Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.

A µ-opioid receptor modulator that works cooperatively with naloxone.

The µ-opioid receptor (µOR) is a well-established target for analgesia1, yet conventional opioid receptor agonists cause serious adverse effects, notably addiction and respiratory depression. These factors have contributed to the current opioid overdose epidemic driven by fentanyl2, a highly potent synthetic opioid. µOR negative allosteric modulators (NAMs) may serve as useful tools in preventing opioid overdose deaths, but promising chemical scaffolds remain elusive. Here we screened a large DNA-encoded chemical library against inactive µOR, counter-screening with active, G-protein and agonist-bound receptor to 'steer' hits towards conformationally selective modulators. We discovered a NAM compound with high and selective enrichment to inactive µOR that enhances the affinity of the key opioid overdose reversal molecule, naloxone. The NAM works cooperatively with naloxone to potently block opioid agonist signalling. Using cryogenic electron microscopy, we demonstrate that the NAM accomplishes this effect by binding a site on the extracellular vestibule in direct contact with naloxone while stabilizing a distinct inactive conformation of the extracellular portions of the second and seventh transmembrane helices. The NAM alters orthosteric ligand kinetics in therapeutically desirable ways and works cooperatively with low doses of naloxone to effectively inhibit various morphine-induced and fentanyl-induced behavioural effects in vivo while minimizing withdrawal behaviours. Our results provide detailed structural insights into the mechanism of negative allosteric modulation of the µOR and demonstrate how this can be exploited in vivo.

Ligand and G-protein selectivity in the κ-opioid receptor.

The κ-opioid receptor (KOR) represents a highly desirable therapeutic target for treating not only pain but also addiction and affective disorders1. However, the development of KOR analgesics has been hindered by the associated hallucinogenic side effects2. The initiation of KOR signalling requires the Gi/o-family proteins including the conventional (Gi1, Gi2, Gi3, GoA and GoB) and nonconventional (Gz and Gg) subtypes. How hallucinogens exert their actions through KOR and how KOR determines G-protein subtype selectivity are not well understood. Here we determined the active-state structures of KOR in a complex with multiple G-protein heterotrimers-Gi1, GoA, Gz and Gg-using cryo-electron microscopy. The KOR-G-protein complexes are bound to hallucinogenic salvinorins or highly selective KOR agonists. Comparisons of these structures reveal molecular determinants critical for KOR-G-protein interactions as well as key elements governing Gi/o-family subtype selectivity and KOR ligand selectivity. Furthermore, the four G-protein subtypes display an intrinsically different binding affinity and allosteric activity on agonist binding at KOR. These results provide insights into the actions of opioids and G-protein-coupling specificity at KOR and establish a foundation to examine the therapeutic potential of pathway-selective agonists of KOR.

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.

Insights into distinct signaling profiles of the µOR activated by diverse agonists.

Drugs targeting the µ-opioid receptor (µOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two µOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and ß-arrestin recruitment. Cryo-EM structures of µOR-Gi1 complex with MP (2.5 Å) and LFT (3.2 Å) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and ß-arrestins bind. These observations highlight how drugs engaging different parts of the µOR orthosteric pocket can lead to distinct signaling outcomes.

IUPHAR themed review: Opioid efficacy, bias, and selectivity.

Drugs acting at the opioid receptor family are clinically used to treat chronic and acute pain, though they represent the second line of treatment behind GABA analogs, antidepressants and SSRI's. Within the opioid family mu and kappa opioid receptor are commonly targeted. However, activation of the mu opioid receptor has side effects of constipation, tolerance, dependence, euphoria, and respiratory depression; activation of the kappa opioid receptor leads to dysphoria and sedation. The side effects of mu opioid receptor activation have led to mu receptor drugs being widely abused with great overdose risk. For these reasons, newer safer opioid analgesics are in high demand. For many years a focus within the opioid field was finding drugs that activated the G protein pathway at mu opioid receptor, without activating the ß-arrestin pathway, known as biased agonism. Recent advances have shown that this may not be the way forward to develop safer analgesics at mu opioid receptor, though there is still some promise at the kappa opioid receptor. Here we discuss recent novel approaches to develop safer opioid drugs including efficacy vs bias and fine-tuning receptor activation by targeting sub-pockets in the orthosteric site, we explore recent works on the structural basis of bias, and we put forward the suggestion that Gα subtype selectivity may be an exciting new area of interest.

Natural Products for the Treatment of Pain: Chemistry and Pharmacology of Salvinorin A, Mitragynine, and Collybolide.

Pain remains a very pervasive problem throughout medicine. Classical pain management is achieved through the use of opiates belonging to the mu opioid receptor (MOR) class, which have significant side effects that hinder their utility. Pharmacologists have been trying to develop opioids devoid of side effects since the isolation of morphine from papaver somniferum, more commonly known as opium by Sertürner in 1804. The natural products salvinorin A, mitragynine, and collybolide represent three nonmorphinan natural product-based targets, which are potent selective agonists of opioid receptors, and emerging next-generation analgesics. In this work, we review the phytochemistry and medicinal chemistry efforts on these templates and their effects on affinity, selectivity, analgesic actions, and a myriad of other opioid-receptor-related behavioral effects.

Predicted Mode of Binding to and Allosteric Modulation of the µ-Opioid Receptor by Kratom's Alkaloids with Reported Antinociception In Vivo.

Pain management devoid of serious opioid adverse effects is still far from reach despite vigorous research and development efforts. Alternatives to classical opioids have been sought for years, and mounting reports of individuals finding pain relief with kratom have recently intensified research on this natural product. Although the composition of kratom is complex, the pharmacological characterization of its most abundant alkaloids has drawn attention to three molecules in particular, owing to their demonstrated antinociceptive activity and limited side effects in vivo. These three molecules are mitragynine (MG), its oxidized active metabolite, 7-hydroxymitragynine (7OH), and the indole-to-spiropseudoindoxy rearrangement product of MG known as mitragynine pseudoindoxyl (MP). Although these three alkaloids have been shown to preferentially activate the G protein signaling pathway by binding and allosterically modulating the µ-opioid receptor (MOP), a molecular level understanding of this process is lacking and yet important for the design of improved therapeutics. The molecular dynamics study and experimental validation reported here provide an atomic level description of how MG, 7OH, and MP bind and allosterically modulate the MOP, which can eventually guide structure-based drug design of improved therapeutics.

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.

Kratom Alkaloids, Natural and Semi-Synthetic, Show Less Physical Dependence and Ameliorate Opioid Withdrawal.

Chronic administration of opioids produces physical dependence and opioid-induced hyperalgesia. Users claim the Thai traditional tea "kratom" and component alkaloid mitragynine ameliorate opioid withdrawal without increased sensitivity to pain. Testing these claims, we assessed the combined kratom alkaloid extract (KAE) and two individual alkaloids, mitragynine (MG) and the analog mitragynine pseudoindoxyl (MP), evaluating their ability to produce physical dependence and induce hyperalgesia after chronic administration, and as treatments for withdrawal in morphine-dependent subjects. C57BL/6J mice (n = 10/drug) were administered repeated saline, or graded, escalating doses of morphine (intraperitoneal; i.p.), kratom alkaloid extract (orally, p.o.), mitragynine (p.o.), or MP (subcutaneously, s.c.) for 5 days. Mice treated chronically with morphine, KAE, or mitragynine demonstrated significant drug-induced hyperalgesia by day 5 in a 48 °C warm-water tail-withdrawal test. Mice were then administered naloxone (10 mg/kg, s.c.) and tested for opioid withdrawal signs. Kratom alkaloid extract and the two individual alkaloids demonstrated significantly fewer naloxone-precipitated withdrawal signs than morphine-treated mice. Additional C57BL/6J mice made physically dependent on morphine were then used to test the therapeutic potential of combined KAE, mitragynine, or MP given twice daily over the next 3 days at either a fixed dose or in graded, tapering descending doses. When administered naloxone, mice treated with KAE, mitragynine, or MP under either regimen demonstrated significantly fewer signs of precipitated withdrawal than control mice that continued to receive morphine. In conclusion, while retaining some liabilities, kratom, mitragynine, and mitragynine pseudoindoxyl produced significantly less physical dependence and ameliorated precipitated withdrawal in morphine-dependent animals, suggesting some clinical value.

The antinociceptive effects of a dual kappa-delta opioid receptor agonist in the mouse formalin test.

Pain management is a challenging and unmet medical need. Despite their demonstrated efficacy, currently used opioid drugs and nonsteroidal anti-inflammatory drugs are frequently associated with several adverse events. The identification of new and safe analgesics is therefore needed. MP1104, an analogue of 3'-iodobenzoyl naltrexamine, is a potent nonselective full agonist at mu (MOR), kappa (KOR), and delta (DOR) opioid receptors, respectively. It was shown to possess potent antinociceptive effects in acute thermal pain assays without aversion in mice. In this study, we investigated MP1104 in the formalin test, a model of tonic pain. MP1104 (0.05, 0.1, and 1.0 mg/kg) reduced pain-like behaviors in phases I and II of the formalin test in male and female ICR mice. Pretreatment with KOR antagonist (norbinaltorphimine 10 mg/kg) and DOR antagonist (naltrindole 10 mg/kg) abolished the antinociceptive effects of MP1104 in the formalin test. These findings support the development of MP1104 for further testing in other pain models.

Biased Opioid Ligands.

Achieving effective pain management is one of the major challenges associated with modern day medicine. Opioids, such as morphine, have been the reference treatment for moderate to severe acute pain not excluding chronic pain modalities. Opioids act through the opioid receptors, the family of G-protein coupled receptors (GPCRs) that mediate pain relief through both the central and peripheral nervous systems. Four types of opioid receptors have been described, including the µ-opioid receptor (MOR), κ-opioid receptor (KOR), δ-opioid receptor (DOR), and the nociceptin opioid peptide receptor (NOP receptor). Despite the proven success of opioids in treating pain, there are still some inherent limitations. All clinically approved MOR analgesics are associated with adverse effects, which include tolerance, dependence, addiction, constipation, and respiratory depression. On the other hand, KOR selective analgesics have found limited clinical utility because they cause sedation, anxiety, dysphoria, and hallucinations. DOR agonists have also been investigated but they have a tendency to cause convulsions. Ligands targeting NOP receptor have been reported in the preclinical literature to be useful as spinal analgesics and as entities against substance abuse disorders while mixed MOR/NOP receptor agonists are useful as analgesics. Ultimately, the goal of opioid-related drug development has always been to design and synthesize derivatives that are equally or more potent than morphine but most importantly are devoid of the dangerous residual side effects and abuse potential. One proposed strategy is to take advantage of biased agonism, in which distinct downstream pathways can be activated by different molecules working through the exact same receptor. It has been proposed that ligands not recruiting ß-arrestin 2 or showing a preference for activating a specific G-protein mediated signal transduction pathway will function as safer analgesic across all opioid subtypes. This review will focus on the design and the pharmacological outcomes of biased ligands at the opioid receptors, aiming at achieving functional selectivity.

Pharmacological Characterization of Levorphanol, a G-Protein Biased Opioid Analgesic.

BACKGROUND: Levorphanol is a potent analgesic that has been used for decades. Most commonly used for acute and cancer pain, it also is effective against neuropathic pain. The recent appreciation of the importance of functional bias and the uncovering of multiple µ opioid receptor splice variants may help explain the variability of patient responses to different opioid drugs. METHODS: Here, we evaluate levorphanol in a variety of traditional in vitro receptor binding and functional assays. In vivo analgesia studies using the radiant heat tail flick assay explored the receptor selectivity of the responses through the use of knockout (KO) mice, selective antagonists, and viral rescue approaches. RESULTS: Receptor binding studies revealed high levorphanol affinity for all the µ, δ, and κ opioid receptors. In S-GTPγS binding assays, it was a full agonist at most µ receptor subtypes, with the exception of MOR-1O, but displayed little activity in ß-arrestin2 recruitment assays, indicating a preference for G-protein transduction mechanisms. A KO mouse and selective antagonists confirmed that levorphanol analgesia was mediated through classical µ receptors, but there was a contribution from 6 transmembrane targets, as illustrated by a lower response in an exon 11 KO mouse and its rescue with a virally transfected 6 transmembrane receptor splice variant. Compared to morphine, levorphanol had less respiratory depression at equianalgesic doses. CONCLUSIONS: While levorphanol shares many of the same properties as the classic opioid morphine, it displays subtle differences that may prove helpful in its clinical use. Its G-protein signaling bias is consistent with its diminished respiratory depression, while its incomplete cross tolerance with morphine suggests it may prove valuable clinically with opioid rotation.

Truncated mu opioid GPCR variant involvement in opioid-dependent and opioid-independent pain modulatory systems within the CNS.

The clinical management of severe pain depends heavily on opioids acting through mu opioid receptors encoded by the Oprm1 gene, which undergoes extensive alternative splicing. In addition to generating a series of prototypic seven transmembrane domain (7TM) G protein-coupled receptors (GPCRs), Oprm1 also produces a set of truncated splice variants containing only six transmembrane domains (6TM) through which selected opioids such as IBNtxA (3'-iodobenzoyl-6ß-naltrexamide) mediate a potent analgesia without many undesirable effects. Although morphine analgesia is independent of these 6TM mu receptor isoforms, we now show that the selective loss of the 6TM variants in a knockout model eliminates the analgesic actions of delta and kappa opioids and of α2-adrenergic compounds, but not cannabinoid, neurotensin, or muscarinic drugs. These observations were confirmed by using antisense paradigms. Despite their role in analgesia, loss of the 6TM variants were not involved with delta opioid-induced seizure activity, aversion to the kappa drug U50, 488H, or α2-mediated hypolocomotion. These observations support the existence of parallel opioid and nonopioid pain modulatory systems and highlight the ability to dissociate unwanted delta, kappa1, and α2 actions from analgesia.

Truncated µ-Opioid Receptors With 6 Transmembrane Domains Are Essential for Opioid Analgesia.

BACKGROUND: Most clinical opioids act through µ-opioid receptors. They effectively relieve pain but are limited by side effects, such as constipation, respiratory depression, dependence, and addiction. Many efforts have been made toward developing potent analgesics that lack side effects. Three-iodobenzoyl-6ß-naltrexamide (IBNtxA) is a novel class of opioid active against thermal, inflammatory, and neuropathic pain, without respiratory depression, physical dependence, and reward behavior. The µ-opioid receptor (OPRM1) gene undergoes extensive alternative precursor messenger ribonucleic acid splicing, generating multiple splice variants that are conserved from rodents to humans. One type of variant is the exon 11 (E11)-associated truncated variant containing 6 transmembrane domains (6TM variant). There are 5 6TM variants in the mouse OPRM1 gene, including mMOR-1G, mMOR-1M, mMOR-1N, mMOR-1K, and mMOR-1L. Gene-targeting mouse models selectively removing 6TM variants in E11 knockout (KO) mice eliminated IBNtxA analgesia without affecting morphine analgesia. Conversely, morphine analgesia is lost in an exon 1 (E1) KO mouse that lacks all 7 transmembrane (7TM) variants but retains 6TM variant expression, while IBNtxA analgesia remains intact. Elimination of both E1 and E11 in an E1/E11 double KO mice abolishes both morphine and IBNtxA analgesia. Reconstituting expression of the 6TM variant mMOR-1G in E1/E11 KO mice through lentiviral expression rescued IBNtxA but not morphine analgesia. The aim of this study was to investigate the effect of lentiviral expression of the other 6TM variants in E1/E11 KO mice on IBNtxA analgesia. METHODS: Lentiviruses expressing 6TM variants were packaged in HEK293T cells, concentrated by ultracentrifugation, and intrathecally administered 3 times. Opioid analgesia was determined using a radiant-heat tail-flick assay. Expression of lentiviral 6TM variant messenger ribonucleic acids was examined by polymerase chain reaction (PCR) or quantitative PCR. RESULTS: All the 6TM variants restored IBNtxA analgesia in the E1/E11 KO mouse, while morphine remained inactive. Expression of lentiviral 6TM variants was confirmed by PCR or quantitative PCR. IBNtxA median effective dose values determined from cumulative dose-response studies in the rescued mice were indistinguishable from wild-type animals. IBNtxA analgesia was maintained for up to 33 weeks in the rescue mice and was readily antagonized by the opioid antagonist levallorphan. CONCLUSIONS: Our study demonstrated the pharmacological relevance of mouse 6TM variants in IBNtxA analgesia and established that a common functional core of the receptors corresponding to the transmembrane domains encoded by exons 2 and 3 is sufficient for activity. Thus, 6TM variants offer potential therapeutic targets for a distinct class of analgesics that are effective against broad-spectrum pain models without many side effects associated with traditional opioids.

Synthetic and Receptor Signaling Explorations of the Mitragyna Alkaloids: Mitragynine as an Atypical Molecular Framework for Opioid Receptor Modulators.

Mu-opioid receptor agonists represent mainstays of pain management. However, the therapeutic use of these agents is associated with serious side effects, including potentially lethal respiratory depression. Accordingly, there is a longstanding interest in the development of new opioid analgesics with improved therapeutic profiles. The alkaloids of the Southeast Asian plant Mitragyna speciosa, represented by the prototypical member mitragynine, are an unusual class of opioid receptor modulators with distinct pharmacological properties. Here we describe the first receptor-level functional characterization of mitragynine and related natural alkaloids at the human mu-, kappa-, and delta-opioid receptors. These results show that mitragynine and the oxidized analogue 7-hydroxymitragynine, are partial agonists of the human mu-opioid receptor and competitive antagonists at the kappa- and delta-opioid receptors. We also show that mitragynine and 7-hydroxymitragynine are G-protein-biased agonists of the mu-opioid receptor, which do not recruit ß-arrestin following receptor activation. Therefore, the Mitragyna alkaloid scaffold represents a novel framework for the development of functionally biased opioid modulators, which may exhibit improved therapeutic profiles. Also presented is an enantioselective total synthesis of both (-)-mitragynine and its unnatural enantiomer, (+)-mitragynine, employing a proline-catalyzed Mannich-Michael reaction sequence as the key transformation. Pharmacological evaluation of (+)-mitragynine revealed its much weaker opioid activity. Likewise, the intermediates and chemical transformations developed in the total synthesis allowed the elucidation of previously unexplored structure-activity relationships (SAR) within the Mitragyna scaffold. Molecular docking studies, in combination with the observed chemical SAR, suggest that Mitragyna alkaloids adopt a binding pose at the mu-opioid receptor that is distinct from that of classical opioids.

Mediation of buprenorphine analgesia by a combination of traditional and truncated mu opioid receptor splice variants.

Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended on the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of (35) S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit ß-arrestin-2 binding at doses as high as 10 µM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes but other actions may involve alternative receptors.

Mild, Pd-catalyzed stannylation of radioiodination targets.

Trialkylstannanes are versatile precursors for chemical transformations, including radiolabeling with a variety of halogens, particularly iodine. In the present work a convenient, Pd-mediated stannylation method is presented that can be performed in an open flask. The method is selective for aryl iodides allowing selective stannylations in the presence of other halogen atoms. The reaction conditions are mild, making the method compatible with chemically sensitive bioactive compounds.