Richard M. van Rijn

Opioid-receptor-heteromer-specific trafficking and pharmacology

Homomerization and heteromerization of 7 transmembrane spanning (7TM)/G-protein-coupled receptors (GPCRs) have been an important field of study. Whereas initial studies were performed in artificial cell systems, recent publications are shifting the focus to the in vivo relevance of heteromerization. This is especially apparent for the field of opioid receptors. Drugs have been identified that selectively target opioid heteromers of the delta-opioid receptor with the kappa and the mu-opioid receptors that influence nociception and ethanol consumption, respectively. In addition, in several cases, the specific physiological response produced by the heteromer may be directly attributed to a difference in receptor trafficking properties of the heteromers compared with their homomeric counterparts. This review attempts to highlight some of the latest developments with regard to opioid receptor heteromer trafficking and pharmacology.

The δ1 opioid receptor is a heterodimer that opposes the actions of the δ2 receptor on alcohol intake

Background Opioid receptors are clinically important targets for both pain and alcohol abuse. Three opioid receptors have been cloned: μ, δ, and κ, all of which effect alcohol consumption in animal models. Naltrexone is a nonselective opioid antagonist used for alcoholism, the clinical utility of which is limited by poor efficacy and adverse side effects. Here, we demonstrate that the therapeutic limitations of naltrexone may reflect its poor selectivity. Despite decades of research, several mysteries surround the pharmacology of these receptors. For example, two pharmacologically defined subtypes of δ receptors exist in vivo. Methods Effects of δ subtype-selective ligands (naltrindole, naltriben, tan-67, 7-benzylidene naltrexone) were measured on ethanol consumption in C57BL/6 wildtype and opioid receptor knockout mice using a limited access two-bottle choice paradigm. Affinity and efficacy of naltriben, 7-benzylidenenaltrexone and tan-67 was measured in vitro using radioligand binding and Ca 2+ -mobilizationa assays. Results We show that the subtypes of the δ receptor, δ 1 and δ 2 , have opposing effects on ethanol consumption. We find that these effects are synergistic; thereby suggesting that δ 1 and δ 2 receptors are distinct molecular targets. Indeed, we provide both in vitro as well as in vivo evidence that the δ 1 subtype is a μ-δ heterodimer and that the δ 2 subtype is most likely a δ homomer. Conclusions Together these data provide insight into the limited actions of the clinically important drug naltrexone and identify a novel target with improved specificity and efficacy for the development of new therapeutics for the treatment of alcoholism.BACKGROUND Opioid receptors are clinically important targets for both pain and alcohol abuse. Three opioid receptors have been cloned: mu, delta, and kappa, all of which effect alcohol consumption in animal models. Naltrexone is a nonselective opioid antagonist used for alcoholism, the clinical utility of which is limited by poor efficacy and adverse side effects. Here, we demonstrate that the therapeutic limitations of naltrexone may reflect its poor selectivity. Despite decades of research, several mysteries surround the pharmacology of these receptors. For example, two pharmacologically defined subtypes of delta receptors exist in vivo. METHODS Effects of delta subtype-selective ligands (naltrindole, naltriben, tan-67, 7-benzylidene naltrexone) were measured on ethanol consumption in C57BL/6 wildtype and opioid receptor knockout mice using a limited access two-bottle choice paradigm. Affinity and efficacy of naltriben, 7-benzylidenenaltrexone and tan-67 was measured in vitro using radioligand binding and Ca(2+)-mobilizationa assays. RESULTS We show that the subtypes of the delta receptor, delta(1) and delta(2), have opposing effects on ethanol consumption. We find that these effects are synergistic; thereby suggesting that delta(1) and delta(2) receptors are distinct molecular targets. Indeed, we provide both in vitro as well as in vivo evidence that the delta(1) subtype is a micro-delta heterodimer and that the delta(2) subtype is most likely a delta homomer. CONCLUSIONS Together these data provide insight into the limited actions of the clinically important drug naltrexone and identify a novel target with improved specificity and efficacy for the development of new therapeutics for the treatment of alcoholism.

Dual Efficacy of Delta Opioid Receptor-Selective Ligands for Ethanol Drinking and Anxiety

Alcoholism and anxiety disorders have a huge impact on society and afflict 17.6 million and 40 million people in the United States, respectively. A strong comorbidity exists between alcoholism and anxiety disorders. Indeed, alcohol withdrawal-induced anxiety is a primary contributing factor for relapse, and anxiolytics are a common adjuvant therapy prescribed for treatment-seeking alcoholics. It is thought that the use of alcohol to self-medicate and relieve anxiety contributes to the development of addiction. Treatment for anxiety disorders and alcoholism exist but are not universally effective. The delta opioid receptor (DOR) plays a role in both alcohol consumption and anxiety, making it a very interesting clinical target. Two pharmacologically distinct DORs have been described: DOR1 and DOR2. We find here that the relative specificity of DOR agonists for DOR1 or DOR2 can greatly affect the effects they exert on ethanol consumption and anxiety. The DOR1 agonist 2-methyl-4aα-(3-hydroxyphenyl)-1,2,3,4,4a,5,12,12aα-octahydro-quinolino[2,3,30g]isoquinoline (TAN-67), although not effective in decreasing anxiety-like behavior in naive mice, has anxiolytic-like properties in ethanol-withdrawn mice. In contrast, a less subtype-selective agonist, (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80), while also reducing anxiety-like behavior, increases ethanol consumption. In addition, we found that the conical anxiolytic diazepam [DZ; 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2(1H)-one] is a less effective anxiolytic in ethanol-withdrawn mice than in naive mice. Together, our findings suggest that selective DOR agonists can decrease anxiety-like behavior and are more effective than diazepam at reducing ethanol consumption. We believe the dual efficacy of DOR1 agonists makes these receptors an interesting therapeutic target for treatment-seeking alcoholics.

Emergence of Functional Spinal Delta Opioid Receptors After Chronic Ethanol Exposure

BACKGROUND The delta opioid receptor (DOR) is a promising target to treat multiple indications, including alcoholism, anxiety, and nonmalignant pain. The potential of the DORs has been underappreciated, in part, due to relatively low functional expression of these receptors in naïve states. However, chronic exposure to stress, opioids, and inflammation can induce a redistribution of DORs to the cell surface where they can be activated. Previously, DORs were shown to be selectively/exclusively present in spinal cord circuits mediating mechanical sensitivity but not those mediating thermal nociception under naïve conditions. METHODS We spinally administered DOR and mu opioid receptor (MOR) selective agonists ([D-Pen2,D-Pen5]-Enkephalin, deltorphin II, SNC80, and DAMGO) and antagonists (naltriben and CTAP) and determined thermal antinociception and mechanical sensitivity in wild-type mice or mice with a genetic disruption of DOR or MOR. Thermal antinociception was measured using a radiant heat tail-flick assay; mechanical sensitivity was measured using von Frey filaments. Dose response curves were generated in naïve mice and mice exposed to ethanol in a model of voluntary consumption. RESULTS We show that prolonged exposure to ethanol can promote an upregulation of functional DORs in the spinal cord in thermal pain-mediating circuits but not in those mediating mechanical sensitivity. The upregulated DORs either modulate MOR-mediated analgesia through convergence of circuits or signal transduction pathways and/or interact directly with MORs to form a new functional (heteromeric) unit. CONCLUSIONS Our findings suggest that DORs could be a novel target in conditions in which DORs are redistributed.

Pharmacological traits of delta opioid receptors: pitfalls or opportunities?

RationaleDelta opioid receptors (DORs) have been considered as a potential target to relieve pain as well as treat depression and anxiety disorders and are known to modulate other physiological responses, including ethanol and food consumption. A small number of DOR-selective drugs are in clinical trials, but no DOR-selective drugs have been approved by the Federal Drug Administration and some candidates have failed in phase II clinical trials, highlighting current difficulties producing effective delta opioid-based therapies. Recent studies have provided new insights into the pharmacology of the DOR, which is often complex and at times paradoxical.ObjectiveThis review will discuss the existing literature focusing on four aspects: (1) Two DOR subtypes have been postulated based on differences in pharmacological effects of existing DOR-selective ligands. (2) DORs are expressed ubiquitously throughout the body and central nervous system and are, thus, positioned to play a role in a multitude of diseases. (3) DOR expression is often dynamic, with many reports of increased expression during exposure to chronic stimuli, such as stress, inflammation, neuropathy, morphine, or changes in endogenous opioid tone. (4) A large structural variety in DOR ligands implies potential different mechanisms of activating the receptor.ConclusionThe reviewed features of DOR pharmacology illustrate the potential benefit of designing tailored or biased DOR ligands.

Methadone antinociception is dependent on peripheral opioid receptors

UNLABELLED Morphine and methadone are both high-affinity, potent mu-opioid peptide (MOP) receptor analgesics. In this report, we compared the antinociceptive potencies of these 2 drugs when administered subcutaneously (s.c.), intrathecally (i.t.), or intracerebroventricularly (i.c.v.) in both rat and mouse, using the tail-flick assay. We found that both morphine and methadone were potently antinociceptive when the drugs were administered s.c., showing comparable AD50 values in both species. However, the antinociception produced by methadone, when it was administered centrally, was much weaker than that produced by centrally administered morphine. Specifically, the AD50 value for methadone antinociception was more than 30-fold higher at both the i.t. and i.c.v. sites in mouse and not measurable in rat. Naloxone methiodide (NLX-M), a peripherally restricted antagonist, was used to further examine the relative contribution of central versus peripheral sites to morphine and methadone antinociception. NLX-M, when administered s.c., blocked the antinociceptive effect of either systemically or centrally administered methadone but had little effect on the antinociception produced by centrally administered morphine. Furthermore, centrally administered NLX-M significantly blocked antinociception produced by centrally administered morphine but not that produced by centrally administered methadone. Together, these results suggest that methadone antinociception is significantly dependent on an action of the drug at peripheral sites and could provide novel insight into the neural mechanisms that distinguish morphine versus methadone antinociception. PERSPECTIVE Methadone is often used as an alternative for pain management. The present study shows that a peripheral action plays a crucial role in methadone antinociception. This finding could have significant clinical relevance for the use of methadone versus morphine for the treatment of certain types of pain.

Novel screening assay for the selective detection of G protein-coupled receptor heteromer signaling

Drugs targeting G-protein-coupled receptors (GPCRs) make up more than 25% of all prescribed medicines. The ability of GPCRs to form heteromers with unique signaling properties suggests an entirely new and unexplored pool of drug targets. However, current in vitro assays are ill equipped to detect heteromer-selective compounds. We have successfully adapted an approach, using fusion proteins of GPCRs and chimeric G proteins, to create an in vitro screening assay (in human embryonic kidney cells) in which only activated heteromers are detectable. Here we show that this assay can demonstrate heteromer-selective G-protein bias as well as measure transinhibition. Using this assay, we reveal that the δ-opioid receptor agonist ADL5859, which is currently in clinical trials, has a 10-fold higher potency against δ-opioid receptor homomers than δ/μ-opioid receptor heteromers (pEC50 = 6.7 ± 0.1 versus 5.8 ± 0.2). The assay enables the screening of large compound libraries to identify heteromer-selective compounds that could then be used in vivo to determine the functional role of heteromers and develop potential therapeutic agents.

Distinctive modulation of ethanol place preference by delta opioid receptor-selective agonists

BACKGROUND Naltrexone is one of the few drugs approved by the Federal Drug Administration for the treatment of alcoholism. However, naltrexone is only effective in a subpopulation of treatment-seeking alcohol abusers, and suffers from compliance issues. The non-selective nature of this opioid antagonist likely contributes to its side effects and poor therapeutic efficacy. Drugs selectively targeting delta opioid receptor subtypes offer a potential way to treat alcohol abuse disorders. We have recently shown that delta subtype-selective agonists TAN-67 and SNC80 can have opposing effects on alcohol consumption, while having similar effects on alcohol withdrawal-induced anxiety. METHODS We studied the ability of TAN-67 and SNC80 to induce place preference in naïve and ethanol exposed C57BL/6 mice and determined the effect of these agonists on the expression of ethanol place preference. RESULTS We show that TAN-67 and SNC80 have opposing actions on ethanol place preference. However, neither of the drugs induces place preference by themselves at doses that are therapeutically effective in mice. Interestingly, SNC80, like naltrexone reduces ethanol place preference, however we have previously shown that SNC80 increases ethanol consumption at the tested dose. Similar to naltrexone, TAN-67 reduces alcohol consumption, but we show here that it may be due to an increase in ethanol place preference. Importantly, we found that chronic ethanol exposure does not increase the rewarding properties of the DOR subtype selective agonists. CONCLUSIONS Our results provide a better understanding of how DOR subtype selective drugs could potentially be used for treatment of alcohol abuse disorders.

Identification of a selective small-molecule inhibitor of type 1 adenylyl cyclase activity with analgesic properties

ST034307 is a selective AC1 inhibitor with analgesic activity. Pain relief through AC1 inhibition Brust et al. identified a small-molecule inhibitor of adenylyl cyclase 1 (AC1), which is a potential target for treating pain and reducing the dependency on opioids for pain management. The challenge has been that there are many AC isoforms and their function is crucial to most physiological processes, so isoform specificity is key to any chance of therapeutic efficacy. The authors identified two compounds that inhibited AC1 in a screen of a chemical library of natural compounds that reduced the production of adenosine 3′,5′-monophosphate (cAMP), the product of AC activity. One of the compounds, ST034307, showed selective inhibition of AC1 over all eight other AC isoforms. This compound produced analgesia in a mouse model of inflammatory pain, blocked cellular changes associated with opioid dependency in transfected cells, and inhibited cAMP accumulation in both transfected cells and hippocampal tissue samples. This compound should not only be a useful tool for investigating AC1-specific physiology but also provide support for the development of AC1-selective pain relievers. Adenylyl cyclase 1 (AC1) belongs to a group of adenylyl cyclases (ACs) that are stimulated by calcium in a calmodulin-dependent manner. Studies with AC1 knockout mice suggest that inhibitors of AC1 may be useful for treating pain and opioid dependence. However, nonselective inhibition of AC isoforms could result in substantial adverse effects. We used chemical library screening to identify a selective AC1 inhibitor with a chromone core structure that may represent a new analgesic agent. After demonstrating that the compound (ST034307) inhibited Ca2+-stimulated adenosine 3′,5′-monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably transfected with AC1 (HEK-AC1 cells), we confirmed selectivity for AC1 by testing against all isoforms of membrane-bound ACs. ST034307 also inhibited AC1 activity stimulated by forskolin- and Gαs-coupled receptors in HEK-AC1 cells and showed inhibitory activity in multiple AC1-containing membrane preparations and mouse hippocampal homogenates. ST034307 enhanced μ-opioid receptor (MOR)–mediated inhibition of AC1 in short-term inhibition assays in HEK-AC1 cells stably transfected with MOR; however, the compound blocked heterologous sensitization of AC1 caused by chronic MOR activation in these cells. ST034307 reduced pain responses in a mouse model of inflammatory pain. Our data indicate that ST034307 is a selective small-molecule inhibitor of AC1 and suggest that selective AC1 inhibitors may be useful for managing pain.

Anti-analgesic effect of the mu/delta opioid receptor heteromer revealed by ligand-biased antagonism

Delta (DOR) and mu opioid receptors (MOR) can complex as heteromers, conferring functional properties in agonist binding, signaling and trafficking that can differ markedly from their homomeric counterparts. Because of these differences, DOR/MOR heteromers may be a novel therapeutic target in the treatment of pain. However, there are currently no ligands selective for DOR/MOR heteromers, and, consequently, their role in nociception remains unknown. In this study, we used a pharmacological opioid cocktail that selectively activates and stabilizes the DOR/MOR heteromer at the cell surface by blocking its endocytosis to assess its role in antinociception. We found that mice treated chronically with this drug cocktail showed a significant right shift in the ED50 for opioid-mediated analgesia, while mice treated with a drug that promotes degradation of the heteromer did not. Furthermore, promoting degradation of the DOR/MOR heteromer after the right shift in the ED50 had occurred, or blocking signal transduction from the stabilized DOR/MOR heteromer, shifted the ED50 for analgesia back to the left. Taken together, these data suggest an anti-analgesic role for the DOR/MOR heteromer in pain. In conclusion, antagonists selective for DOR/MOR heteromer could provide an avenue for alleviating reduced analgesic response during chronic pain treatment.