The endogenous opioid system in the medial prefrontal cortex mediates ketamine's antidepressant-like actions.

Recent studies have implicated the endogenous opioid system in the antidepressant actions of ketamine, but the underlying mechanisms remain unclear. We used a combination of pharmacological, behavioral, and molecular approaches in rats to test the contribution of the prefrontal endogenous opioid system to the antidepressant-like effects of a single dose of ketamine. Both the behavioral actions of ketamine and their molecular correlates in the medial prefrontal cortex (mPFC) are blocked by acute systemic administration of naltrexone, a competitive opioid receptor antagonist. Naltrexone delivered directly into the mPFC similarly disrupts the behavioral effects of ketamine. Ketamine treatment rapidly increases levels of ß-endorphin and the expression of the µ-opioid receptor gene (Oprm1) in the mPFC, and the expression of gene that encodes proopiomelanocortin, the precursor of ß-endorphin, in the hypothalamus, in vivo. Finally, neutralization of ß-endorphin in the mPFC using a specific antibody prior to ketamine treatment abolishes both behavioral and molecular effects. Together, these findings indicate that presence of ß-endorphin and activation of opioid receptors in the mPFC are required for the antidepressant-like actions of ketamine.

Long-Term Effects of Low-Dose Naltrexone on Immunomodulatory Properties of Human Adipose-Derived Mesenchymal Stem Cells.

Background: Low-dose naltrexone (LDN) is involved in the treatment of inflammatory and immune system diseases and can affect immune cells. Mesenchymal stem cells (MSCs) are known for their immunomodulatory effects and the potential for the treatment of certain types of autoimmune diseases. Objective: To investigate the long-term effects of LDN on human adipose-derived mesenchymal stem cells (ASCs) to see how their immunomodulatory properties are affected and also how LDN-treated ASCs interact with other immune cells present in peripheral blood mononuclear cells (PBMCs). Methods: After 14 days of treatment, the ability of LDN-treated ASCs to modulate PBMC proliferation in a two-way mixed lymphocyte reaction (MLR) model was assessed using XTT. The relative expression of IDO, PD-L1, COX-2, HGF genes, and the level of IL-6 and TGF-ß cytokines were measured in IFN-γ stimulated and unstimulated ASCs (treated and not treated cells) using real-time PCR and ELISA respectively. Results: Unstimulated ASCs treated with 10-8 M Naltrexone (10-8 M NTX) showed higher levels of TGF-ß, compared with the controls (P<0.05). Stimulated ASCs treated with 10-6 M NTX showed elevated expression of IDO, PD-L1 genes, and IL-6 level (P<0.05). Conclusion: Our results demonstrated that various LDN concentrations have dissimilar effects on ASCs' immunomodulatory properties. A higher LDN concentration induced an alteration in the immunomodulatory features of ASCs.

Naltrexone Transport by a Proton-Coupled Organic Cation Antiporter in hCMEC/D3 Cells, an in Vitro Human Blood-Brain Barrier Model.

Naltrexone is a mu-opioid receptor antagonist used in the treatment of opioid and alcohol dependence. The blood-brain barrier (BBB) transport characteristics of naltrexone was investigated by means of hCMEC/D3 cells, a human immortalized brain capillary endothelial cell line. In hCMEC/D3 cells, naltrexone is taken up in a concentration-dependent manner. Furthermore, naltrexone uptake significantly decreased in the presence of H+/organic cation (OC) antiporter substrates, during the little alteration exhibited by substrates of well-identified OC transporters classified into SLC22A family. Although naltrexone uptake by hCMEC/D3 cells was partially affected by changes of ionic conditions, it was markedly decreased in the presence of the metabolic inhibitor sodium azide. Furthermore, when treated by ammonium chloride, naltrexone uptake by hCMEC/D3 cells was altered by intracellular acidification and alkalization, suggesting the involvement of oppositely directed proton gradient in naltrexone transport across the BBB. The results obtained in the present in vitro study suggest the active transport of naltrexone from blood to the brain across the BBB by the H+/OC antiporter.

Naltrexone protects against BDL-induced cirrhosis in Wistar rats by attenuating thrombospondin-1 and enhancing antioxidant defense system via Nrf-2.

AIMS: It is well-established that thrombospondin-1 (THBS-1), vascular endothelial growth factor-A (VEGF-A), nuclear factor-erythroid 2-related factor 2 (Nrf-2), Kelch-like ECH-associated protein 1 (Keap-1), and transforming growth factor-beta 1 (TGF-ß1) are the pivotal players of liver fibrosis. Recent studies have shown that endogenous opioid levels increase during liver cirrhosis. Therefore, the present study aimed to clarify the effect of naltrexone (NTX), an opioid antagonist, on the alteration of these factors following bile duct ligation (BDL)-induced liver cirrhosis. MAIN METHODS: Wistar male rats (n = 50) were categorized equally into 5 groups (baseline, sham+saline, BDL + saline, sham+NTX (10 mg/kg of body weight (BW)), and BDL + NTX (10 mg/kg of BW)). At the end of the experiment, H&E staining was used to assess necrosis and lobular damage of hepatic tissue. The gene expression of THBS-1 and NADPH oxidase 1 (NOX-1) was measured by real time-PCR and VEGF-A, Nrf-2, Keap-1, and TGF-ß1 protein levels were assessed by western blot. The antioxidant enzymes activity, total oxidant status (TOS) and MDA level were measured by commercial kits. KEY FINDINGS: Hepatic necrosis and lobular damage increased substantially and NTX reduced them markedly in the BDL group. Gene expression of hepatic THBS-1 and NOX-1, TOS and MDA levels increased markedly in the BDL + saline group, and Nrf-2 and VEGF-A values decreased significantly in the BDL + NTX group. NTX recovered THBS-1, NOX-1 and Nrf-2 in the BDL + NTX group, substantially (p-value ≤ 0.05). SIGNIFICANCE: Data showed that NTX treatment attenuates liver fibrosis mainly by lowering THBS-1 and NOX-1 and increasing Nrf-2 protein level and antioxidant enzymes.

Exploring the putative mechanism of allosteric modulations by mixed-action kappa/mu opioid receptor bitopic modulators.

The modulation and selectivity mechanisms of seven mixed-action kappa opioid receptor (KOR)/mu opioid receptor (MOR) bitopic modulators were explored. Molecular modeling results indicated that the 'message' moiety of seven bitopic modulators shared the same binding mode with the orthosteric site of the KOR and MOR, whereas the 'address' moiety bound with different subdomains of the allosteric site of the KOR and MOR. The 'address' moiety of seven bitopic modulators bound to different subdomains of the allosteric site of the KOR and MOR may exhibit distinguishable allosteric modulations to the binding affinity and/or efficacy of the 'message' moiety. Moreover, the 3-hydroxy group on the phenolic moiety of the seven bitopic modulators induced selectivity to the KOR over the MOR.

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.

Methylnaltrexone crosses the blood-brain barrier and attenuates centrally-mediated behavioral effects of morphine and oxycodone in mice.

BACKGROUND: Antagonism of peripheral opioid receptors by methylnaltrexone (MNTX) was recently proposed as a potential mechanism to attenuate the development of opioid analgesic tolerance based on experiments conducted in mice. However, reports indicate that MNTX is demethylated to naltrexone (NTX) in mice, and NTX may subsequently cross the blood-brain barrier to antagonize centrally-mediated opioid effects. The goal of this study was to determine whether MNTX alters centrally-mediated behaviors elicited by the opioid analgesics, morphine and oxycodone, and to quantify concentrations of MNTX and NTX in blood and brain following their administration in mice. METHODS: Combinations of MNTX and morphine were tested under acute and chronic conditions in thermal nociceptive assays. Effects of MNTX and NTX pretreatment were assessed in an oxycodone discrimination operant procedure. Blood and brain concentrations of these antagonists were quantified after their administration using liquid chromatography-mass spectrometry. RESULTS: MNTX dose-dependently attenuated acute and chronic morphine antinociception. MNTX and NTX dose-dependently antagonized the discriminative stimulus effects of oxycodone. MNTX and NTX were detected in both blood and brain after administration of MNTX, confirming its demethylation and demonstrating that MNTX itself can cross the blood-brain barrier. CONCLUSIONS: These results provide converging behavioral and analytical evidence that MNTX administration in mice attenuates centrally-mediated effects produced by opioid analgesics and results in functional concentrations of MNTX and NTX in blood and brain. Collectively, these findings indicate that MNTX cannot be administered systemically in mice for making inferences that its effects are peripherally restricted.

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.

Preventive effects of naldemedine, peripherally acting µ-opioid receptor antagonist, on morphine-induced nausea and vomiting in ferrets.

AIMS: Naldemedine is a peripherally acting µ-opioid receptor antagonists (PAMORAs) indicated for the treatment of opioid-induced constipation (OIC). We investigated the preventive effect of naldemedine on morphine-induced nausea and vomiting in ferrets and conducted a pharmacokinetic/pharmacodynamic (PK/PD) analysis. MAIN METHODS: The antiemetic effect of naldemedine was evaluated as the frequency and time of retching (rhythmic abdominal contractile motion) and vomiting (throwing up vomit or similar reactions) caused by morphine in ferrets. After a single oral administration of naldemedine to ferrets, the plasma concentrations of naldemedine and morphine were measured by liquid chromatography-tandem mass spectrometry. KEY FINDINGS: Naldemedine showed a potent and dose-dependent anti-emetic effects against morphine-induced emetic responses, for up to 6 h. The dose of naldemedine that produced half the maximal effect (ED50) value for anti-emetic effect of naldemedine in the morphine-treated ferrets was 0.033 mg/kg. The PK/PD analysis revealed that the antiemetic effect was related to the plasma naldemedine concentration, with a half maximal effective concentration that produces half the maximal effect (EC50) of 3.51 ng/mL. The plasma concentration producing an antiemetic effect was almost 200-fold lower than that inducing an anti-analgesic effect in rats. SIGNIFICANCE: Naldemedine showed potent inhibition of morphine-induced vomiting for up to 6 h after dosing. These data suggest that naldemedine possesses antiemetic properties and could be effective against opioid-induced nausea and vomiting (OINV).

Effects of naltrexone on alcohol, sucrose, and saccharin binge-like drinking in C57BL/6J mice: a study with a multiple bottle choice procedure.

Chronic alcohol (ethyl alcohol, EtOH) binging has been associated with long-term neural adaptations that lead to the development of addiction. Many of the neurobiological features of EtOH abuse are shared with other forms of binging, like pathological feeding. The drinking-in-the-dark (DID) paradigm has been used extensively to study the neurobiology of EtOH binge-like drinking due to its ability to promote high intakes relevant to human behavior. DID can also generate high consumption of other tastants, but this procedure has not been fully adapted to study forms of binging behavior that are not alcohol-driven. In the present study, we used a modified version of DID that uses multiple bottle availability to promote even higher levels of EtOH drinking in male C57BL/6J mice and allows a thorough investigation of tastant preferences. We assessed whether administration of systemic naltrexone could reduce binging on EtOH, sucrose, and saccharin separately as well as in combination. Our multiple bottle DID procedure resulted in heightened levels of consumption compared with previously reported data using this task. We found that administration of the opioid receptor antagonist naltrexone reduced intakes of preferred, highly concentrated EtOH, sucrose, and saccharin. We also report that naltrexone was able to reduce overall intakes when animals were allowed to self-administer EtOH, sucrose, or saccharin in combination. Our modified DID procedure provides a novel approach to study binging behavior that extends beyond EtOH to other tastants (i.e. sucrose and artificial sweeteners), and has implications for the study of the neuropharmacology of binge drinking.

Opioid receptor blockade inhibits self-disclosure during a closeness-building social interaction.

Social ties are critical to human health and well-being; thus, it is important to gain a better understanding of the neurobiological mechanisms involved in the development of interpersonal closeness. Prior research indicates that endogenous opioids may play a role in social affiliation by elaborating feelings of social connection and warmth; however, it is not currently known whether opioids mediate affiliative behavior and emerging feelings of closeness in humans at the relationship initiation stage. This randomized, double-blind study examined opioidergic processes in the context of a naturalistic, face-to-face social interaction. Eighty pairs of unacquainted participants (final N = 159 due to removal of one dyad member from analysis) received either 50 mg of the opioid receptor antagonist naltrexone or placebo prior to completing a closeness-building exercise centered on escalating self-disclosure (sharing of personal information about the self). Compared to the placebo group, naltrexone participants held lower social reward expectations prior to the interaction, engaged in less intimacy-fostering behavior (self-disclosure) during the interaction, and reported wanting less closeness with their partner. Feelings of social connection were not significantly lower in the naltrexone group. However, placebo participants experienced improvements in mood after the closeness-building task whereas naltrexone participants did not. These findings suggest that endogenous opioids may contribute to behavioral, affective, and motivational processes related to the development of initial closeness.

Naltrexone and Its Noroxymorphone Minor Metabolite - A Case Report.

As part of substance use maintenance programs, individuals are monitored for sobriety through urine drug screens. A positive screen, and its confirmation and interpretation, can have devastating consequences, sometimes even leading to termination from the program and relapse. Naltrexone metabolism involves several steps and metabolites - one minor metabolite with very little mention in medical literature being noroxymorphone. This is also the final intermediate in the metabolic pathway of oxycodone; hence, detection is naturally presumed by clinicians to be attributed to oxycodone use. Through this case report, we alert clinicians that, depending on individual pharmacogenomics, it is possible to obtain a positive confirmation of this component alone (without any oxycodone pathway intermediates) with naltrexone administration.

Clinically utilized kappa-opioid receptor agonist nalfurafine combined with low-dose naltrexone prevents alcohol relapse-like drinking in male and female mice.

Alcohol relapse is a treatment goal for alcohol dependence and the target for medications' development. Clinically utilized nalfurafine (NFF) is a potent and selective kappa- opioid receptor (KOP-r) agonist, with fewer side effects (e.g., sedation or anhedonia) than classic KOP-r full agonists. We have recently found that NFF reduces excessive alcohol drinking in mice via a KOP-r-mediated mechanism. Here, we further investigated whether NFF alone (1-10 µg/kg) or in combination with naltrexone (NTX, mu-opioid receptor [MOP-r] antagonist) altered alcohol relapse-like drinking using a mouse alcohol deprivation effect (ADE) paradigm to mimic the relapse episodes in human alcoholics. Nalmefene (NMF, clinically utilized KOP-r partial agonist with MOP-r antagonism) was used as a reference compound for the effects on mouse ADE of new NFF + NTX combination. After exposed to 3-week intermittent- access alcohol drinking (two-bottle choice, 24-h access every other day), both male and female mice displayed excessive alcohol intake and then pronounced ADE after 1-week abstinence. NFF prevented the ADE in a dose-dependent manner in both male and female mice. A combination of NFF with NTX reduced the ADE without sex differences at doses lower than those individual effective ones, suggesting synergistic effects between the two compounds. NMF prevented the ADE in both sexes, while selective KOP-r antagonist nor-BNI had no effect. Our new study suggests that a combination of clinically-utilized, potent KOP-r agonist NFF with low-dose NTX has therapeutic potential in alcohol "relapse" treatment.

Study on structural insight of the analysis of negative effects of opioids analgesics in naltrexone with TLR4 Mutations.

Chronic pain has been defined as the persistence that remained for more than three months. The extent of previous time duration with the normal time of natural healing phase becomes poor and results in reduced life quality and morbidity. Opioids are well recognized therapy for pain management and the clinical prescriptions based on opioids have been defined with increasing implicating behavior among patients suffering with chronic pain. The association between the pain and immunity has long been established since the involvement of interleukin-1ß (IL-1ß) in sickness that is considered with the induced hyperalgesia. In the context of pharmacodynamics Toll like receptors (TLRs) are involved in the negative effects of opioids as analgesics. The soluble factors released by immune cells as well as from the disruptive cells bind to TLRs. This binding leads the pre and post-synaptic ends on endothelial and microglial cells that exhibit the activation of complex inhibitory and excitatory process at the synapses site. In TLRs, TLR4 is mostly reported that is strongly associated in specifically in areas of T cells and macrophages. The current study is designed to investigate the structural insights of the opioids and TLR4 interactions by using computational approach in the aspect of recognizing the chemical combinatorial factors that are involved in the pain management. This study targets that how opioids interact with TLR4 and the process of chemical interaction that leads to negative effects of opioids at neuroimmune interface as well as to investigate the extent of particular naltrexone that mediates with the negative effects of opioids.

New opioid receptor modulators and agonists.

There has been significant research to develop an ideal synthetic opioid. Opioids with variable properties possessing efficacy and with reduced side effects have been synthesized when compared to previously used agents. An opioid modulator is a drug that can produce both agonistic and antagonistic effects by binding to different opioid receptors and therefore cannot be classified as one or the other alone. These compounds can differ in their structures while still possessing opioid-mediated actions. This review will discuss TRV130 receptor modulators and other novel opioid receptor modulators, including Mitragyna "Kratom," Ignavine, Salvinorin-A, DPI-289, UFP-505, LP1, SKF-10,047, Cebranopadol, Naltrexone-14-O-sulfate, and Naloxegol. In summary, the structural elucidation of opioid receptors, allosteric modulation of opioid receptors, new opioid modulators and agonists, the employment of optogenetics, optopharmacology, and next-generation sequencing of opioid receptor genes and related functionality should create exciting new avenues for research and therapeutic development to treat conditions including pain, opioid abuse, and addiction.

Dissecting the Innate Immune Recognition of Opioid Inactive Isomer (+)-Naltrexone Derived Toll-like Receptor 4 (TLR4) Antagonists.

The opioid inactive isomer (+)-naltrexone is one of the rare Toll-like receptor 4 (TLR4) antagonists with good blood-brain barrier (BBB) permeability, which is a lead with promising potential for treating neuropathic pain and drug addiction. (+)-Naltrexone targets the lipopolysaccharides (LPS) binding pocket of myeloid differentiation protein 2 (MD-2) and blocks innate immune TLR4 signaling. However, the details of the molecular interactions of (+)-naltrexone and its derivatives with MD-2 are not fully understood, which hinders the ligand-based drug discovery. Herein, in silico and in vitro assays were performed to elucidate the innate immune recognition of the opioid inactive (+)-isomers. The results showed that the conserved LPS binding pocket of MD-2 accommodated these opioid inactive (+)-isomers. The calculated binding free energies of (+)-naltrexone and its derivatives in complex with MD-2 correlated well with their experimental binding affinities and TLR4 antagonistic activities. Hydrophobic residues in the MD-2 cavity interacted directly with these (+)-naltrexone based TLR4 antagonists and principally participated in ligand binding. Increasing the hydrophobicity of substituted group at N-17 improved its TLR4 antagonistic activity, while charged groups disfavored the binding with MD-2. Molecular dynamics (MD) simulations showed the binding of (+)-naltrexone or its derivatives to MD-2 stabilized the "collapsed" conformation of MD-2, consequently blocking the binding and signaling of TLR4. Thermodynamics and dynamic analysis showed the topology of substituted group at N-17 of (+)-naltrexone affected the binding with MD-2 and TLR4 antagonistic activity. This study provides a molecular insight into the innate immune recognition of opioid inactive (+)-isomers, which would be of great help for the development of next-generation of (+)-opioid based TLR4 antagonists.

Morphine induces changes in the gut microbiome and metabolome in a morphine dependence model.

Opioid analgesics are frequently prescribed in the United States and worldwide. However, serious comorbidities, such as dependence, tolerance, immunosuppression and gastrointestinal disorders limit their long-term use. In the current study, a morphine-murine model was used to investigate the role of the gut microbiome and metabolome as a potential mechanism contributing to the negative consequences associated with opioid use. Results reveal a significant shift in the gut microbiome and metabolome within one day following morphine treatment compared to that observed after placebo. Morphine-induced gut microbial dysbiosis exhibited distinct characteristic signatures, including significant increase in communities associated with pathogenic function, decrease in communities associated with stress tolerance and significant impairment in bile acids and morphine-3-glucuronide/morphine biotransformation in the gut. Moreover, expansion of Enterococcus faecalis was strongly correlated with gut dysbiosis following morphine treatment, and alterations in deoxycholic acid (DCA) and phosphatidylethanolamines (PEs) were associated with opioid-induced metabolomic changes. Collectively, these results indicate that morphine induced distinct alterations in the gut microbiome and metabolome, contributing to negative consequences associated with opioid use. Therapeutics directed at maintaining microbiome homeostasis during opioid use may reduce the comorbidities associated with opioid use for pain management.

To probe interaction of morphine and IBNtxA with 7TM and 6TM variants of the human µ-opioid receptor using all-atom molecular dynamics simulations with an explicit membrane.

IBNtxA, a morphine derivative, is 10-fold more potent and has a better safety profile than morphine. Animal studies indicate that the analgesic effect of IBNtxA appears to be mediated by the activation of truncated splice variants (6TM) of the Mu opioid receptor (MOR-1) where transmembrane helix 1 (TM1) is removed. Interestingly, morphine is unable to activate 6TM variants. To date, a high resolution structure of 6TM variants is missing, and the interaction of 6TM variants with IBNtxA and morphine remains elusive. In this study we used homology modeling, docking and molecular dynamics (MD) simulations to study a representative 6TM variant (G1) and a full-length 7TM variant of human MOR-1 in complex with IBNtxA and morphine respectively. The structural models of human G1 and 7TM were obtained by homology modeling using the X-ray solved crystal structure of the active mouse 7TM bound to an agonist BU72 (PDB id: ) as the template. Our 6000 ns MD data show that either TM1 truncation (i.e. from 7TM to 6TM) or ligand modification (i.e. from morphine to IBNtxA) alone causes the loss of key morphine-7TM interactions that are well-known to be required for MOR-1 activation. Receptor disruptions are mainly located at TMs 2, 3, 6 and 7 in comparison with the active crystal complex. However, when both perturbations occur in the 6TM-IBNtxA complex, the key ligand-receptor interactions and the receptor conformation are recovered to resemble those in the active 7TM-morphine complex. Our molecular switch analysis further explains well why morphine is not able to activate 6TM variants. The close resemblance between 6TM-IBTtxA and 7TM in complex with PZM21, a G-protein biased 7TM agonist, suggests the possible biased agonism of IBNtxA on G1, which is consistent with its reduced side effects.

Computational insights into the G-protein-biased activation and inactivation mechanisms of the µ opioid receptor.

The µ opioid receptor (OR), a member of the class A subfamily of G-protein coupled receptors (GPCRs), is a major target for the treatment of pain. G-protein biased µ-OR agonists promise to be developed as analgesics. Thus, TRV130, the first representative µ-OR ligand with G-protein bias, has entered into phase III clinical trials. To identify the detailed G-protein-biased activation and inactivation mechanisms of the µ-OR, we constructed five µ-OR systems that were in complexes with the G-protein-biased agonists TRV130 and BU72, the antagonists ß-FNA and naltrexone, as well as the free receptor. We performed a series of conventional molecular dynamics simulations and analyses of G-protein-biased activation and inactivation mechanisms of µ-OR. Our results, together with previously reported mutation results, revealed the operating mode of the activation switch composed of residues W6.48 and Y7.43 (Ballesteros/Weinstein numbering), the activity of which was responsible for down- and up-regulation, respectively, of the ß-arrestin signaling, which in turn affected G-protein-biased activation of µ-OR. TRV130 was found to stabilize W6.48 by interacting with Y7.43. In addition, we obtained useful information regarding µ-OR-biased activation, such as strong stabilization of W7.35 through a hydrophobic ring interaction in the TRV130 system. These findings may facilitate understanding of µ-OR biased activation and the design of new biased ligands for GPCRs.

The Glycine Receptor-A Functionally Important Primary Brain Target of Ethanol.

Identification of ethanol's (EtOH) primary molecular brain targets and determination of their functional role is an ongoing, important quest. Pentameric ligand-gated ion channels, that is, the nicotinic acetylcholine receptor, the γ-aminobutyric acid type A receptor, the 5-hydroxytryptamine3 , and the glycine receptor (GlyR), are such targets. Here, aspects of the structure and function of these receptors and EtOH's interaction with them are briefly reviewed, with special emphasis on the GlyR and the importance of this receptor and its ligands for EtOH pharmacology. It is suggested that GlyRs are involved in (i) the dopamine-activating effect of EtOH, (ii) regulating EtOH intake, and (iii) the relapse preventing effect of acamprosate. Exploration of the GlyR subtypes involved and efforts to develop subtype specific agonists or antagonists may offer new pharmacotherapies for alcohol use disorders.