Inflammation-associated changes in DOR expression and function in the mouse colon.

Endogenous opioids activate opioid receptors (ORs) in the enteric nervous system to control intestinal motility and secretion. The µ-OR mediates the deleterious side effects of opioid analgesics, including constipation, respiratory depression, and addiction. Although the δ-OR (DOR) is a promising target for analgesia, the function and regulation of DOR in the colon are poorly understood. This study provides evidence that endogenous opioids activate DOR in myenteric neurons that may regulate colonic motility. The DOR agonists DADLE, deltorphin II, and SNC80 inhibited electrically evoked contractions and induced neurogenic contractions in the mouse colon. Electrical, chemical, and mechanical stimulation of the colon evoked the release of endogenous opioids, which stimulated endocytosis of DOR in the soma and proximal neurites of myenteric neurons of transgenic mice expressing DOR fused to enhanced green fluorescent protein. In contrast, DOR was not internalized in nerve fibers within the circular muscle. Administration of dextran sulfate sodium induced acute colitis, which was accompanied by DOR endocytosis and an increased density of DOR-positive nerve fibers within the circular muscle. The potency with which SNC80 inhibited neurogenic contractions was significantly enhanced in the inflamed colon. This study demonstrates that DOR-expressing neurons in the mouse colon can be activated by exogenous and endogenous opioids. Activated DOR traffics to endosomes and inhibits neurogenic motility of the colon. DOR signaling is enhanced during intestinal inflammation. This study demonstrates functional expression of DOR by myenteric neurons and supports the therapeutic targeting of DOR in the enteric nervous system. NEW & NOTEWORTHY DOR is activated during physiologically relevant reflex stimulation. Agonist-evoked DOR endocytosis is spatially and temporally regulated. A significant proportion of DOR is internalized in myenteric neurons during inflammation. The relative proportion of all myenteric neurons that expressed DOR and the overlap with the nNOS-positive population are increased in inflammation. DOR-specific innervation of the circular muscle is increased in inflammation, and this is consistent with enhanced responsiveness to the DOR agonist SNC80.

Chitosan nanoparticles are efficient carriers for delivering biodegradable drugs to neuronal cells.

Chitosan nanoparticles (NPs) are biocompatible drug carriers able to cross the blood-brain barrier and represent a promising drug delivery system to the central nervous system. We used chitosan NPs to deliver the D-Ala2-D-Leu5-enkephalin (DADLE) to neuronal cells in vitro. DADLE is a hypometabolising synthetic opioid potentially useful for biomedical applications, but its short plasmatic half-life makes its in vivo administration ineffective. Here, we demonstrate by immunoelectron microscopy that (1) chitosan NPs are capable to deliver the opioid to neuronal cells; (2) DADLE is released from the internalised, opioid-loaded NPs up to 48 h; (3) in the nucleus, DADLE binds the transcription/splicing sites; (4) cells treated with DADLE-loaded NPs undergo a decrease in transcription factor amounts and proliferation rate without damage to cell organelles. In this model, chitosan NPs protected the loaded opioid from degradation, thereby prolonging its intracellular effects. These findings suggest that these NPs are efficient for the systemic and tissue administration of opioids in vivo.

DADLE promotes motor function recovery by inhibiting cytosolic phospholipase A2 mediated lysosomal membrane permeabilization after spinal cord injury.

BACKGROUND AND PURPOSE: Autophagy is a protective factor for controlling neuronal damage, while necroptosis promotes neuroinflammation after spinal cord injury (SCI). DADLE (D-Ala2 , D-Leu5 ]-enkephalin) is a selective agonist for delta (δ) opioid receptor and has been identified as a promising drug for neuroprotection. The aim of this study was to investigate the mechanism/s by which DADLE causes locomotor recovery following SCI. EXPERIMENTAL APPROACH: Spinal cord contusion model was used and DADLE was given by i.p. (16 mg·kg-1 ) in mice for following experiments. Motor function was assessed by footprint and Basso mouse scale (BMS) score analysis. Western blotting used to evaluate related protein expression. Immunofluorescence showed the protein expression in each cell and its distribution. Network pharmacology analysis was used to find the related signalling pathways. KEY RESULTS: DADLE promoted functional recovery after SCI. In SCI model of mice, DADLE significantly increased autophagic flux and inhibited necroptosis. Concurrently, DADLE restored autophagic flux by decreasing lysosomal membrane permeabilization (LMP). Additionally, chloroquine administration reversed the protective effect of DADLE to inhibit necroptosis. Further analysis showed that DADLE decreased phosphorylated cPLA2 , overexpression of cPLA2 partially reversed DADLE inhibitory effect on LMP and necroptosis, as well as the promotion autophagy. Finally, AMPK/SIRT1/p38 pathway regulating cPLA2 is involved in the action DADLE on SCI and naltrindole inhibited DADLE action on δ receptor and on AMPK signalling pathway. CONCLUSION AND IMPLICATION: DADLE causes its neuroprotective effects on SCI by promoting autophagic flux and inhibiting necroptosis by decreasing LMP via activating δ receptor/AMPK/SIRT1/p38/cPLA2 pathway.

Crosstalk between delta opioid receptor and nerve growth factor signaling modulates neuroprotection and differentiation in rodent cell models.

Both opioid signaling and neurotrophic factor signaling have played an important role in neuroprotection and differentiation in the nervous system. Little is known about whether the crosstalk between these two signaling pathways will affect neuroprotection and differentiation. Previously, we found that nerve growth factor (NGF) could induce expression of the delta opioid receptor gene (Oprd1, dor), mainly through PI3K/Akt/NF-κB signaling in PC12h cells. In this study, using two NGF-responsive rodent cell model systems, PC12h cells and F11 cells, we found the delta opioid neuropeptide [D-Ala2, D-Leu5] enkephalin (DADLE)-mediated neuroprotective effect could be blocked by pharmacological reagents: the delta opioid antagonist naltrindole, PI3K inhibitor LY294002, MAPK inhibitor PD98059, and Trk inhibitor K252a, respectively. Western blot analysis revealed that DADLE activated both the PI3K/Akt and MAPK pathways in the two cell lines. siRNA Oprd1 gene knockdown experiment showed that the upregulation of NGF mRNA level was inhibited with concomitant inhibition of the survival effects of DADLE in the both cell models. siRNA Oprd1 gene knockdown also attenuated the DADLE-mediated neurite outgrowth in PC12h cells as well as phosphorylation of MAPK and Akt in PC12h and F11 cells, respectively. These data together strongly suggest that delta opioid peptide DADLE acts through the NGF-induced functional G protein-coupled Oprd1 to provide its neuroprotective and differentiating effects at least in part by regulating survival and differentiating MAPK and PI3K/Akt signaling pathways in NGF-responsive rodent neuronal cells.

Angiotensin II type 1 receptor-mediated upregulation of calcineurin activity underlies impairment of cardioprotective signaling in diabetic hearts.

RATIONALE: The diabetic heart is resistant to ischemic preconditioning because of diabetes-associated impairment of phosphatidylinositol 3-kinase (PI3K)-Akt signaling. The mechanism by which PI3K-Akt signaling is impaired by diabetes remains unclear. OBJECTIVE: Here, we examined the hypothesis that phosphorylation of Jak2 upstream of PI3K is impaired in diabetic hearts by an angiotensin II type 1 (AT1) receptor-mediated mechanism. METHODS AND RESULTS: Infarct size (as percentage of risk area) after 20-minute ischemia/2-hour reperfusion was larger in a rat model of type 2 diabetes (Otsuka-Long-Evans-Tokushima fatty [OLETF] rat) than in its control (Long-Evans-Tokushima-Otsuka [LETO] rat) (60.4+/-1.6% versus 48.4+/-1.3%). Activation of Jak2-mediated signaling by erythropoietin or DADLE ([D-Ala2, D-Leu5]-enkephalin acetate), a delta-opioid receptor agonist, limited infarct size in LETO rats (27.7+/-3.4% and 24.8+/-5.0%) but not in OLETF rats (53.9+/-5.3% and 55.0+/-2.2%). Blockade of the AT1 receptor by valsartan or losartan for 2 weeks restored the myocardial response of OLETF rats to erythropoietin-induced infarct size limitation (39.4+/-4.9% and 31.2+/-7.5). In OLETF rats, erythropoietin failed to phosphorylate both Jak2 and Akt, and calcineurin activity was significantly higher than in LETO rats. Two-week treatment with valsartan normalized calcineurin activity in OLETF rats and restored the response of Jak2 to erythropoietin. This effect of AT1 receptor blockade was mimicked by inhibition of calcineurin by FK506. CONCLUSIONS: These results suggest that the diabetic heart is refractory to protection by Jak2-activating ligands because of AT1 receptor-mediated upregulation of calcineurin activity.

Evidence for two different broad-specificity oligopeptide transporters in intestinal cell line Caco-2 and colonic cell line CCD841.

Recently the existence of two different Na(+)-coupled oligopeptide transport systems has been described in mammalian cells. These transport systems are distinct from the previously known H(+)/peptide cotransporters PEPT1 and PEPT2, which transport only dipeptides and tripeptides. To date, the only peptide transport system known to exist in the intestine is PEPT1. Here we investigated the expression of the Na(+)-coupled oligopeptide transporters in intestinal cell lines, using the hydrolysis-resistant synthetic oligopeptides deltorphin II and [d-Ala(2),d-Leu(5)]enkephalin (DADLE) as model substrates. Caco-2 cells and CCD841 cells, both representing epithelial cells from human intestinal tract, were able to take up these oligopeptides. Uptake of deltorphin II was mostly Na(+) dependent, with more than 2 Na(+) involved in the uptake process. In contrast, DADLE uptake was only partially Na(+) dependent. The uptake of both peptides was also influenced by H(+) and Cl(-), although to a varying degree. The processes responsible for the uptake of deltorphin II and DADLE could be differentiated not only by their Na(+) dependence but also by their modulation by small peptides. Several dipeptides and tripeptides stimulated deltorphin II uptake but inhibited DADLE uptake. These modulating small peptides were, however, not transportable substrates for the transport systems that mediate deltorphin II or DADLE uptake. These two oligopeptide transport systems were also able to take up several nonopioid oligopeptides, consisting of 9-17 amino acids. This represents the first report on the existence of transport systems in intestinal cells that are distinct from PEPT1 and capable of transporting oligopeptides consisting of five or more amino acids.

A strategy for delivering peptides into the central nervous system by sequential metabolism.

Most peptides do not enter the central nervous system because of their hydrophilic character and the presence of peptidolytic enzymes in the lipoidal blood-brain barrier. To achieve brain delivery of a peptide conjugate, an opioid peptide (enkephalin) was placed in a molecular environment that disguises its peptide nature and provides biolabile, lipophilic functions to penetrate the blood-brain barrier by passive transport. The strategy also incorporates a 1,4-dihydrotrigonellinate targetor that undergoes an enzymatically mediated oxidation to a hydrophilic, membrane-impermeable trigonellinate salt. The polar targetorpeptide conjugate that is trapped behind the lipoidal blood-brain barrier is deposited in the central nervous system. Analgesia was observed with "packaged" enkephalin but not with the unmodified peptide or lipophilic peptide precursors.

CXCR2 chemokine receptor antagonism enhances DOP opioid receptor function via allosteric regulation of the CXCR2-DOP receptor heterodimer.

Opioid agonists have a broad range of effects on cells of the immune system, including modulation of the inflammatory response, and opioid and chemokine receptors are co-expressed by many white cells. Hetero-oligomerization of the human DOP opioid and chemokine CXCR2 receptors could be detected following their co-expression by each of co-immunoprecipitation, three different resonance energy transfer techniques and the construction of pairs of individually inactive but potentially complementary receptor G-protein alpha subunit fusion proteins. Although DOP receptor agonists and a CXCR2 antagonist had no inherent affinity for the alternative receptor when either receptor was expressed individually, use of cells that expressed a DOP opioid receptor construct constitutively, and in which expression of a CXCR2 receptor construct could be regulated, demonstrated that the CXCR2 antagonist enhanced the function of DOP receptor agonists only in the presence of CXCR2. This effect was observed for both enkephalin- and alkaloid-based opioid agonists, and the effective concentrations of the CXCR2 antagonist reflected CXCR2 receptor occupancy. Entirely equivalent results were obtained in cells in which the native DOP opioid receptor was expressed constitutively and in which expression of the isolated CXCR2 receptor could be induced. These results indicate that a CXCR2 receptor antagonist can enhance the function of agonists at a receptor for which it has no inherent direct affinity by acting as an allosteric regulator of a receptor that is a heterodimer partner for the CXCR2 receptor. These results have novel and important implications for the development and use of small-molecule therapeutics.

Multifaceted Effects of Delta Opioid Receptors and DADLE in Diseases of the Nervous System.

BACKGROUND: The opioid system is considered a potential therapeutic target in a variety of neurological disorders. Delta opioid receptors (DORs) are broadly expressed in the brain, and their activation protects cells from hypoxic/ischemic insults by counteracting disruptions of ionic homeostasis and initiating neuroprotective pathways. The DOR agonist D-Ala2-D-Leu2-Enkephalin (DADLE) promotes neuronal survival, mitigates apoptotic pathways, and protects neurons and glial cells from ischemia-induced cell death, thus making DADLE a promising therapeutic option for stroke. The significant amount of research regarding DORs and DADLE in the last decades also suggests their potential in treating other neurological disorders. METHODS: This review compiled relevant literature detailing the role of DORs and agonists in central nervous system function and neuropathologies. RESULTS: Several studies demonstrate potential mechanisms implicating a key interaction between DORs and DADLE in conferring neuroprotective benefits. A better understanding of DOR function in disease-specific contexts is critical to transitioning DOR agonists into the clinic as a therapy for stroke and other neurological diseases. CONCLUSION: Evidence-based studies support the potential of the delta-opioid family of receptors and its ligands in developing novel therapeutic strategies for stroke and other brain disorders.

A Genetically Encoded Biosensor Reveals Location Bias of Opioid Drug Action.

Opioid receptors (ORs) precisely modulate behavior when activated by native peptide ligands but distort behaviors to produce pathology when activated by non-peptide drugs. A fundamental question is how drugs differ from peptides in their actions on target neurons. Here, we show that drugs differ in the subcellular location at which they activate ORs. We develop a genetically encoded biosensor that directly detects ligand-induced activation of ORs and uncover a real-time map of the spatiotemporal organization of OR activation in living neurons. Peptide agonists produce a characteristic activation pattern initiated in the plasma membrane and propagating to endosomes after receptor internalization. Drugs produce a different activation pattern by additionally driving OR activation in the somatic Golgi apparatus and Golgi elements extending throughout the dendritic arbor. These results establish an approach to probe the cellular basis of neuromodulation and reveal that drugs distort the spatiotemporal landscape of neuronal OR activation.

d-Alanine 2, Leucine 5 Enkephaline (DADLE)-mediated DOR activation augments human hUCB-BFs viability subjected to oxidative stress via attenuation of the UPR.

Human mesenchymal stem cells (hMSCs) although being potent in repairing injured or ischemic tissues, their success regarding tissue-regenerative approaches are hindered by the paucity in their viability. The elevated levels of reactive oxygen species (ROS) in damaged sites provoke the pernicious effects of donor MSC survival. In the present study, the effect of delta-opioid receptor (DOR) activation on human umbilical cord-blood borne fibroblasts (hUCB-BFs) survival under oxidative stress (H2O2) was evaluated. Oxidative stress which is known to trigger pathological conditions of the unfolded protein response (UPR) leads to endoplasmic reticulum stress. Upon its activation by D-Alanine 2, Leucine 5 Enkephaline (DADLE, selective DOR agonist) in hUCB-BFs under oxidative stress, a significant down regulation (~2 folds) of key UPR genes was observed as determined by qPCR, Thioflavin-T protein aggregation assay and western blot analysis. Concomitantly, the oxidative stress-mediated cell-death was ameliorated and the viable-cells' percentage was enhanced following DOR activation. The intracellular ROS production upon H2O2 treatment as determined by CM-H2DCFDA staining was repressed, the anti-apoptotic marker Bcl-2 was upregulated along with a significant suppression in the expression levels of pro-apoptotic proteins Bax and Bad upon DOR activation. Upon subsequent treatment with naltrindole, the effects of DADLE-induced cytoprotection were reverted significantly. These results propound the role of DADLE-mediated DOR-activation on improvement of the viability, which might succour successful hUCB-BFs transplants and greatly absolve the inefficacy of tissue-specific engineered transplants.

Rapid hypoxia preconditioning protects cortical neurons from glutamate toxicity through delta-opioid receptor.

BACKGROUND AND PURPOSE: Hypoxia preconditioning (HPC), rapid or delayed, has been reported to induce neuroprotection against subsequent severe stress. Because delta-opioid receptor (DOR) plays an important role in delayed HPC-induced neuroprotection against severe hypoxic injury, we asked whether DOR is also involved in the rapid HPC-induced neuroprotection. METHODS: Cultured rat cortical neurons at culture days 8 to 9 were exposed to a short-term hypoxia (1% O2 for 30 minutes) to induce HPC followed by 30-minute normoxia before exposing to glutamate toxicity (100 micromol/L; 4 hours). Neuronal viability was assessed by lactate dehydrogenase leakage and morphological assessment. Protein and mRNA levels of DOR were detected by receptor binding and RT-PCR, respectively. Naltrindole was used to block DOR. Developmental changes in NMDA receptor expression was measured by Western blots. RESULTS: HPC significantly reduced the glutamate-induced neuronal injury. Receptor binding showed that HPC increased DADLE (a DOR ligand) binding density in the cultured cortical neurons by >90% over control level (P<0.05), although RT-PCR did not detect any appreciable change in DOR mRNA. DOR inhibition with naltrindole had no effect on neuronal injury and completely abolished the HPC-induced neuroprotection. In contrast to HPC-induced increase in DADLE binding density, prolonged hypoxia caused severe neuronal injury with a significant decrease in DADLE binding density and DOR mRNA level. CONCLUSIONS: DOR is involved in neuroprotection induced by rapid HPC in cortical neurons.

Receptor binding on whole cells can oscillate.

The study of the kinetics of binding of the opiate receptor agonist [3H]DADLE with NG108-15 cell suspensions has revealed a new periodic biological phenomenon, i.e., oscillations of the cellular receptor activity. The absence of oscillations for binding of the receptor antagonist shows that oscillations occur as a result of the transformation of the receptor signal only.

Stability of oxymethyl-modified coumarinic acid cyclic prodrugs of diastereomeric opioid peptides in biological media from various animal species including human.

In vitro stability studies of oxymethyl-modified coumarinic acid (OMCA) cyclic prodrugs of the diastereomeric opioid peptides DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), [Ala2,D-Leu5]-Enk (H-Tyr-Ala-Gly-Phe-D-Leu-OH), [D-Ala2,Leu5]-Enk (H-Tyr-D-Ala-Gly-Phe-Leu-OH), and [Ala2,Leu5]-Enk (H-Tyr-Ala-Gly-Phe-Leu-OH) were conducted to evaluate how the chirality of specific amino acid residues (Ala2 and Leu5) in the peptide portion affects their bioconversion by esterases. The stability studies were conducted at 37 degrees C in plasma and tissue homogenates (liver and brain) from five animal species (rat, mouse, canine, guinea pig, and hamster) and human in an attempt to identify an animal species that had a "prodrug bioconversion profile" comparable to that of humans. Initially, the total esterase activity in these biological media was measured using p-nitrophenyl butyrate (PNPB) as a substrate. By repeating this activity assay in the presence of paraoxon, a potent esterase B inhibitor, it was possible to estimate the relative amounts of esterases B and esterases A/C in a biological sample. Stability studies of the cyclic prodrugs were carried out under identical conditions, that is, in the presence and absence of paraoxon. Significant differences in the rates of hydrolysis of the cyclic prodrugs were observed, particularly between cyclic prodrugs with differences in the chirality of the amino acid on the C-terminus of the peptide portion, for example, L-amino acids at the C-terminus hydrolyzed more rapidly than D-amino acids. This stereoselective hydrolysis was independent of the animal species but tended to be more pronounced in brain and liver homogenates compared to plasma. Increased esterase specific activity, as measured by PNPB, in the biological media did not necessarily correlate with increased bioconversion rates of the cyclic prodrugs. The enzymatic stability profiles of the cyclic prodrugs in biological media from canine and guinea pig most closely resembled the profiles from human biological media. Therefore, canine and guinea pig appear to be the most relevant animal models for conducting pharmacokinetic studies on these cyclic prodrugs of opioid peptides.

Identification and characterization of opioid-binding sites present in the Ishikawa human endometrial adenocarcinoma cell line.

Normal epithelial cells of human endometrium, and Ishikawa human endometrial adenocarcinoma cells (an in vitro model for the study of steroid hormone effects on human endometrium) have been found to express and secrete opioid peptides deriving from proenkephalin, prodynorphin, and proopiomelanocortin. These opioids may act locally, affecting the uterine tissues. In the present study, we identified and characterized opioid-binding sites on the Ishikawa cell line, producing evidence for the mechanism of local opioid action. We used an acid shock before the receptor assay to dissociate any endogenously bound peptide. The acidification improved specific binding by 2- to 4.5-fold. Characterization of opioid binding using different radiolabeled opioids and effectors has shown the existence of a low concentration of delta-sites (Kd, 6.20 nmol/L; 4,890 sites/cell), no mu-sites, low affinity kappa 1-sites (Kd, 10.8 nmol/L; 276,000 sites/cell), kappa 2-sites with high affinity for ethylketocyclazocine (Kd, approximately 1 nmol/L) and low affinity for diprenorphine (Kd, approximately 8 nmol/L) at a concentration of 93,000 sites/cell, and high affinity kappa 3-sites (Kd, 3.6 nmol/L; 77,000 sites/cell). In conclusion, our report characterizes opioid sites in a particular and homogeneous cell type of human endometrium, i.e. in epithelial cells. The coexistence of opioid sites and their endogenous ligands in the Ishikawa cell line makes these cells a good model for the study of autocrine/paracrine interactions of opioids in nonneural tissues.

Purification and reconstitution of the delta opioid receptor.

The delta opioid receptor has been purified, in an active form, by succinylmorphine affinity chromatography. The receptor was purified partially from bovine frontal cortex and to apparent homogeneity from neuroblastoma x glioma hybrid NG108-15 cells as observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining. Antiserum to the purified bovine receptor inhibited ligand binding to membranes and immunoprecipitated a 58 kDa protein from NG108-15 cells. Reconstitution of the receptor with lipids enhanced binding by 9-fold. The 58 kDa band protein after electroelution and reconstitution with lipids also showed specific binding, indicating that the receptor could be renatured even after SDS-PAGE in an appropriate lipid environment.

Kinetic evidence that His-711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state.

Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane-bound Zn-metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc-endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site-directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP-Gln711 and NEP-Phe711). Determination of kinetic parameters showed that both mutants had Km values very similar to that of the non-mutated enzyme but that their kcat values were 25-fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.

Function of negative charge in the "address domain" of deltorphins.

Deltorphins A, B, and C exhibit high delta-affinities (Ki = 0.12-0.31 nM) and very high delta-receptor binding selectivities (Ki mu/Ki delta = 1800-4100). A study of the delta-receptor binding properties of 15 deltorphin analogues focused primarily on the influence of the anionic group in the C-terminal tetrapeptide. Amidation of the beta-carboxyl groups of [Asp7], [Glu4], or [Asp4] in deltorphins A, B, and C, respectively, yielded peptides with enhanced mu-receptor affinities and minor changes in delta-affinities (Ki delta = 0.20-0.65 nM), but 5-8-fold diminished delta-selectivities. A free C-terminal carboxyl group markedly reduced delta-affinities and decreased delta-selectivities 6-11-fold; thus, the C-terminal amide group critically facilitates delta-affinity. Modifications in the anionic charged group or hydrophobic residues in the C-terminal tetrapeptide address domain of deltorphin A altered spatial distributions critical for delta-affinity and selectivity; e.g., [Nle6]deltorphin A enhanced mu-affinity and lowered delta-selectivity by two-thirds; the progressive, step-wise repositioning of Asp in deltorphin C (from position 4 to 7) was accompanied by linear decreases in delta-affinities and -selectivities, and increased mu-affinities of these peptides; enhancement of the charge density to -2, in [Asp6, Asp-OH7]deltorphin A, decreased delta-affinity and -selectivity, while [Asp4,5,His7]deltorphin A bound to neither mu- nor delta-sites. These results demonstrate that while an anionic group may occasionally facilitate high delta-receptor affinity, it represents an absolute requirement for the high delta-binding selectivity of these peptides. The locations of the charged groups relative to hydrophobic residues in the address domain of the peptide are also critical determinants for both delta-affinity and -selectivity.

Probes for narcotic receptor mediated phenomena. 23. Synthesis, opioid receptor binding, and bioassay of the highly selective delta agonist (+)-4-[(alpha R)-alpha-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]- N,N-diethylbenzamide (SNC 80) and related novel nonpeptide delta opioid receptor ligands.

The highly selective delta (delta) opioid receptor agonist SNC 80 [(+)-4- [(alpha R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N ,N- diethylbenzamide, (+)-21] and novel optically pure derivatives were synthesized from the enantiomers of 1-allyl-trans-2,5-dimethylpiperazine (2). The piperazine (+/-)-2 was synthesized, and its enantiomers were obtained on a multigram scale in > 99% optical purity by optical resolution of the racemate with the camphoric acids. The absolute configuration of (+)-2 was determined to be 2S,5R by X-ray analysis of the salt with (+)-camphoric acid. Since the chirality of the starting material was known, and the relative configuration of compounds (-)-21, (-)-22, and (+)-23 were obtained by single-crystal X-ray analysis, the assignment of the absolute stereochemistry of the entire series could be made. Radioreceptor binding studies in rat brain preparations showed that methyl ethers (+)-21 (SNC 80) and (-)-25 exhibited strong selectivity for rat delta receptors with low nanomolar affinity to delta receptors and only micromolar affinity for rat mu (mu) opioid receptors. Compounds (-)-21, (-)-22, and (-)-23 showed micromolar affinities for delta opioid receptors. The unsubstituted derivative (+)-22 and the fluorinated derivative (-)-27 showed > 2659- and > 2105-fold delta/mu binding selectivity, respectively. The latter derivatives are the most selective ligands described in the new series. Studies with some of the compounds described in the isolated mouse vas deferens and guinea pig ileum bioassays revealed that all were agonists with different degrees of selectivity for the delta opioid receptor. These data show that (+)-21 and (+)-22 are potent delta receptor agonists and suggest that these compounds will be valuable tools for further study of the delta opioid receptor at the molecular level, including its function and role in analgesia and drug abuse.

Synthesis, biological evaluation, and quantitative receptor docking simulations of 2-[(acylamino)ethyl]-1,4-benzodiazepines as novel tifluadom-like ligands with high affinity and selectivity for kappa-opioid receptors.

The synthesis and biological evaluation of a series of 2-substituted 5-phenyl-1,4-benzodiazepines, structurally related to tifluadom (5), the only benzodiazepine that acts simultaneously as a kappa-opioid agonist and a cholecystokinin-A (CCK-A) antagonist, are reported. The radioligand binding models used in these studies were [(125)I](BH)-CCK-8 in rat pancreas (CCK-A), [(3)H]-(MENLE(28,31))-cck-8 in guinea pig cerebral cortex (CCK-B), and [(3)H]U-69593 (kappa(1)), [(3)H]DAMGO (mu), and [(3)H]DADLE (delta) in guinea pig brain. All the title compounds were devoid of significant affinity for both CCK-A and CCK-B receptors, while some of them bound with nanomolar affinity and high selectivity for kappa-opioid receptors. In particular, the 2-thienyl derivative 7A(X = H) with a K(i) = 0.50 nM represents a clear improvement with respect to tifluadom, showing a comparable potency but higher selectivity. The application of computational simulations and linear regression analysis techniques to the complexes between guinea pig kappa (kappa(1))-receptor and the title compounds allowed the identification of the structural determinants for recognition and quantitative elucidation of the structure-affinity relationships in this class of receptors.