Elevating levels of the endocannabinoid 2-arachidonoylglycerol blunts opioid reward but not analgesia.

Converging findings have established that the endocannabinoid (eCB) system serves as a possible target for the development of new treatments for pain as a complement to opioid-based treatments. Here we show in male and female mice that enhancing levels of the eCB, 2-arachidonoylglycerol (2-AG), through pharmacological inhibition of its catabolic enzyme, monoacylglycerol lipase (MAGL), either systemically or in the ventral tegmental area (VTA) with JZL184, leads to a substantial attenuation of the rewarding effects of opioids in male and female mice using conditioned place preference and self-administration paradigms, without altering their analgesic properties. These effects are driven by CB1 receptors (CB1Rs) within the VTA as VTA CB1R conditional knockout, counteracts JZL184's effects. Conversely, pharmacologically enhancing the levels of the other eCB, anandamide (AEA), by inhibition of fatty acid amide hydrolase (FAAH) has no effect on opioid reward or analgesia. Using fiber photometry with fluorescent sensors for calcium and dopamine (DA), we find that enhancing 2-AG levels diminishes opioid reward-related nucleus accumbens (NAc) activity and DA neurotransmission. Together these findings reveal that 2-AG counteracts the rewarding properties of opioids and provides a potential adjunctive therapeutic strategy for opioid-related analgesic treatments.

Photo-affinity and Metabolic Labeling Probes Based on the Opioid Alkaloids.

The opioids are powerful analgesics yet possess contingencies that can lead to opioid-use disorder. Chemical probes derived from the opioid alkaloids can provide deeper insight into the molecular interactions in a cellular context. Here, we designed and developed photo-click morphine (PCM-2) as a photo-affinity probe based on morphine and dialkynyl-acetyl morphine (DAAM) as a metabolic acetate reporter based on heroin. Application of these probes to SH-SY5Y, HEK293T, and U2OS cells revealed that PCM-2 and DAAM primarily localize to the lysosome amongst other locations inside the cell by confocal microscopy and chemical proteomics. Interaction site identification by mass spectrometry revealed the mitochondrial phosphate carrier protein, solute carrier family 25 member 3, SLC25A3, and histone H2B as acylation targets of DAAM. These data illustrate the utility of chemical probes to measure localization and protein interactions in a cellular context and will inform the design of probes based on the opioids in the future.

Binding preference at the µ-opioid receptor underlies distinct pharmacology of cyclopropyl versus valeryl analogs of fentanyl.

Illicitly manufactured fentanyl is driving the current opioid crisis, and various fentanyl analogs are appearing in recreational drug markets worldwide. To assess the potential health risks posed by fentanyl analogs, it is necessary to understand structure-activity relationships for these compounds. Here we compared the pharmacology of two structurally related fentanyl analogs implicated in opioid overdose: cyclopropylfentanyl and valerylfentanyl. Cyclopropylfentanyl has a three-carbon ring attached to the carbonyl group on the fentanyl scaffold, whereas valerylfentanyl has a four-carbon chain at the same position. In vitro assays examining µ-opioid receptor (MOR) coupling to G proteins in CHO cells showed that cyclopropylfentanyl is a full agonist (EC50 = 8.6 nM, %Emax = 113%), with potency and efficacy similar to fentanyl (EC50 = 10.3 nM, %Emax = 113%). By contrast, valerylfentanyl is a partial agonist at MOR (EC50 = 179.8 nM, %Emax = 60%). Similar results were found in assays assessing MOR-mediated ß-arrestin recruitment in HEK cells. In vivo studies in male CD-1 mice demonstrated that both fentanyl analogs induce naloxone-reversible antinociception and respiratory suppression, but cyclopropylfentanyl is 100-times more potent as an antinociceptive agent (ED50 = 0.04 mg/kg, s. c.) than valerylfentanyl (ED50 = 4.0 mg/kg, s. c.). Molecular simulation results revealed that the alkyl chain of valerylfentanyl cannot be well accommodated by the active state of MOR and may transition the receptor toward an inactive state, converting the fentanyl scaffold to a partial agonist. Taken together, our results suggest that cyclopropylfentanyl presents much greater risk of adverse effects when compared to valerylfentanyl. Moreover, the summed findings may provide clues to the design of therapeutic opioids with reduced adverse side effects.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.

Exploring Pharmacological Functions of Alternatively Spliced Variants of the Mu Opioid Receptor Gene, Oprm1, via Gene-Targeted Animal Models.

The mu opioid receptor has a distinct place in the opioid receptor family, since it mediates the actions of most opioids used clinically (e.g., morphine and fentanyl), as well as drugs of abuse (e.g., heroin). The single-copy mu opioid receptor gene, OPRM1, goes through extensive alternative pre-mRNA splicing to generate numerous splice variants that are conserved from rodents to humans. These OPRM1 splice variants can be classified into three structurally distinct types: (1) full-length 7 transmembrane (TM) carboxyl (C)-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Distinct pharmacological functions of these splice variants have been demonstrated by both in vitro and in vivo studies, particularly by using several unique gene-targeted mouse models. These studies provide new insights into our understanding of the complex actions of mu opioids with regard to OPRM1 alternative splicing. This review provides an overview of the studies that used these gene-targeted mouse models for exploring the functional importance of Oprm1 splice variants.

Dysregulated expression of the alternatively spliced variant mRNAs of the mu opioid receptor gene, OPRM1, in the medial prefrontal cortex of male human heroin abusers and heroin self-administering male rats.

Heroin, a mu agonist, acts through the mu opioid receptor. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating an array of splice variants that are conserved from rodent to humans. Increasing evidence suggests that these OPRM1 splice variants are pharmacologically important in mediating various actions of mu opioids, including analgesia, tolerance, physical dependence, rewarding behavior, as well as addiction. In the present study, we examine expression of the OPRM1 splice variant mRNAs in the medial prefrontal cortex (mPFC), one of the major brain regions involved in decision-making and drug-seeking behaviors, of male human heroin abusers and male rats that developed stable heroin-seeking behavior using an intravenous heroin self-administration (SA) model. The results show similar expression profiles among multiple OPRM1 splice variants in both human control subjects and saline control rats, illustrating conservation of OPRM1 alternative splicing from rodent to humans. Moreover, the expressions of several OPRM1 splice variant mRNAs were dysregulated in the postmortem mPFCs from heroin abusers compared to the control subjects. Similar patterns were observed in the rat heroin SA model. These findings suggest potential roles of the OPRM1 splice variants in heroin addiction that could be mechanistically explored using the rat heroin SA model.

Oxidative Metabolism as a Modulator of Kratom's Biological Actions.

The leaves of Mitragyna speciosa (kratom), a plant native to Southeast Asia, are increasingly used as a pain reliever and for attenuation of opioid withdrawal symptoms. Using the tools of natural products chemistry, chemical synthesis, and pharmacology, we provide a detailed in vitro and in vivo pharmacological characterization of the alkaloids in kratom. We report that metabolism of kratom's major alkaloid, mitragynine, in mice leads to formation of (a) a potent mu opioid receptor agonist antinociceptive agent, 7-hydroxymitragynine, through a CYP3A-mediated pathway, which exhibits reinforcing properties, inhibition of gastrointestinal (GI) transit and reduced hyperlocomotion, (b) a multifunctional mu agonist/delta-kappa antagonist, mitragynine pseudoindoxyl, through a CYP3A-mediated skeletal rearrangement, displaying reduced hyperlocomotion, inhibition of GI transit and reinforcing properties, and (c) a potentially toxic metabolite, 3-dehydromitragynine, through a non-CYP oxidation pathway. Our results indicate that the oxidative metabolism of the mitragynine template beyond 7-hydroxymitragynine may have implications in its overall pharmacology in vivo.

Alternative Pre-mRNA Splicing of the Mu Opioid Receptor Gene, OPRM1: Insight into Complex Mu Opioid Actions.

Most opioid analgesics used clinically, including morphine and fentanyl, as well as the recreational drug heroin, act primarily through the mu opioid receptor, a class A Rhodopsin-like G protein-coupled receptor (GPCR). The single-copy mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating multiple splice variants or isoforms via a variety of alternative splicing events. These OPRM1 splice variants can be categorized into three major types based on the receptor structure: (1) full-length 7 transmembrane (TM) C-terminal variants; (2) truncated 6TM variants; and (3) single TM variants. Increasing evidence suggests that these OPRM1 splice variants are pharmacologically important in mediating the distinct actions of various mu opioids. More importantly, the OPRM1 variants can be targeted for development of novel opioid analgesics that are potent against multiple types of pain, but devoid of many side-effects associated with traditional opiates. In this review, we provide an overview of OPRM1 alternative splicing and its functional relevance in opioid pharmacology.

Kratom Alkaloids as Probes for Opioid Receptor Function: Pharmacological Characterization of Minor Indole and Oxindole Alkaloids from Kratom.

Dry leaves of kratom (mitragyna speciosa) are anecdotally consumed as pain relievers and antidotes against opioid withdrawal and alcohol use disorders. There are at least 54 alkaloids in kratom; however, investigations to date have focused around mitragynine, 7-hydroxy mitragynine (7OH), and mitragynine pseudoindoxyl (MP). Herein, we probe a few minor indole and oxindole based alkaloids, reporting the receptor affinity, G-protein activity, and ßarrestin-2 signaling of corynantheidine, corynoxine, corynoxine B, mitraciliatine, and isopaynantheine at mouse and human opioid receptors. We identify corynantheidine as a mu opioid receptor (MOR) partial agonist, whereas its oxindole derivative corynoxine was an MOR full agonist. Similarly, another alkaloid mitraciliatine was found to be an MOR partial agonist, while isopaynantheine was a KOR agonist which showed reduced ßarrestin-2 recruitment. Corynantheidine, corynoxine, and mitraciliatine showed MOR dependent antinociception in mice, but mitraciliatine and corynoxine displayed attenuated respiratory depression and hyperlocomotion compared to the prototypic MOR agonist morphine in vivo when administered supraspinally. Isopaynantheine on the other hand was identified as the first kratom derived KOR agonist in vivo. While these minor alkaloids are unlikely to play the majority role in the biological actions of kratom, they represent excellent starting points for further diversification as well as distinct efficacy and signaling profiles with which to probe opioid actions in vivo.

Understanding Opioid Actions, Pain and Analgesia: A Tribute to Dr. Gavril Pasternak.

This special issue is a tribute to our mentor, colleague and friend, Gavril W. Pasternak, MD, PhD. Homage to the breadth and depth of his work (~ 450 publications) over a 40 career in pharmacology and medicine cannot be captured fully in one special issue, but the 22 papers collected herein represent seven of the topics near and dear to Gav's heart, and the colleagues, friends and mentees who held him near to theirs. The seven themes include: (1) sites and mechanisms of opioid actions in vivo; (2) development of novel analgesic agents; (3) opioid tolerance, withdrawal and addiction: mechanisms and treatment; (4) opioid receptor splice variants; (5) novel research tools and approaches; (6) receptor signaling and crosstalk in vitro; and (7) mentorship. This introduction to the issue summarizes contributions and includes formal and personal remembrances of Gav that illustrate his personality, warmth, and dedication to making a difference in patient care and people's lives.

Endogenous Opioid Peptides and Alternatively Spliced Mu Opioid Receptor Seven Transmembrane Carboxyl-Terminal Variants.

There exist three main types of endogenous opioid peptides, enkephalins, dynorphins and ß-endorphin, all of which are derived from their precursors. These endogenous opioid peptides act through opioid receptors, including mu opioid receptor (MOR), delta opioid receptor (DOR) and kappa opioid receptor (KOR), and play important roles not only in analgesia, but also many other biological processes such as reward, stress response, feeding and emotion. The MOR gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms. One type of these splice variants, the full-length 7 transmembrane (TM) Carboxyl (C)-terminal variants, has the same receptor structures but contains different intracellular C-terminal tails. The pharmacological functions of several endogenous opioid peptides through the mouse, rat and human OPRM1 7TM C-terminal variants have been considerably investigated together with various mu opioid ligands. The current review focuses on the studies of these endogenous opioid peptides and summarizes the results from early pharmacological studies, including receptor binding affinity and G protein activation, and recent studies of ß-arrestin2 recruitment and biased signaling, aiming to provide new insights into the mechanisms and functions of endogenous opioid peptides, which are mediated through the OPRM1 7TM C-terminal splice variants.

From Pharmacology to Physiology: Endocrine Functions of µ-Opioid Receptor Networks.

The steady rise in opioid users and abusers has uncovered multiple detrimental health consequences of perturbed opioid receptor signaling, thereby creating the need to better understand the biology of these systems. Among endogenous opioid networks, µ-receptors have received special attention due to their unprecedented biological complexity and broad implications in homeostatic functions. Here, we review the origin, molecular biology, and physiology of endogenous opioids with a special focus on µ-opioid receptor networks within the endocrine system. Moreover, we summarize the current evidence supporting an involvement of the latter in regulating distinct endocrine functions. Finally, we combine these insights to present an integrated perspective on µ-opioid receptor biology and provide an outlook on future studies and unresolved questions in this field.

Controlling opioid receptor functional selectivity by targeting distinct subpockets of the orthosteric site.

Controlling receptor functional selectivity profiles for opioid receptors is a promising approach for discovering safer analgesics; however, the structural determinants conferring functional selectivity are not well understood. Here, we used crystal structures of opioid receptors, including the recently solved active state kappa opioid complex with MP1104, to rationally design novel mixed mu (MOR) and kappa (KOR) opioid receptor agonists with reduced arrestin signaling. Analysis of structure-activity relationships for new MP1104 analogs points to a region between transmembrane 5 (TM5) and extracellular loop (ECL2) as key for modulation of arrestin recruitment to both MOR and KOR. The lead compounds, MP1207 and MP1208, displayed MOR/KOR Gi-partial agonism with diminished arrestin signaling, showed efficient analgesia with attenuated liabilities, including respiratory depression and conditioned place preference and aversion in mice. The findings validate a novel structure-inspired paradigm for achieving beneficial in vivo profiles for analgesia through different mechanisms that include bias, partial agonism, and dual MOR/KOR agonism.

Synthesis and Characterization of Azido Aryl Analogs of IBNtxA for Radio-Photoaffinity Labeling Opioid Receptors in Cell Lines and in Mouse Brain.

Mu opioid receptors (MOR-1) mediate the biological actions of clinically used opioids such as morphine, oxycodone, and fentanyl. The mu opioid receptor gene, OPRM1, undergoes extensive alternative splicing, generating multiple splice variants. One type of splice variants are truncated variants containing only six transmembrane domains (6TM) that mediate the analgesic action of novel opioid drugs such as 3'-iodobenzoylnaltrexamide (IBNtxA). Previously, we have shown that IBNtxA is a potent analgesic effective in a spectrum of pain models but lacks many side-effects associated with traditional opiates. In order to investigate the targets labeled by IBNtxA, we synthesized two arylazido analogs of IBNtxA that allow photolabeling of mouse mu opioid receptors (mMOR-1) in transfected cell lines and mMOR-1 protein complexes that may comprise the 6TM sites in mouse brain. We demonstrate that both allyl and alkyne arylazido derivatives of IBNtxA efficiently radio-photolabeled mMOR-1 in cell lines and MOR-1 protein complexes expressed either exogenously or endogenously, as well as found in mouse brain. In future, design and application of such radio-photolabeling ligands with a conjugated handle will provide useful tools for further isolating or purifying MOR-1 to investigate site specific ligand-protein contacts and its signaling complexes.

Mu Opioids Induce Biased Signaling at the Full-Length Seven Transmembrane C-Terminal Splice Variants of the mu Opioid Receptor Gene, Oprm1.

The biased signaling has been extensively studied in the original mu opioid receptor (MOR-1), particularly through G protein and ß-arrestin2 signaling pathways. The concept that the G protein pathway is often linked to the therapeutic effect of the drug, while the ß-arrestin pathway is associated to the side effects has been proposed to develop biased analgesic compounds with limited side-effects associated with traditional opiates. The mu opioid receptor gene, OPRM1, undergoes extensive alternative pre-mRNA splicing, generating multiple splice variants or isoforms that are conserved from rodent to human. One type of the Oprm1 splice variants are the full-length 7 transmembrane (7TM) C-terminal splice variants, which have identical receptor structures including entire binding pocket, but contain a different intracellular C-terminal tail resulted from 3' alternative splicing. Increasing evidence suggest that these full-length 7TM C-terminal variants play important roles in mu opioid pharmacology, raising questions regarding biased signaling at these multiple C-terminal variants. In the present study, we investigated the effect of different C-terminal variants on mu agonist-induced G protein coupling, ß-arrestin2 recruitment, and ultimately, signaling bias. We found that mu agonists produced marked differences in G protein activation and ß-arrestin2 recruitment among various C-terminal variants, leading to biased signaling at various level. Particularly, MOR-1O, an exon 7-associated variant, showed greater ß-arrestin2 bias for most mu agonists than MOR-1, an exon 4-associated variant. Biased signaling of G protein-coupled receptors has been defined by evidences that different agonists can produce divergent signaling transduction pathways through a single receptor. Our findings that a single mu agonist can induce differential signaling through multiple 7TM splice variants provide a new perspective on biased signaling at least for Oprm1, which perhaps is important for our understanding of the complex mu opioid actions in vivo where all the 7TM splice variants co-exist.

Synthesis and Pharmacology of a Novel µ-δ Opioid Receptor Heteromer-Selective Agonist Based on the Carfentanyl Template.

In this work, we studied a series of carfentanyl amide-based opioid derivatives targeting the mu opioid receptor (µOR) and the delta opioid receptor (δOR) heteromer as a credible novel target in pain management therapy. We identified a lead compound named MP135 that exhibits high G-protein activity at µ-δ heteromers compared to the homomeric δOR or µOR and low ß-arrestin2 recruitment activity at all three. Furthermore, MP135 exhibits distinct signaling profile, as compared to the previously identified agonist targeting µ-δ heteromers, CYM51010. Pharmacological characterization of MP135 supports the utility of this compound as a molecule that could be developed as an antinociceptive agent similar to morphine in rodents. In vivo characterization reveals that MP135 maintains untoward side effects such as respiratory depression and reward behavior; together, these results suggest that optimization of MP135 is necessary for the development of therapeutics that suppress the classical side effects associated with conventional clinical opioids.

A Truncated Six Transmembrane Splice Variant MOR-1G Enhances Expression of the Full-Length Seven Transmembrane µ-Opioid Receptor through Heterodimerization.

The µ-opioid receptor gene undergoes extensive alternative splicing to generate an array of splice variants. One group of splice variants excludes the first transmembrane (TM) domain and contains six TM domains. These 6TM variants are essential for the action of a novel class of analgesic drugs, including 3-iodobenzoyl-6ß-naltrexamide, which is potent against a spectrum of pain models without exhibiting the adverse side effects of traditional opiates. The 6TM variants are also involved in analgesic action through other drug classes, including δ-opioid and κ-opioids and α 2-adrenergic drugs. Of the five 6TM variants in mouse, mouse µ-opioid receptor (mMOR)-1G is abundant and conserved from rodent to human. In the present study, we demonstrate a new function of mMOR-1G in enhancing expression of the full-length 7TM µ-opioid receptor, mMOR-1. When coexpressed with mMOR-1 in a Tet-Off inducible CHO cell line, mMOR-1G has no effect on mMOR-1 mRNA expression but greatly increases mMOR-1 protein expression in a dose-dependent manner determined by opioid receptor binding and [35S] guanosine 5'-3-O-(thio)triphosphate binding. Subcellular fractionation analysis using OptiPrep density gradient centrifugation shows an increase of functional mMOR-1 receptor in plasma membrane-enriched fractions. Using a coimmunoprecipitation approach, we further demonstrate that mMOR-1G physically associates with mMOR-1 starting at the endoplasmic reticulum, suggesting a chaperone-like function. These data provide a molecular mechanism for how mMOR-1G regulates expression and function of the full-length 7TM µ-opioid receptor. SIGNIFICANCE STATEMENT: The current study establishes a novel function of mouse µ-opioid receptor (mMOR)-1G, a truncated splice variant with six transmembrane (TM) domains of the mouse µ-opioid receptor gene, in enhancing expression of the full-length 7TM mMOR-1 through a chaperone-like function.

Morphine modulates the expression of mu-opioid receptor exon 5-associated full-length C-terminal splice variants by upregulating miR-378a-3p.

The mu-opioid receptor gene, OPRM1, undergoes extensive alternative splicing, creating an array of splice variants that are conserved from rodent to human. Both mouse and human OPRM1 have five exon 5-associated seven transmembrane full-length carboxyl terminal variants, MOR-1B1, MOR-1B2, MOR-1B3, MOR-1B4, and MOR-1B5, all of which are derived from alternative 3' splicing from exon 3 to alternative sites within exon 5. The functional relevance of these exon 5-associated MOR-1Bs has been demonstrated in mu agonist-induced G protein coupling, adenylyl cyclase activity, receptor internalization and desensitization, and post-endocytic sorting, as well as region-specific expression at the mRNA level. In the present study, we mapped a polyadenylation site for both mouse and human MOR-1Bs that defines the 3'-untranslated regions (3'-UTR) of MOR-1Bs and stabilizes mMOR-1Bs mRNAs. We identified a conserved miR378a-3p sequence in the 3'-UTR of both mouse and human MOR-1BS transcripts through which miR-378a-3p can regulate the expression of MOR-1Bs at the mRNA level. Chronic morphine treatment significantly increased the miR-378-3p level in Be(2)C cells and the brainstem of the morphine tolerant mice, contributing to the decreased expression of the mouse and human MOR-1B3 and MOR-1B4. Our study provides new insights into the role of miRNAs and Oprm1 splice variants in morphine tolerance.