DPPG), compared to the zwitterionic lipid, 1,2 dihexadecanoyl-sn-glycerol-3-phosphocholine (DPPC). All three peptides have been shown to penetrate DPPC lipid layers by surface pressure, and this was confirmed for the melittin-DPPC interaction by neutron reflectivity measurements. Adsorption of peptide was, however, minimal, with a maximum of 0.4 mg m(-2) seen for melittin adsorption compared to 2.1 mg m(-2) for adsorption to DPPG (from 0.7 microM solution). The mode of binding to DPPG was shown to depend on the distribution of basic residues within the peptide alpha-helix, although in all cases adsorption below the lipid layer was shown to dominate over insertion within the layer. Melittin adsorption to DPPG altered the lipid layer structure observed through changes in the external reflection-Fourier transform infrared lipid spectra and neutron reflectivity. This lipid disruption was not observed for magainin or cecropin. In addition, melittin binding to both lipids was shown to be 50% greater than for either magainin or cecropin. Adsorption to the bare air-water interface was also investigated and surface activity followed the trend melittin>magainin>cecropin. External reflection-Fourier transform infrared amide spectra revealed that melittin adopted a helical structure only in the presence of lipid, whereas magainin and cecropin adopted helical structure also at an air-water interface. This behavior has been related to the different charge distributions on the peptide amino acid sequences.     

Receptor selectivity of enkephalin analogs carrying artificial address peptides

The synthetic [Leu]enkephalin analogs YGGFLGP(KS'S'S')2-OMe (S' represents Sar residue, ENK-S'8), YGGFLGP(KPPP)2-OMe (ENK-P8), and YGGFLGP(KA'LA')2-OMe (A' represents 2-aminoisobutyric acid (Aib), ENK-A'8) were designed to investigate the role of membrane affinity in opioid receptor binding. CD measurement in ethanol revealed that ENK-S'8 took a random coil conformation, while ENK-P8 and ENK-A'8 partly adopted a 3(1)- and alpha-helical conformation, respectively. ENK-A'8 was shown to be distributed to lipid bilayer membrane due to the amphiphilic alpha-helical structure, while ENK-S'8 and ENK-P8 stayed in aqueous phase. Opioid receptor affinities ENK-S'8 and ENK-P8 were similar to [Leu]enkephalinamide, indicating that the connection of hydrophilic peptide segments to the C-terminal of [Leu]enkephalinamide did not affect the receptor affinity. On the other hand, delta- and mu-receptor affinities of ENK-A'8 were about 1/20 and 1/6 times, respectively, those of [Leu]enkephalinamide. Therefore, ENK-A'8 has higher selectivity for mu-receptor than [Leu]enkephalinamide. These results are explained in terms of the membrane compartment concept proposed by Schwyzer.     

Novel trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines as mu opioid receptor antagonists with improved opioid receptor selectivity profiles

A series of N-substituted trans-3,4-dimethyl-4-(3-hydroxyphenyl)piperidines, mu opioid receptor antagonists, analogs of alvimopan, were prepared using solid phase methodology. This study led to the identification of a highly selective mu opioid receptor antagonist, which interacts selectively with mu peripheral receptors.     

Conformationally constrained opioid peptide analogs with novel activity profiles

Novel conformationally constrained opioid peptide analogs with antagonist, mixed agonist/ antagonist or agonist properties were developed. TIP(P)-related antagonists showed unprecedented antagonist potency and receptor selectivity, and may have potential for use in analgesia in combination with agonists. A definitive model of their receptor-bound conformation was developed. Three prototype mixed agonist/ antagonists were discovered. They represent the only known compounds with this pharmacological profile and, as expected, one of them was shown to be a potent analgesic and to produce no dependence and less tolerance than morphine. Novel dipeptide derivatives turned out to be potent and selective agonists. Because of their low molecular weight and lipophilic character, these compounds may cross the blood-brain barrier and, thus, may have potential as centrally acting analgesics.     

Synthesis and opioid activity of partial retro-inverso analogs of dermorphin

We studied the effect of partial retro-inverso modification of selected peptide bonds of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2. The modifications concern two consecutive peptide bonds (Phe3-Cly4-Tyr5, I) or a single one (Gly4-Tyr5-, II or Phe3-Gly4, III). All pseudoheptapeptides showed low opioid activity in the in vitro and in vivo tests. Compound III has a biological potency comparable to that of morphine but only 2-5% of original dermorphin when tested in guinea pig ileum preparation and in mice tail-flick assay after intracerebro or subcutaneous administration.     

Conformationally constrained opioid peptide analogs with novel activity profiles

Summary Novel conformationally constrained opioid peptide analogs with δ antagonist, mixed μ agonist/δ antagonist or δ agonist properties were developed. TIP(P)-related δ antagonists showed unprecedented δ antagonist potency and δ receptor selectivity, and may have potential for use in analgesia in combination with μ agonists. A definitive model of their δ receptor-bound conformation was developed. Three prototype mixed μ agonist/δ antagonists were discovered. They represent the only known compounds with this pharmacological profile and, as expected, one of them was shown to be a potent analgesic and to produce no dependence and less tolerance than morphine. Novel dipeptide derivatives turned out to be potent and selective δ agonists. Because of their low molecular weight and lipophilic character, these compounds may cross the blood-brain barrier and, thus, may have potential as centrally acting analgesics.     

Probing the binding pocket of the active site of aromatase with 6-ether or 6-ester substituted androst-4-ene-3,17-diones and their diene and triene analogs

A series of 6-ester- (3 and 4) and 6-ether- (7 and 8) substituted androst-4-ene-3,17-diones (androstenediones) and their 1,4-diene analogs (5 and 6, and 9 and 10) as well as C6-substituted 4,6-diene and 1,4,6-triene steroids 11 and 12 were synthesized as aromatase inhibitors to gain insight into the structure-activity relationship between various substituents and inhibitory activity. All of the inhibitors synthesized blocked aromatase in a competitive manner. The inhibitory activities of all of the steroids, except for the 6beta-benzoates 4g and 6h and the 6beta-acetate 6a, were fairly effective to very powerful (K(i): 7.0-320 nM). The 6alpha-n-hexanoyloxy- and 6alpha-benzyloxyandrostenediones (3e and 7e) were the most potent inhibitors (K(i): 7.0 nM each). In the series of 4-ene and 1,4-diene steroids, the 6alpha-substituted steroids had higher affinity for the enzyme than the corresponding 6beta-isomers. In the 1,4-diene steroid series, 6beta-substituted steroids 6a, e, g, and 10a, b, e caused a time-dependent inactivation of aromatase, whereas their 6alpha-isomers 5 and 9 essentially did not. The ether-substituted 1,4,6-trienes 12 inactivated the enzyme in a time-dependent manner; in contrast, their 4,6-diene analogs 11 did not. The substrate androstenedione blocked the inactivation, but no significant effect of L-cysteine was observed. Based on molecular modeling with the PM3 method, along with the present inhibition and inactivation results, it is thought that both the steric effects of the 6-substituents as well as the electronic effects of the C-6 oxygen functions play a critical role in the binding of inhibitors to the active site of aromatase.     

Delta opioid receptor selectivity induced by conformational constraints in linear enkephalin-related peptides: 1H 400-MHz NMR study and theoretical calculations

Introduction into the structure of the linear hexapeptide DSLET (Tyr-D-Ser-Gly-Phe-Leu-Thr) or DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr) of tert-butyl groups as constraints different from cyclization leads to a large increase in the selectivity for delta opioid binding site in the case of DSTBULET [Tyr-D-Ser-(OtBu)-Gly-Phe-Leu-Thr] (Ki delta = 6.14 nM; Ki mu = 374 nM) and BUBU [Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu)] (Ki delta = 4.68 nM; Ki mu = 475 nM) or a loss of affinity for DTTBULET [Tyr-D-Thr(OtBu)-Gly-Phe-Leu-Thr] (Ki delta = 866 nM; Ki mu = 4500 nM). This puzzling behavior is studied here by 400-MHz 1H NMR spectroscopy in DMSO-d6 solution and by theoretical calculations. When DSLET and DTLET are compared, the reduction in energetically accessible phi and psi angles induced by the tert-butyl group in the D-Ser2 residue decreases the degree of freedom in the N-terminal part of the peptides. For DSTBULET and BUBU, the rigidification of the backbone evidenced by the appearance of the large NOE's of Phe4 NH-Gly3 alpha and Gly3 NH-alpha and by the loss of the C7 folding around the D-Ser2 residue found in DSLET could explain the drastic loss of affinity for mu opioid receptors. In DTTBULET, a large change in the spatial orientation around the D-Thr2 (OtBu) residue forces the aromatic rings far from each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Respiratory depression after intravenous administration of delta-selective opioid peptide analogs

We compared the effects of three micro-(DAMGO, DALDA, TNPO) and three delta-(DPDPE, DELT, SNC-80) opioid agonists on arterial blood gas after IV administration in awake sheep. None of the mu agonists altered pO2, pCO2 or pH. All three mu agonists decreased pO2 increased pCO2 and decreased pO2, and this effect was not sensitive to naloxone or TIPPpsi, a delta-antagonist, suggesting that it is not mediated by beta-opioid receptors. When administered to pregnant animals, there were significant changes in fetal pCO2 and pH. It may be possible to develop delta-selective opioid agonists which do not produce respiratory depression.     

Recognition of opioid agonist and antagonist in the opioid receptor binding site

By treating the rat crude synaptosomal fraction with 5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB, a marked decrease of stereo-specific binding of opioid agonist (dihydromorphine or D-Ala-D-Leu-enkephalin) was observed, but there was no effect in the case of the binding of opioid antagonist (naloxone or diprenorphine). The decrease of the agonist binding in the presence of 500 microM of DTNB was nearly equal to that of 100 mM of NaCl. The ability of opioids to inhibit 3H-naloxone binding in the absence of DTNB was compared to their inhibitory potency in the presence of 500 microM of DTNB to obtain DTNB response ratio. This ratio closely correlated with sodium index of each opioid. Potency of the inactivation of the agonist binding by congeners of DTNB changed with net charge of the reagents, and 2,2'-dithiobis-(5-nitropyridine), bearing a positive charge, was most effective. These results suggest that an aliphatic sulfhydryl group, being sensitive to DTNB is located to the active center of an anionic binding site for the agonist, and controls opioid agonist binding through a proton transfer mechanism.