Neuroprotective effects of miRNA-326 knockout in neonatal hypoxic-ischemic brain damage mice via the δ-opioid receptor.

Hypoxic-ischemic brain damage (HIBD) in the perinatal period is an important cause of cerebral damage and long-term neurological sequelae, and can place much pressure on families and society. Our previous study demonstrated that miRNA-326 reduces neuronal apoptosis by up-regulating the δ-opioid receptor (DOR) under oxygen-glucose deprivation in vitro. In the present study, we aimed to explore the neuroprotective effects of the miRNA-326/DOR axis by inhibiting apoptosis in HIBD using neonatal miRNA-326 knockout mice. Neonatal C57BL/6 mice, neonatal miRNA-326 knockout mice, and neonatal miRNA-326 knockout mice intraperitoneally injected with the DOR inhibitor naltrindole were treated with hypoxic-ischemia (HI). Neurological deficit scores, magnetic resonance imaging, terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end labeling, and Caspase-3, Bax, and B-cell lymphoma 2 (Bcl-2) expression were evaluated on day 2 after HI. Neurobehavioral analyses were performed on days 2 and 28 after HI. Additionally, the Morris water maze test was conducted on days 28. Compared with HI-treated neonatal C57BL/6 mice, HI-treated neonatal miRNA-326 knockout mice had higher neurological deficit scores, smaller cerebral infarction areas, and improved motor function, reaction ability, and long-term spatial learning and memory. These effects were likely the result of inhibiting apoptosis; the DOR inhibitor reversed these neuroprotective effects. Our findings indicate that miRNA-326 knockout plays a neuroprotective effect in neonatal HIBD by inhibiting apoptosis via the target gene DOR.

Expression profile and immunomodulatory roles of methionine-enkephalin and delta opioid receptor in Octopus ocellatus.

In this study, the expressions and distributions of methionine-enkephalin (Met-enk) and δ opioid receptor in the nervous system of Octopus ocellatus, and the immune regulatory mechanisms of Met-enk on O. ocellatus were explored. The distributions and expressions of Met-enk and δ opioid receptor were assessed by immunohistochemistry and enzyme-linked immunosorbent assay. UV-spectrophotometer, microplate reader, and flow cytometer were used to examine the effects of different concentrations of Met-enk on phagocytosis, antioxidant effects, and body surface mucus immunity of O. ocellatus hemocytes. The data were used to study the mechanisms of Met-enk immunity regulation in O. ocellatus. According to the results, the expression levels of Met-enk and δ opioid receptor in O. ocellatus lymphocytes were higher than those in hemocytes. The expression levels of Met-enk in the ganglia of O. ocellatus decreased in the following order: pedal ganglia > cerebral ganglia > visceral ganglia > optic ganglia > stellate ganglia. Moreover, the phagocytic activity of O. ocellatus hemocytes was enhanced with increasing Met-enk concentration. With increasing Met-enk concentration, the expressions of nitric oxide, total nitric oxide synthase, inducible nitric oxide synthase, catalase, hydrogen peroxide, myeloperoxidase, reduced glutathione, α-naphthy acetate esterase, and methionine aminopeptidases decreased in serums of O. ocellatus in the experimental group compared to the blank group. Similarly, the content of reduced glutathione in the hemocytes of O. ocellatus was also lower in the experimental group than in the blank group; however, the expressions of other substances were higher compared to the blank group. Furthermore, α-naphthy acetate esterase, myeloperoxidase, and hydrogen peroxide expressions in mucus immunity trials of the body surface were lower in the experimental group compared to the blank group. These results indicate that the distributions and expressions of Met-enk and δ opioid receptor in the nervous system of O. ocellatus were related to axoplasmic transport and immune regulation mechanisms. Met-enk participates in cellular immunity, humoral immunity, and mucus immunity in the form of neurotransmitters, thereby regulating the immune response of O. ocellatus.

The Role of δ2-Opioid Receptors in the Regulation of Tolerance of Isolated Cardiomyocytes to Hypoxia and Reoxygenation.

Hypoxia (20 min) and reoxygenation (30 min) were simulated on isolated rat cardiomyocytes to evaluate the cytoprotective effect of selective δ2-opioid receptor agonist deltorphin II, opioid receptor antagonist naloxone methiodide, µ-opioid receptor antagonist CTAP, κ-opioid receptor antagonist nor-binaltorphimine, ε1-opioid receptor antagonist BNTX, and δ2-opioid receptors naltriben. Deltorphin II was administered 5 min before reoxygenation, antagonists were administered 10 min before reoxygenation. The cytoprotective effect of deltorphin II was assessed by the number of cardiomyocytes survived after hypoxia/reoxygenation, as well as by the lactate dehydrogenase content in the incubation medium. It has been established that the cytoprotective effect of deltorphin II occurs at a concentration of 64 nmol/liter and is associated with activation of δ2-opioid receptors.

In vivo mapping of a GPCR interactome using knockin mice.

With over 30% of current medications targeting this family of proteins, G-protein-coupled receptors (GPCRs) remain invaluable therapeutic targets. However, due to their unique physicochemical properties, their low abundance, and the lack of highly specific antibodies, GPCRs are still challenging to study in vivo. To overcome these limitations, we combined here transgenic mouse models and proteomic analyses in order to resolve the interactome of the δ-opioid receptor (DOPr) in its native in vivo environment. Given its analgesic properties and milder undesired effects than most clinically prescribed opioids, DOPr is a promising alternative therapeutic target for chronic pain management. However, the molecular and cellular mechanisms regulating its signaling and trafficking remain poorly characterized. We thus performed liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses on brain homogenates of our newly generated knockin mouse expressing a FLAG-tagged version of DOPr and revealed several endogenous DOPr interactors involved in protein folding, trafficking, and signal transduction. The interactions with a few identified partners such as VPS41, ARF6, Rabaptin-5, and Rab10 were validated. We report an approach to characterize in vivo interacting proteins of GPCRs, the largest family of membrane receptors with crucial implications in virtually all physiological systems.

Switching of delta opioid receptor subtypes in central amygdala microcircuits is associated with anxiety states in pain.

Anxiety is often comorbid with pain. Delta opioid receptors (DORs) are promising targets for the treatment of pain and mental disorders with little addictive potential. However, their roles in anxiety symptoms at different stages of pain are unclear. In the current study, mice with inflammatory pain at the fourth hour following complete Freund's adjuvant (CFA) injection displayed significant anxiety-like behavior, which disappeared at the seventh day. Combining electrophysiology, optogenetics, and pharmacology, we found that activation of delta opioid receptor 1 (DOR1) in the central nucleus amygdala (CeA) inhibited both the anxiolytic excitatory input from the basolateral amygdala (BLA) and the anxiogenic excitatory input from the parabrachial nucleus (PBN). In contrast, activation of delta opioid receptor 2 (DOR2) did not affect CeA excitatory synaptic transmission in normal and 4-h CFA mice but inhibited the excitatory projection from the PBN rather than the BLA in 7-day CFA mice. Furthermore, the function of both DOR1 and DOR2 was downregulated to the point of not being detectable in the CeA of mice at the 21st day following CFA injection. Taken together, these results suggest that functional switching of DOR1 and DOR2 is associated with anxiety states at different stages of pain via modulating the activity of specific pathways (BLA-CeA and PBN-CeA).

Distribution of delta and mu opioid receptor mRNA in rodent dorsal root ganglia neurons.

Primary afferents are responsible for transmitting signals produced by noxious stimuli from the periphery to the spinal cord. Mu and delta opioid receptors (MOP and DOP) have analgesic properties and are highly expressed in dorsal root ganglia (DRG) neurons. In humans, spinal DOP is almost exclusively located on central terminals of DRG neurons, whereas in rodents, it is expressed both on presynaptic terminals and spinal neurons. In this study, we aimed to assess the distribution of MOP and DOP in the DRGs of mice and rats. Using in situ hybridization and immunofluorescence, we visualized MOP and DOP mRNA together with various neuronal markers. In rats and mice, we show that both receptors are expressed, albeit to different extents, in all types of neurons, namely, large and medium myelinated neurons (NF200-positive), small nonpeptidergic (IB4- or P2X3R-positive) and peptidergic C fibres (Tac1-positive). Overall, DOP mRNA was found to be mainly expressed in large and medium myelinated neurons, whereas MOP mRNA was mainly found in C fibres. The distribution of MOP and DOP, however, slightly differs between rats and mice, with a higher proportion of small nonpeptidergic C fibres expressing DOP mRNA in mice than in rats. We further found that neither morphine nor inflammation affected the distribution of the receptor mRNA. Because of their location, our results confirm that MOP and DOP have the potential to alleviate similar types of pain and that this effect could slightly differ between species.

Delta opioid receptors on nociceptive sensory neurons mediate peripheral endogenous analgesia in colitis.

BACKGROUND: Inflammatory visceral pain is endogenously controlled by enkephalins locally released by mucosal CD4+ T lymphocytes in mice. The present study aimed at identifying opioid receptor(s) expressed on nociceptive sensory nerves involved in this peripheral opioid-mediated analgesia. METHODS: The peripheral analgesia associated with the accumulation of CD4+ T lymphocytes within the inflamed colonic mucosa was assessed in conditional knockout mice specifically deleted for either of the two opioid receptors for enkephalins (i.e., µ (MOR) and δ (DOR) receptors) in Nav1.8-expressing sensory neurons in the dextran sulfate sodium (DSS)-induced colitis model. RESULTS: Endogenous analgesia is lost in conditional knockout mice for DOR, but not MOR at the later phase of the DSS-induced colitis. The absence of either of the opioid receptors on sensory nerves had no impact on both the colitis severity and the rate of T lymphocytes infiltrating the inflamed colonic mucosa. CONCLUSION: The key role of DOR on primary afferents in relieving intestinal inflammatory pain opens new therapeutic opportunities for peripherally restricted DOR analgesics to avoid most of the side effects associated with MOR-targeting drugs used in intestinal disorders.

Selective N-glycan editing on living cell surfaces to probe glycoconjugate function.

Cell surfaces are glycosylated in various ways with high heterogeneity, which usually leads to ambiguous conclusions about glycan-involved biological functions. Here, we describe a two-step chemoenzymatic approach for N-glycan-subtype-selective editing on the surface of living cells that consists of a first 'delete' step to remove heterogeneous N-glycoforms of a certain subclass and a second 'insert' step to assemble a well-defined N-glycan back onto the pretreated glyco-sites. Such glyco-edited cells, carrying more homogeneous oligosaccharide structures, could enable precise understanding of carbohydrate-mediated functions. In particular, N-glycan-subtype-selective remodeling and imaging with different monosaccharide motifs at the non-reducing end were successfully achieved. Using a combination of the expression system of the Lec4 CHO cell line and this two-step glycan-editing approach, opioid receptor delta 1 (OPRD1) was investigated to correlate its glycostructures with the biological functions of receptor dimerization, agonist-induced signaling and internalization.

FBNTI, a DOR-Selective Antagonist That Allosterically Activates MOR within a MOR-DOR Heteromer.

This report describes the unique pharmacological profile of FBNTI, a potent DOR antagonist that acts as a MOR agonist via an allosteric mechanism. Binding of FBNTI to opioid receptors expressed in HEK 293 cells revealed a 190-fold greater affinity for DOR (Ki = 0.84 nM) over MOR (Ki = 160 nM). In mice, intrathecal FBNTI produced potent antinociception (ED50 = 46.9 pmol/mouse), which was antagonized by selective MOR antagonists (CTOP, ß-FNA). Autoantagonism of the MOR agonism by FBNTI was observed above the ED75 dose, suggesting antagonism of activated MOR. That FBNTI is devoid of agonism in DOR knockout mice is consistent with allosteric activation of the MOR protomer via FBNTI bound to within a MOR-DOR heteromer. This proposed mechanism is supported by calcium mobilization assays, which indicate that FBNTI selectively activates the MOR-DOR heteromer and functionally antagonizes the MOR protomer at >ED75. The unprecedented mode of MOR activation by FBNTI may be responsible for the lack of tolerance after intrathecal (i.t.) administration. FBNTI was highly effective upon topical administration to the ipsolateral hind paw in the Hargreaves assay (EC50 = 0.17 ± 0.08 µM) and without significant contralateral activity, suggesting a lack of systemic exposure.

A Delta-Opioid Receptor Gene Polymorphism Moderates the Therapeutic Response to Extended-Release Buprenorphine in Opioid Use Disorder.

BACKGROUND: Buprenorphine treatment is not equally effective in all patients with opioid use disorder (OUD). Two retrospective studies showed that, among African Americans (AAs), rs678849, a polymorphism in the delta-opioid receptor gene, moderated the therapeutic effect of sublingual buprenorphine. METHODS: We examined rs678849 as a moderator of the response to an extended-release subcutaneous buprenorphine formulation (BUP-XR) in a 24-week OUD treatment study of 127 AAs and 327 European Americans (EAs). Participants were randomly assigned to receive: (1) BUP-XR as 2 monthly injections of 300 mg followed by either 300 mg monthly or 100 mg monthly for 4 months, or (2) monthly volume-matched placebo injections. Generalized estimating equations logistic regression analyses tested, per population group, the main and interaction effects of treatment (BUP-XR vs placebo) and genotype group (rs678849*CC vs CT/TT) on weekly urine drug screens (UDS). RESULTS: Among AAs, the placebo group had higher rates of opioid-positive UDS than the BUP-XR group (log odds ratio = 1.67, 95% CI = 0.36, 2.98), but no genotype by treatment effect (P = .80). Among EAs, the placebo group also showed higher rates of opioid-positive UDS than the BUP-XR group (log odds ratio = 1.97, 95% CI = 1.14, 2.79) but a significant genotype by treatment interaction (χ 2(1) = 4.33, P = .04). CONCLUSION: We found a moderating effect of rs678849 on the response to buprenorphine treatment of OUD in EAs, but not AAs. These findings require replication in well-powered, prospective studies of both AA and EA OUD patients treated with BUP-XR and stratified on rs678849 genotype.

Alternative Splicing of Opioid Receptor Genes Shows a Conserved Pattern for 6TM Receptor Variants.

The opioid receptor (OPR) family comprises the mu-, delta-, and kappa-opioid, and nociceptin receptors that belong to the superfamily of 7-transmembrane spanning G protein-coupled receptors (GPCRs). The mu-opioid receptor is the main target for clinically used opioid analgesics, and its biology has been extensively studied. The N-terminally truncated 6TM receptors isoform produced through alternative splicing of the OPRM1 gene displays unique signaling and analgesic properties, but it is unclear if other OPRs have the same ability. In this study, we have built a comprehensive map of alternative splicing events that produce 6TM receptor variants in all the OPRs and demonstrated their evolutionary conservation. We then obtained evidence for their translation through ribosomal footprint analysis. We discovered that N-terminally truncated 6TM GPCRs are rare in the human genome and OPRs are overrepresented in this group. Finally, we also observed a significant enrichment of 6TM GPCR genes among genes associated with pain, psychiatric disorders, and addiction. Understanding the biology of 6TM receptors and leveraging this knowledge for drug development should pave the way for novel therapies.

Differential expression and transcription factor binding associated with genotype at a pharmacogenetic variant in OPRD1.

BACKGROUND: The functional mechanism is unknown for many genetic variants associated with substance use disorder phenotypes. Rs678849, an intronic variant in the delta-opioid receptor gene (OPRD1), has been found to predict regional brain volume, addiction risk, and the efficacy of buprenorphine/naloxone in treating opioid use disorder. The variant has also been implicated as an expression quantitative trait locus (eQTL) for several genes. OBJECTIVES: The objective of this study was to identify functional differences between the two alleles of rs678849 in vitro. We hypothesized that the two alleles of rs678849 would have different effects on transcriptional activity due to differential interactions with transcription factors. METHODS: 15bp regions containing the C or T alleles of rs678849 were cloned into luciferase constructs and transfected into BE(2)C neuroblastoma cells to test the effect on transcription. Electrophoretic mobility shift assays (EMSA) using nuclear lysates from BE(2)C cell or human postmortem medial prefrontal cortex were used to identify proteins that differentially bound the two alleles. RESULTS: At 24 hours post-transfection, the C allele construct had significantly lower luciferase expression than the T allele construct and empty vector control (ANOVA p < .001). Proteomic analysis and supershift assays identified XRCC6 as a transcription factor specifically binding the C allele, whereas hnRNP D0 was found to specifically bind the T allele. CONCLUSION: These functional differences between the C and T alleles may help explain the psychiatric and neurological phenotype differences predicted by rs678849 genotype and the potential role of the variant as an eQTL.

Opioid Addiction and Opioid Receptor Dimerization: Structural Modeling of the OPRD1 and OPRM1 Heterodimer and Its Signaling Pathways.

Opioid addiction is a complex phenomenon with genetic, social, and other components. Due to such complexity, it is difficult to interpret the outcome of clinical studies, and thus, mutations found in individuals with these addictions are still not indisputably classified as opioid addiction-causing variants. Here, we computationally investigated two such mutations, A6V and N40D, found in the mu opioid receptor gene OPRM1. The mutations are located in the extracellular domain of the corresponding protein, which is important to the hetero-dimerization of OPRM1 with the delta opioid receptor protein (OPRD1). The hetero-dimerization of OPRD1-OPRM1 affects the signaling pathways activated by opioids and natural peptides and, thus, could be considered a factor contributing to addiction. In this study, we built four 3D structures of molecular pathways, including the G-protein signaling pathway and the ß-arrestin signaling pathway of the heterodimer of OPRD1-OPRM1. We also analyzed the effect of mutations of A6V and N40D on the stability of individual OPRM1/OPRD1 molecules and the OPRD1-OPRM1 heterodimer with the goal of inferring their plausible linkage with opioid addiction. It was found that both mutations slightly destabilize OPRM1/OPRD1 monomers and weaken their association. Since hetero-dimerization is a key step for signaling processes, it is anticipated that both mutations may be causing increased addiction risk.

Genetic polymorphisms in the opioid receptor delta 1 (OPRD1) gene are associated with methadone dose in methadone maintenance treatment for heroin dependence.

Delta opioid receptor (DOR) is well known to be involved in heroin dependence. This study tested the hypothesis that single nucleotide polymorphisms (SNPs) in the opioid receptor delta 1 (OPRD1) gene coding region are associated with treatment responses in a methadone maintenance therapy (MMT) cohort in Taiwan. Three hundred forty-four MMT patients were recruited. Diastolic/systolic blood pressure, heart rate, methadone dosage, and plasma concentrations of methadone were recorded. Twenty-five SNPs located within the OPRD1 genetic region were selected and genotyped from the genomic DNA of all 344 participants. After pairwise tagger analyses, tagger SNP rs204047 showed a significant association with methadone dosage (P = 0.0019), and tagger SNPs rs204047 and rs797397 were significantly associated with plasma R, S-methadone concentrations (P < 0.0006) in patients tested negative in the urine morphine test, which indicated patients with a better response to MMT. The major genotype carriers showed a higher methadone dosage and higher plasma concentrations of R, S-methadone than the minor genotype carriers. The results indicated that OPRD1 genetic variants were associated with methadone dosage and methadone plasma concentration in MMT patients with a negative morphine test result.

Molecular control of δ-opioid receptor signalling.

Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8 Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion in mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the seven-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn 131 to an alanine or a valine augments constitutive ß-arrestin-mediated signalling. Asp95Ala, Asn310Ala and Asn314Ala mutations transform classical δ-opioid antagonists such as naltrindole into potent ß-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signalling, revealing that sodium-coordinating residues act as 'efficacy switches' at a prototypic G-protein-coupled receptor.

Effects of N-Substituents on the Functional Activities of Naltrindole Derivatives for the δ Opioid Receptor: Synthesis and Evaluation of Sulfonamide Derivatives.

We have recently reported that N-alkyl and N-acyl naltrindole (NTI) derivatives showed activities for the δ opioid receptor (DOR) ranging widely from full inverse agonists to full agonists. We newly designed sulfonamide-type NTI derivatives in order to investigate the effects of the N-substituent on the functional activities because the side chain and S=O part in the sulfonamide moiety located in spatially different positions compared with those in the alkylamine and amide moieties. Among the tested compounds, cyclopropylsulfonamide 9f (SYK-839) was the most potent full inverse agonist for the DOR, whereas phenethylsulfonamide 9e (SYK-901) showed full DOR agonist activity with moderate potency. These NTI derivatives are expected to be useful compounds for investigation of the molecular mechanism inducing these functional activities.

A Structure-Activity Relationship Comparison of Imidazodiazepines Binding at Kappa, Mu, and Delta Opioid Receptors and the GABAA Receptor.

Analgesic and anti-inflammatory properties mediated by the κ opioid receptor (KOR) have been reported for oxadiazole imidazodiazepines. Affinities determined by radioligand competition assays of more than seventy imidazodiazepines using cell homogenates from HEK293 cells that overexpress KOR, µ opioid receptor (MOR), and δ opioid receptor (DOR) are presented. Affinities to synaptic, benzodiazepine-sensitive receptors (BZR) were determined with rat brain extract. The highest affinity for KOR was recorded for GL-I-30 (Ki of 27 nM) and G-protein recruitment was observed with an EC50 of 32 nM. Affinities for MOR and DOR were weak for all compounds. Ester and amide imidazodiazepines were among the most active KOR ligands but also competed with 3H-flunitrazepam for brain extract binding, which is mediated predominately by gamma aminobutyric acid type A receptors (GABAAR) of the α1-3ß2-3γ1-2 subtypes. Imidazodiazepines with carboxylic acid and primary amide groups did not bind KOR but interacted strongly with GABAARs. Pyridine substitution reduced KOR affinity. Oxadiazole imidazodiazepines exhibited good KOR binding and interacted weakly with BZR, whereas oxazole imidazodiazepines were more selective towards BZR. Compounds that lack the imidazole moiety, the pendent phenyl, or pyridine substitutions exhibited insignificant KOR affinities. It can be concluded that a subset of imidazodiazepines represents novel KOR ligands with high selectivity among opioid receptors.

Novel miRNA expression in the delta opioid signaling pathway mediated cell survivability in an in vitro model of ER stress.

Micro RNAs (miRNAs) are small non-coding RNAs which bind to the 3'-untranslated region of a mature mRNA to induce degradation; thereby regulating gene expression. It is reported that dysregulated miRNAs involved in neurodegenerative diseases including Parkinson's disease, could play a significant role as prognostic markers and therapeutic targets. Neuroprotective effect of delta opioid receptors (DOR) and its known miRNA regulation against endoplasmic reticulum (ER) stress have been reported previously by our lab. Current study focuses on understanding the regulation of novel miRNAs by DOR under ER stress. Novel miRNAs were identified for three different samples; control, tunicamycin (ER stress inducer), and tunicamycin+DADLE (DOR agonist). Differentially regulated miRNAs between the different samples were identified and pathway/target genes analysis was carried out. The results suggest that following DOR activation novel miRNAs like xxx-m0073-3p, xxx-m0225-3p, xxx-m0088-3p, xxx-m0098-5p etc. could regulate cell survival mechanisms in neuronal cells (SH-SY5Y) under ER stress.

Clathrin light chains' role in selective endocytosis influences antibody isotype switching.

Clathrin, a cytosolic protein composed of heavy and light chain subunits, assembles into a vesicle coat, controlling receptor-mediated endocytosis. To establish clathrin light chain (CLC) function in vivo, we engineered mice lacking CLCa, the major CLC isoform in B lymphocytes, generating animals with CLC-deficient B cells. In CLCa-null mice, the germinal centers have fewer B cells, and they are enriched for IgA-producing cells. This enhanced switch to IgA production in the absence of CLCa was attributable to increased transforming growth factor ß receptor 2 (TGFßR2) signaling resulting from defective endocytosis. Internalization of C-X-C chemokine receptor 4 (CXCR4), but not CXCR5, was affected in CLCa-null B cells, and CLC depletion from cell lines affected endocytosis of the δ-opioid receptor, but not the ß2-adrenergic receptor, defining a role for CLCs in the uptake of a subset of signaling receptors. This instance of clathrin subunit deletion in vertebrates demonstrates that CLCs contribute to clathrin's role in vivo by influencing cargo selectivity, a function previously assigned exclusively to adaptor molecules.

The Meta-Position of Phe4 in Leu-Enkephalin Regulates Potency, Selectivity, Functional Activity, and Signaling Bias at the Delta and Mu Opioid Receptors.

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 ß-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.