Descriptive molecular pharmacology of the δ opioid receptor (DOR): A computational study with structural approach.

This work focuses on the δ receptor (DOR), a G protein-coupled receptor (GPCR) belonging to the opioid receptor group. DOR is expressed in numerous tissues, particularly within the nervous system. Our study explores computationally the receptor's interactions with various ligands, including opiates and opioid peptides. It elucidates how these interactions influence the δ receptor response, relevant in a wide range of health and pathological processes. Thus, our investigation aims to explore the significance of DOR as an incoming drug target for pain relief and neurodegenerative diseases and as a source for novel opioid non-narcotic analgesic alternatives. We analyze the receptor's structural properties and interactions using Molecular Dynamics (MD) simulations and Gaussian-accelerated MD across different functional states. To thoroughly assess the primary differences in the structural and conformational ensembles across our different simulated systems, we initiated our study with 1 µs of conventional Molecular Dynamics. The strategy was chosen to encompass the full activation cycle of GPCRs, as activation processes typically occur within this microsecond range. Following the cMD, we extended our study with an additional 100 ns of Gaussian accelerated Molecular Dynamics (GaMD) to enhance the sampling of conformational states. This simulation approach allowed us to capture a comprehensive range of dynamic interactions and conformational changes that are crucial for GPCR activation as influenced by different ligands. Our study includes comparing agonist and antagonist complexes to uncover the collective patterns of their functional states, regarding activation, blocking, and inactivation of DOR, starting from experimental data. In addition, we also explored interactions between agonist and antagonist molecules from opiate and opioid classifications to establish robust structure-activity relationships. These interactions have been systematically quantified using a Quantitative Structure-Activity Relationships (QSAR) model. This research significantly contributes to our understanding of this significant pharmacological target, which is emerging as an attractive subject for drug development.

In-silico screening of missense nsSNPs in Delta-opioid receptor protein and their restoring tendency on MCRT interaction; focusing on dynamic nature.

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.

Key Residues in δ Opioid Receptor Allostery Explored by the Elastic Network Model and the Complex Network Model Combined with the Perturbation Method.

Opioid receptors, a kind of G protein-coupled receptors (GPCRs), mainly mediate an analgesic response via allosterically transducing the signal of endogenous ligand binding in the extracellular domain to couple to effector proteins in the intracellular domain. The δ opioid receptor (DOP) is associated with emotional control besides pain control, which makes it an attractive therapeutic target. However, its allosteric mechanism and key residues responsible for the structural stability and signal communication are not completely clear. Here we utilize the Gaussian network model (GNM) and amino acid network (AAN) combined with perturbation methods to explore the issues. The constructed fcfGNMMD, where the force constants are optimized with the inverse covariance estimation based on the correlated fluctuations from the available DOP molecular dynamics (MD) ensemble, shows a better performance than traditional GNM in reproducing residue fluctuations and cross-correlations and in capturing functionally low-frequency modes. Additionally, fcfGNMMD can consider implicitly the environmental effects to some extent. The lowest mode can well divide DOP segments and identify the two sodium ion (important allosteric regulator) binding coordination shells, and from the fastest modes, the key residues important for structure stabilization are identified. Using fcfGNMMD combined with a dynamic perturbation-response method, we explore the key residues related to the sodium ion binding. Interestingly, we identify not only the key residues in sodium ion binding shells but also the ones far away from the perturbation sites, which are involved in binding with DOP ligands, suggesting the possible long-range allosteric modulation of sodium binding for the ligand binding to DOP. Furthermore, utilizing the weighted AAN combined with attack perturbations, we identify the key residues for allosteric communication. This work helps strengthen the understanding of the allosteric communication mechanism in δ opioid receptor and can provide valuable information for drug design.

Discovery of Novel Delta Opioid Receptor (DOR) Inverse Agonist and Irreversible (Non-Competitive) Antagonists.

The delta opioid receptor (DOR) is a crucial receptor system that regulates pain, mood, anxiety, and similar mental states. DOR agonists, such as SNC80, and DOR-neutral antagonists, such as naltrindole, were developed to investigate the DOR in vivo and as potential therapeutics for pain and depression. However, few inverse agonists and non-competitive/irreversible antagonists have been developed, and none are widely available. This leaves a gap in our pharmacological toolbox and limits our ability to investigate the biology of this receptor. Thus, we designed and synthesized the novel compounds SRI-9342 as an irreversible antagonist and SRI-45128 as an inverse agonist. These compounds were then evaluated in vitro for their binding affinity by radioligand binding, their functional activity by 35S-GTPγS coupling, and their cAMP accumulation in cells expressing the human DOR. Both compounds demonstrated high binding affinity and selectivity at the DOR, and both displayed their hypothesized molecular pharmacology of irreversible antagonism (SRI-9342) or inverse agonism (SRI-45128). Together, these results demonstrate that we have successfully designed new inverse agonists and irreversible antagonists of the DOR based on a novel chemical scaffold. These new compounds will provide new tools to investigate the biology of the DOR or even new potential therapeutics.

Kappa but not delta or mu opioid receptors form homodimers at low membrane densities.

Opioid receptors (ORs) have been observed as homo- and heterodimers, but it is unclear if the dimers are stable under physiological conditions, and whether monomers or dimers comprise the predominant fraction in a cell. Here, we use three live-cell imaging approaches to assess dimerization of ORs at expression levels that are 10-100 × smaller than in classical biochemical assays. At membrane densities around 25/µm2, a split-GFP assay reveals that κOR dimerizes, while µOR and δOR stay monomeric. At receptor densities < 5/µm2, single-molecule imaging showed no κOR dimers, supporting the concept that dimer formation depends on receptor membrane density. To directly observe the transition from monomers to dimers, we used a single-molecule assay to assess membrane protein interactions at densities up to 100 × higher than conventional single-molecule imaging. We observe that κOR is monomeric at densities < 10/µm2 and forms dimers at densities that are considered physiological. In contrast, µOR and δOR stay monomeric even at the highest densities covered by our approach. The observation of long-lasting co-localization of red and green κOR spots suggests that it is a specific effect based on OR dimerization and not an artefact of coincidental encounters.

Synthesis and Antinociceptive Effect of Some Thiazole-Piperazine Derivatives: Involvement of Opioidergic System in the Activity.

In this study, we aimed to design and synthesize novel molecules carrying both the thiazole and piperazine rings in their structures and to investigate their antinociceptive activity. Targeted compounds were obtained by reacting thiosemicarbazide derivative and appropriate 2-bromoacetophenone in ethanol. The structures of the obtained compounds were determined using data from various spectroscopic methods (IR, 1H-NMR, 13C-NMR, and LCMSMS). Experimental data from in vivo tests showed that test compounds 3a-3c, 3f, and 3g (50 mg/kg) significantly prolonged reaction times of animals in tail-clip and hot-plate tests compared to the controls, indicating that these compounds possess centrally mediated antinociceptive activities. Furthermore, these compounds reduced the number of writhing behaviors in the acetic acid-induced writhing tests, showing that the compounds also possess peripheral antinociceptive activity. In the mechanistic studies, naloxone pre-treatments abolished the antinociceptive activities of compounds 3a-3c, 3f, and 3g, indicating that opioidergic mechanisms were involved in their antinociceptive effects. Molecular docking studies demonstrating significant interactions between the active compounds and µ- and δ-opioid receptor proteins supported the pharmacological findings. This study is the first showing that molecules designed to bear thiazole and piperazine moieties together on their structure exert centrally and peripherally mediated antinociceptive effects by activating the opioid system.

Conformational specificity of opioid receptors is determined by subcellular location irrespective of agonist.

The prevailing model for the variety in drug responses is that different drugs stabilize distinct active states of their G protein-coupled receptor (GPCR) targets, allowing coupling to different effectors. However, whether the same ligand generates different GPCR active states based on the immediate environment of receptors is not known. Here we address this question using spatially resolved imaging of conformational biosensors that read out distinct active conformations of the δ-opioid receptor (DOR), a physiologically relevant GPCR localized to Golgi and the surface in neuronal cells. We have shown that Golgi and surface pools of DOR both inhibit cAMP, but engage distinct conformational biosensors in response to the same ligand in rat neuroendocrine cells. Further, DOR recruits arrestins on the surface but not on the Golgi. Our results suggest that the local environment determines the active states of receptors for any given drug, allowing GPCRs to couple to different effectors at different subcellular locations.

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.

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.

ß2-Homo-Amino Acid Scan of µ-Selective Opioid Tetrapeptide TAPP.

TAPP (H-Tyr-d-Ala-Phe-Phe-NH2) is a potent, µ-selective opioid ligand. In order to gain further insights into pharmacophoric features of this tetrapeptide, we have performed a ß2-Homo-amino acid (ß2hAA) scan of the TAPP sequence. To this aim, 10 novel analogues have been synthesized and evaluated for µ-opioid and δ-opioid receptor affinity as well as for stability in human plasma. The derivatives included compounds in which a (R)- or (S)-ß2-Homo-Homologue replaced the amino acids in the TAPP sequence. The derivatives with (R)- or (S)-ß2hPhe4 turned out to bind µOR with affinities equal to that of the parent. ß2hAAs in position 1 and 3 resulted in rather large affinity decreases, but the change differed depending on the stereochemistry. ß2-Homologation in the second position gave derivatives with very poor µOR binding. According to molecular modelling, the presented α/ß-peptides adopt a variety of binding poses with their common element being an ionic interaction between a protonable amine of the first residue and Asp147. A feature required for high µOR affinity seems the ability to accommodate the ring in the fourth residue in a manner similar to that found for TAPP. Contrary to what might be expected, several compounds were significantly less stable in human plasma than the parent compound.

A highly conserved δ-opioid receptor region determines RGS4 interaction.

The δ-opioid receptor (δ-OR) couples to Gi/Go proteins to modulate a variety of responses in the nervous system. Τhe regulator of G protein signalling 4 (RGS4) was previously shown to directly interact within the C-terminal region of δ-OR using its N-terminal domain to negatively modulate opioid receptor signalling. Herein, using molecular dynamics simulations and in vitro pull-down experiments we delimit this interaction to 12 helix 8 residues of δ-ΟR and to the first 17 N-terminal residues (NT) of RGS4. Monitoring the complex arrangement and stabilization between RGS4 and δ-OR by molecular dynamics simulations combined with mutagenesis studies, we defined that two critical interactions are formed: one between Phe329 of helix8 of δ-ΟR and Pro9 of the NT of RGS4 and the other a salt bridge between Glu323 of δ-ΟR and Lys17 of RGS4. Our observations allow drafting for the first time a structural model of a ternary complex including the δ-opioid receptor, a G protein and a RGS protein. Furthermore, the high degree of conservation among opioid receptors of the RGS4-binding region, points to a conserved interaction mode between opioid receptors and this important regulatory protein.

Elucidating the active δ-opioid receptor crystal structure with peptide and small-molecule agonists.

Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to µ-opioid receptor (MOP) agonists that are in the spotlight of the current "opioid crisis." Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects.

Molecular aspects of delta opioid receptors.

The delta opioid receptor (DOP) belongs to the Class A, rhodopsin-like family of G protein-coupled receptors. Although this receptor has a high level of similarity with the other opioid receptors, it displays unique aspects and functions. Indeed, as opposed to most membrane receptors, DOP is poorly addressed to the plasma membrane. In this chapter, we first review the molecular and cellular mechanisms regulating the expression and the cellular trafficking/sorting of DOP. We then summarize the structural insights of this receptor through the analysis of the existing crystal structures, with a particular focus on the role of the sodium binding site. Finally, we review the current signaling mechanisms mediating receptor function and desensitization.

Multivalent peptide and peptidomimetic ligands for the treatment of pain without toxicities and addiction.

The current opioid crisis has created a tragic problem in medicine and society. Pain is the most ubiquitous and costly disease in society and yet all of our "treatments" have toxicities, especially for prolonged use. However, there are several alternatives that have been discovered in the past fifteen years that have been demonstrated in animals to have none of the toxicities of current drugs. Many of the compounds are multivalent and have novel biological activity profiles. Unfortunately, none of these have been in clinical trials in humans, perhaps because they were discovered in academic laboratories. A review of these novel chemicals are given in this paper.

Dual RXR motifs regulate nerve growth factor-mediated intracellular retention of the delta opioid receptor.

The delta opioid receptor (DOR), a physiologically relevant prototype for G protein-coupled receptors, is retained in intracellular compartments in neuronal cells. This retention is mediated by a nerve growth factor (NGF)-regulated checkpoint that delays the export of DOR from the trans-Golgi network. How DOR is selectively retained in the Golgi, in the midst of dynamic membrane transport and cargo export, is a fundamental unanswered question. Here we address this by investigating sequence elements on DOR that regulate DOR surface delivery, focusing on the C-terminal tail of DOR that is sufficient for NGF-mediated regulation. By systematic mutational analysis, we define conserved dual bi-arginine (RXR) motifs that are required for NGF- and phosphoinositide-regulated DOR export from intracellular compartments in neuroendocrine cells. These motifs were required to bind the coatomer protein I (COPI) complex, a vesicle coat complex that mediates primarily retrograde cargo traffic in the Golgi. Our results suggest that interactions of DOR with COPI, via atypical COPI motifs on the C-terminal tail, retain DOR in the Golgi. These interactions could provide a point of regulation of DOR export and delivery by extracellular signaling pathways.

Rubiscolins are naturally occurring G protein-biased delta opioid receptor peptides.

The impact that ß-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 ß-arrestin. These findings have led to the development of biased opioid small molecule agonists that do not recruit ß-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 ß-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 ß-arrestin 2 and have undetectable recruitment of ß-arrestin 1 at the δ opioid receptor.

Computational systems pharmacology analysis of cannabidiol: a combination of chemogenomics-knowledgebase network analysis and integrated in silico modeling and simulation.

With treatment benefits in both the central nervous system and the peripheral system, the medical use of cannabidiol (CBD) has gained increasing popularity. Given that the therapeutic mechanisms of CBD are still vague, the systematic identification of its potential targets, signaling pathways, and their associations with corresponding diseases is of great interest for researchers. In the present work, chemogenomics-knowledgebase systems pharmacology analysis was applied for systematic network studies to generate CBD-target, target-pathway, and target-disease networks by combining both the results from the in silico analysis and the reported experimental validations. Based on the network analysis, three human neuro-related rhodopsin-like GPCRs, i.e., 5-hydroxytryptamine receptor 1 A (5HT1A), delta-type opioid receptor (OPRD) and G protein-coupled receptor 55 (GPR55), were selected for close evaluation. Integrated computational methodologies, including homology modeling, molecular docking, and molecular dynamics simulation, were used to evaluate the protein-CBD binding modes. A CBD-preferred pocket consisting of a hydrophobic cavity and backbone hinges was proposed and tested for CBD-class A GPCR binding. Finally, the neurophysiological effects of CBD were illustrated at the molecular level, and dopamine receptor 3 (DRD3) was further predicted to be an active target for CBD.

Synthesis and Evaluation of a Novel Bivalent Selective Antagonist for the Mu-Delta Opioid Receptor Heterodimer that Reduces Morphine Withdrawal in Mice.

A major limitation in the study of the mu-delta opioid receptor heterodimer (MDOR) is that few selective pharmacological tools exist and no heteromer-selective antagonists. We thus designed a series of variable-length (15-41 atoms) bivalent linked peptides with selective but moderate/low-affinity pharmacophores for the mu and delta opioid receptors. We observed a U-shaped MDOR potency/affinity profile in vitro, with the 24-atom spacer length (D24M) producing the highest MDOR potency/affinity (<1 nM) and selectivity (≥89-fold). We further evaluated D24M in mice and observed that D24M dose-dependently antagonized tail flick antinociception produced by the MDOR agonists CYM51010 and Deltorphin-II, without antagonizing the monomer agonists DAMGO and DSLET. We also observed that D24M sharply reduced withdrawal behavior in models of acute and chronic morphine dependence. These findings suggest that D24M is a first-in-class high-potency MDOR-selective antagonist both in vitro and in vivo.

Pharmacologic Evidence for a Putative Conserved Allosteric Site on Opioid Receptors.

Allosteric modulators of G protein-coupled receptors, including opioid receptors, have been proposed as possible therapeutic agents with enhanced selectivity. BMS-986122 is a positive allosteric modulator (PAM) of the µ-opioid receptor (µ-OR). BMS-986187 is a structurally distinct PAM for the δ-opioid receptor (δ-OR) that has been reported to exhibit 100-fold selectivity in promoting δ-OR over µ-OR agonism. We used ligand binding and second-messenger assays to show that BMS-986187 is an effective PAM at the µ-OR and at the κ-opioid receptor (κ-OR), but it is ineffective at the nociceptin receptor. The affinity of BMS-986187 for δ-ORs and κ-ORs is approximately 20- to 30-fold higher than for µ-ORs, determined using an allosteric ternary complex model. Moreover, we provide evidence, using a silent allosteric modulator as an allosteric antagonist, that BMS-986187 and BMS-986122 bind to a similar region on all three traditional opioid receptor types (µ-OR, δ-OR, and κ-OR). In contrast to the dogma surrounding allosteric modulators, the results indicate a possible conserved allosteric binding site across the opioid receptor family that can accommodate structurally diverse molecules. These findings have implications for the development of selective allosteric modulators.

Synthesis of novel norsufentanil analogs via a four-component Ugi reaction and in vivo, docking, and QSAR studies of their analgesic activity.

Novel substituted amino acid tethered norsufentanil derivatives were synthesized by the four-component Ugi reaction. Norsufentanil was reacted with succinic anhydride to produce the corresponding carboxylic acid. The resulting carboxylic acid has undergone a multicomponent reaction with different aldehydes, amines, and isocyanides to produce a library of the desired compounds. In all cases, amide bond rotation was observed in the NMR spectra. In vivo analgesic activity of the synthesized compounds was evaluated by a tail flick test. Very encouraging results were obtained for a number of the synthesized products. Some of the synthesized compounds such as 5a, 5b, 5h, 5j, and 5r were found to be more potent than sufentanil, sufentanil citrate, and norsufentanil. Binding modes between the compounds and mu and delta-opioid receptors were studied by molecular docking method. The relationship between the molecular structural features and the analgesic activity was investigated by a quantitative structure-activity relationship model. The results of the molecular modeling studies and the in vivo analgesic activity suggested that the majority of the synthesized compounds were more potent than sufentanil and norsufentanil.