Search Results (6)

Stroke is one of the leading causes of death. (D-ala2, D-leu5) enkephalin (DADLE) is a synthetic peptide and highly selective delta opioid receptor (δOR) agonist that has exhibited protective properties in ischemia. However, the specific target and mechanism are still unclear. The present study explores the expression of δOR on brain microvascular endothelial cells (BMECs) and whether DADLE could relieve I/R-induced injury by reducing apoptosis. A lateral ventricular injection of DADLE for pretreatment, the neurofunctional behavior score, and TTC staining, were used to evaluate the protective effect of DADLE. Immunofluorescence technology was used to label different types of cells with apoptosis-positive signals to test co-localization status. Primary cultured BMECs were separated and treated with DADLE, accompanied by OGD/R. The CCK-8 test was conducted to evaluate cell viability and TdT-mediated dUTP Nick-end Labelling (TUNEL) staining to test apoptosis levels. The levels of apoptosis-related proteins were analyzed by Western blotting. The co-localization results showed that BMECs, but not astrocytes, microglia, or neurons, presented mostly TUNEL-positive signals, especially in the Dentate gyrus (DG) area of the hippocampus. Either activation of δORs on rats’ brains or primary BMECs mainly reduce cellular apoptosis and relieve the injury. Interference with the expression δOR could block this effect. DADLE also significantly increased levels of Bcl-2 and reduced levels of Bax. δOR’s expressions can be detected on the BMECs, but not on the HEK293 cells, by Western blotting and IFC. Therefore, DADLE exerts a cytoprotective effect, primarily under hypoxia-ischemic injury/reperfusion conditions, by targeting BMECs to inhibit apoptosis. Full article

To develop potent and safer analgesics, we designed and synthesized a novel enantiomerically enriched ethereal analog of (R)-iso-moramide, namely 2-[(2R)-2-(morpholin-4-yl)propoxy]-2,2-diphenyl-1-(pyrrolidin-1-yl)ethan-1-one. The titled active agent can potentially serve as a powerful synthetic opiate with an improved affinity and selectivity toward opioid receptors (ORs). This hypothesis was postulated based on docking studies regarding the respective complexes between the designed ligand and µ-OR, δ-OR, and κ-OR. The key step of the elaborated asymmetric synthesis of novel analog involves lipase-catalyzed kinetic resolution of racemic 1-(morpholin-4-yl)propan-2-ol, which was accomplished on a 10 g scale via an enantioselective transesterification employing vinyl acetate as an irreversible acyl donor in tert-butyl methyl ether (MTBE) as the co-solvent. Next, the obtained homochiral (S)-(+)-morpholino-alcohol (>99% ee) was functionalized into corresponding chloro-derivative using thionyl chloride (SOCl₂) or the Appel reaction conditions. Further transformation with N-diphenylacetyl-1-pyrrolidine under phase-transfer catalysis (PTC) conditions using O₂-saturated DMSO/NaOH mixture as an oxidant furnished the desired levorotatory isomer of the title product isolated in 26% total yield after three steps, and with 89% ee. The absolute configuration of the key-intermediate of (R)-(–)-iso-moramide was determined using a modified form of Mosher’s methodology. The preparation of the optically active dextrorotatory isomer of the titled product (87% ee) was carried out essentially by the same route, utilizing (R)-(–)-1-(morpholin-4-yl)propan-2-ol (98% ee) as a key intermediate. The spectroscopic characterization of the ethereal analog of iso-moramide and the enantioselective retention relationship of its enantiomers using HPLC on the cellulose-based chiral stationary phase were performed. Moreover, as a proof-of-principle, single-crystal X-ray diffraction (XRD) analysis of the synthesized 2-[(2R)-2-(morpholin-4-yl)propoxy]-2,2-diphenyl-1-(pyrrolidin-1-yl)ethan-1-one is reported. Full article

The δ-opioid receptor (δOR) holds great potential as a therapeutic target. Yet, clinical drug development, which has focused on δOR agonists that mimic the potent and selective tool compound SNC80 have largely failed. It has increasingly become apparent that the SNC80 scaffold carries with it potent and efficacious β-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit β-arrestin, as it has been suggested that compounds that efficaciously recruit β-arrestin are proconvulsant. The screen identified a hit compound and further characterization using cellular binding and signaling assays revealed that this molecule (R995045, compound 1) exhibited ten-fold selectivity over µ- and κ-opioid receptors. Compound 1 represents a novel chemotype at the δOR. A subsequent characterization of fourteen analogs of compound 1, however did not identify a more potent δOR agonist. Computational modeling and in vitro characterization of compound 1 in the presence of the endogenous agonist leu-enkephalin suggest compound 1 may also bind allosterically and negatively modulate the potency of Leu-enkephalin to inhibit cAMP, acting as a ‘NAM-agonist’ in this assay. The potential physiological utility of such a class of compounds will need to be assessed in future in vivo assays. Full article

Oxytocin (OT) influences various physiological functions such as uterine contractions, maternal/social behavior, and analgesia. Opioid signaling pathways are involved in one of the analgesic mechanisms of OT. We previously showed that OT acts as a positive allosteric modulator (PAM) and enhances μ-opioid receptor (MOR) activity. In this study, which focused on other opioid receptor (OR) subtypes, we investigated whether OT influences opioid signaling pathways as a PAM for δ-OR (DOR) or κ-OR (KOR) using human embryonic kidney-293 cells expressing human DOR or KOR, respectively. The CellKey^TM results showed that OT enhanced impedance induced by endogenous/exogenous KOR agonists on KOR-expressing cells. OT did not affect DOR activity induced by endogenous/exogenous DOR agonists. OT potentiated the KOR agonist-induced Gi/o protein-mediated decrease in intracellular cAMP, but did not affect the increase in KOR internalization caused by the KOR agonists dynorphin A and (-)-U-50488 hydrochloride (U50488). OT did not bind to KOR orthosteric binding sites and did not affect the binding affinities of dynorphin A and U50488 for KOR. These results suggest that OT is a PAM of KOR and MOR and enhances G protein signaling without affecting β-arrestin signaling. Thus, OT has potential as a specific signaling-biased PAM of KOR. Full article

As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu⁵-enkephalin and the more metabolically stable synthetic peptide (d-Ala², d-Leu⁵)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR’s protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe⁴ position of Leu⁵-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit β-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe⁴ of Leu⁵-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe⁴ may be combined with other modifications to Leu⁵-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties. Full article

The discovery of endogenous peptide ligands for morphine binding sites occurred in parallel with the identification of three subclasses of opioid receptor (OR), traditionally designated as μ, δ, and κ, along with the more recently defined opioid-receptor-like (ORL1) receptor. Early efforts in opioid receptor radiochemistry focused on the structure of the prototype agonist ligand, morphine, although N-[methyl-¹¹C]morphine, -codeine and -heroin did not show significant binding in vivo. [¹¹C]Diprenorphine ([¹¹C]DPN), an orvinol type, non-selective OR antagonist ligand, was among the first successful PET tracers for molecular brain imaging, but has been largely supplanted in research studies by the μ-preferring agonist [¹¹C]carfentanil ([¹¹C]Caf). These two tracers have the property of being displaceable by endogenous opioid peptides in living brain, thus potentially serving in a competition-binding model. Indeed, many clinical PET studies with [¹¹C]DPN or [¹¹C]Caf affirm the release of endogenous opioids in response to painful stimuli. Numerous other PET studies implicate μ-OR signaling in aspects of human personality and vulnerability to drug dependence, but there have been very few clinical PET studies of μORs in neurological disorders. Tracers based on naltrindole, a non-peptide antagonist of the δ-preferring endogenous opioid enkephalin, have been used in PET studies of δORs, and [¹¹C]GR103545 is validated for studies of κORs. Structures such as [¹¹C]NOP-1A show selective binding at ORL-1 receptors in living brain. However, there is scant documentation of δ-, κ-, or ORL1 receptors in healthy human brain or in neurological and psychiatric disorders; here, clinical PET research must catch up with recent progress in radiopharmaceutical chemistry. Full article