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Delta-opioid receptor protein (OPRD1) is one of the potential targets for treating pain. The presently available opioid agonists are known to cause unnecessary side effects. To discover a novel opioid agonist, our research group has synthesized a chimeric peptide MCRT and proved its potential activity through in vivo analysis. Non-synonymous SNPs (nsSNPs) missense mutations affect the functionality and stability of proteins leading to diseases. The current research was focused on understanding the role of MCRT in restoring the binding tendency of OPRD1 nsSNPs missense mutations on dynamic nature in comparison with Deltorphin-II and morphiceptin. The deleterious effects of nsSNPs were analyzed using various bioinformatics tools for predicting structural, functional, and oncogenic influence. The shortlisted nine nsSNPs were predicted for allergic reactions, domain changes, post-translation modification, multiple sequence alignment, secondary structure, molecular dynamic simulation (MDS), and peptide docking influence. Further, the docked complex of three shortlisted deleterious nsSNPs was analyzed using an MDS study, and the highly deleterious shortlisted nsSNP A149T was further analyzed for higher trajectory analysis. MCRT restored the binding tendency influence caused by nsSNPs on the dynamics of stability, functionality, binding affinity, secondary structure, residues connection, motion, and folding of OPRD1 protein., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The impact that β-arrestin proteins have on G protein-coupled receptor trafficking, signaling and physiological behavior has gained much appreciation over the past decade. A number of studies have attributed the side effects associated with the use of naturally occurring and synthetic opioids, such as respiratory depression and constipation, to excessive recruitment of β-arrestin. These findings have led to the development of biased opioid small molecule agonists that do not recruit β-arrestin, activating only the canonical G protein pathway. Similar G protein-biased small molecule opioids have been found to occur in nature, particularly within kratom, and opioids within salvia have served as a template for the synthesis of other G protein-biased opioids. Here, we present the first report of naturally occurring peptides that selectively activate G protein signaling pathways at δ opioid receptors, but with minimal β-arrestin recruitment. Specifically, we find that rubiscolin peptides, which are produced as cleavage products of the plant protein rubisco, bind to and activate G protein signaling at δ opioid receptors. However, unlike the naturally occurring δ opioid peptides leu-enkephalin and deltorphin II, the rubiscolin peptides only very weakly recruit β-arrestin 2 and have undetectable recruitment of β-arrestin 1 at the δ opioid receptor., (Copyright © 2018. Published by Elsevier B.V.)

The anti-hyperalgesic effect of the complex containing α-terpineol (αTPN) and β-cyclodextrin (βCD) was analyzed in a non-inflammatory chronic muscle pain model, as well as its mechanism of action through docking study for a possible interaction with receptors. The αTPN-βCD complex was prepared and characterized through the thermogravimetry/derivate thermogravimetry (TG/DTG), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscope (SEM). The model of chronic muscle pain was induced by two injections of pH 4.0 saline (20 μl) into the left gastrocnemius 5 days apart. After confirming hyperalgesia, male mice were treated with αTPN-βCD (25, 50 or 100 mg/kg; p.o.) or vehicle (saline 0.9%, p.o.) daily for 10 days. 1 h after the mechanical hyperalgesia, motor performance was evaluated. In addition, the systemic administration of naloxone and ondansetron tested the analgesic effect on the active opioid and serotonin receptors, respectively. The characterization tests indicated that αTPN was efficiently incorporated into βCD. The oral treatment with αTPN-βCD, at all doses tested, produced a significant (p < 0.001) decrease in the mechanical hyperalgesia, without causing any alteration in the force and in motor performance. This analgesic effect was reversed by the systemic administration of naloxone or ondansetron. These findings are corroborated by the docking study described in the present study, which verified a possible interaction of αTPN-βCD with opioid (MU, Kappa, Delta) and 5-HT receptors. Thus, it can be concluded that αTPN-βCD reduced the hyperalgesia followed by the chronic muscle pain model, probably evoked by the descending inhibitory pain system, specifically by opioid and serotoninergic receptors., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The crystal structure of the mu and kappa opioid receptors has revealed dimeric structural arrangements. Mu-delta receptors heteromers also exist and we have identified discrete cytoplasmic regions in each receptor required for oligomer formation. In the carboxyl tail of the delta receptor we identified three glycine residues (-GGG), substitution of any of these residues prevented heteromer formation. In intracellular loop 3 of both mu and delta receptors we identified three residues (-SVR), substitution of any of these residues prevented heteromer formation., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Calcium (Ca(2+)) plays a pivotal role in both cellular signaling and protein synthesis. However, it is not well understood how calcium metabolism and synthesis of secreted and membrane-bound proteins are related. Here we demonstrate that the sarco(endo)plasmic reticulum Ca(2+) ATPase 2b (SERCA2b), which maintains high Ca(2+) concentration in the lumen of the endoplasmic reticulum, interacts specifically with the human delta opioid receptor during early steps of receptor biogenesis in human embryonic kidney 293 cells. The interaction involves newly synthesized incompletely folded receptor precursors, because the association between the delta opioid receptor and SERCA2b (i) was short-lived and took place soon after receptor translation, (ii) was not affected by misfolding of the receptor, and (iii) decreased if receptor folding was enhanced by opioid receptor pharmacological chaperone. The physical association with SERCA2b was found to be a universal feature among G protein-coupled receptors within family A and was shown to occur also between the endogenously expressed luteinizing hormone receptor and SERCA2b in rat ovaries. Importantly, active SERCA2b rather than undisturbed Ca(2+) homeostasis was found to be essential for delta opioid receptor biogenesis, as inhibition of its Ca(2+) pumping activity by thapsigargin reduced the interaction and impaired the efficiency of receptor maturation, two phenomena that were not affected by a Ca(2+) ionophore A23187. Nevertheless, inhibition of SERCA2b did not compromise the functionality of receptors that were able to mature. Thus, we propose that the association with SERCA2b is required for efficient folding and/or membrane integration of G protein-coupled receptors.

A lanthanide-based assay for ligand-receptor interactions provides an attractive alternative to the traditional radiolabeled determinations in terms of sensitivity, throughput, and biohazards. We designed and tested five peptide ligands for the delta-opioid receptor that were modified with a europium (Eu)-containing chelate. These labeled ligands were tested for their binding affinities and compared with the unlabeled parental ligands. The Eu-diethylenetriaminepentaacetic acid (DTPA)-[D-Pen(2),l-Cys(5)] enkephalin (DPLCE) ligand bound to Chinese hamster ovary (CHO) cells overexpressing the human delta-opioid receptor with affinity similar to the unlabeled ligand. This ligand was used in competitive binding assays with results comparable to those obtained using the traditional radiolabeled binding assays. These lanthanide-based assays provide superior results with higher throughput and eliminate the need for radioactive waste disposal; hence, they are appropriate for high-throughput screening of ligand libraries.

It has previously been suggested that exogenous growth hormone (GH) affect quality of life and higher brain functions through the endogenous opioid system. Recently, we showed that GH down-regulate 72 and 48 kDa delta opioid receptor (DOR) proteins in the adult rat cerebral cortex and cerebellum. In the present study, we found that an antiserum raised against the N-terminus of the DOR also recognizes a 36 kDa protein, not recognized by a C-terminus-directed antiserum. We aimed to investigate the identity of the 72, 48 and 36 kDa proteins and to further study the effects of GH on their expression in different brain regions. The expression was studied in hypophysectomized (Hx) and untreated normal female rats. One subgroup of Hx rats received GH as a daily subcutaneous injection for 19 days. Our data show that treatment with GH in Hx rats normalized the expression of the 72 kDa protein in the cerebral cortex, whereas no significant effect were observed for the 48 or 36 kDa proteins. However, GH significantly reduced the ratio between the 72 and 36 kDa proteins in different brain regions of Hx rats. Our data suggest that GH reduces the levels of a 72 kDa DOR that likely represents a dimeric form of a 36 kDa DOR post-translationally truncated at the C-terminus, and that altered receptor dimerization may be involved in GH induced effects in the central nervous system.

The agonist-dependent activation and desensitization of a recombinant analog of delta-opioid receptor lacking the carboxyl-terminal (C-terminal) 31 residue peptide segment were discussed. The cDNA of the C-truncated delta-opioid receptor was created by polymerase chain reaction (PCR), stably expressed in Chinese hamster ovary cells and characterized by binding assay and function assay. Agonist [D-Pen2, D-Pen5]-enkephalin (DPDPE) stimulated the specific [35S]GTPgammaS binding to membrane fragments of cells expressing the C-truncated and the wild-type delta-opioid receptors in different levels dose-dependently. EC50 values of DPDPE on truncated and wild-types in stimulation were very similar. Agonist-dependent desensitization, which could be blocked by protein kinase inhibitor staurosporine (Stau), was observed after pretreating truncated receptors with 1 microM DPDPE for 10 min, the same as wild-types. The results reveal that the C-terminal of the delta-opioid receptor is not involved in controlling the G-protein activation and phosphorylation-related functional desensitization., (Copyright 1998 Academic Press.)

A full-length cDNA, ZFOR1, has been isolated from the teleost zebrafish (Danio rerio) using a probe from rat mu opioid receptor. ZFOR1 encodes a 373 amino acid protein with seven potential transmembrane domains that shows a high degree of homology to mammalian delta opioid receptor. We have also isolated a genomic clone which contains two exons of ZFOR1, homologous to exons 2 and 3 in mouse and human delta opioid receptor. Expression of ZFOR1 appears to be restricted to nervous tissue as assessed by Northern blot. In situ hybridization in zebrafish brain with specific probes revealed several discrete areas of ZFOR1 expression; higher levels are detected in dorsal telencephalic areas, the periventricular layer of the optic tectum, and the granular layer of the cerebellum. This is the first molecular evidence of the presence of the delta opioid receptor in a non-mammalian species and suggests that it has been highly conserved throughout vertebrate evolution.

The pharmacological effects caused by use of opiate are exerted through the opioid receptors (ORs). ORs couple to the inhibitory G protein (Gi) and result in decreased cAMP levels upon activation by specific agonists. To initiate study of the structure-function relationship during this process, we first ectopically expressed the wild-type delta OR and a C-terminally truncated mutant in CHO cells to investigate the necessity of its C-terminus. The binding potency of both the wild-type and truncated delta ORs to ligands including DPDPE, DSLET, DAGO, and U-50488 was compared. Their membrane localization and ability to mediate signal transduction were also studied. We conclude that the C-terminus of delta OR is not essential for plasma membrane targeting, ligand specificity, and agonist-dependent activation.

The delta opioid receptor is an important target for analgesic drug development. This report describes the identification of delta opioid receptor clones from human cDNA libraries and the preparation of a human delta receptor cDNA in the pcDNA3 expression vector for transfection studies. The cDNA encodes a 372 amino acid protein that has 93% amino acid identity to mouse and rat delta receptors. COS-7 cells transfected with this clone express over 1.0 pmol receptor/mg protein when measured by saturation binding with [3H]naltrindole. The delta receptor selective ligands NTB, BNTX, [4'-Cl-Phe4]DPDPE and [D-Ala2,Glu4]deltorphin all have Ki values under 10 nM while the affinities of the mu and kappa opioid receptor ligands CTAP and U-69593, respectively, are over 4.0 microM. Agonists show binding to multiple affinity states of the receptor consistent with the presence of G-protein coupled and uncoupled forms of the expressed receptor. The 8-fold higher affinity of NTB relative to BNTX suggests that the human delta receptor is of the delta 2 subtype.

Naltrexone-derived antagonists which are highly selective for delta opioid receptors have been designed using the message-address concept. The prototypical member of this series, naltrindole 1, possesses very high affinity and selectivity for delta receptors, and a closely related benzofuran analogue, NTB 2, is a selective delta 2 antagonist. The fact that 7-benzylidenenaltrexone (BNTX) was found to be a delta 1 antagonist suggests that the conformation of the putative delta address component (phenyl group) of BNTX plays a role in modulating delta subtype selectivity.