The mu opioid receptor (MOR) is a diversely regulated target for the alleviation of pain in the clinical setting. However, untoward side effects such as tolerance, dependence, respiratory ...suppression, constipation, and abuse liability detract from the general activation of these receptors. Studies in genetically modified rodent models suggest that activating G protein signaling pathways while avoiding phosphorylation of the receptor or recruitment of β-arrestin scaffolding proteins could preserve the analgesic properties of MOR agonists while avoiding certain side effects. With the development of novel MOR “biased” agonists, which lead to preferential activation of G protein pathways over receptor phosphorylation, internalization, or interaction with other effectors, this hypothesis can be tested in a native, physiological setting. Overall, it is clear that the MOR is not a simple on-off switch and that the diverse means by which the receptor can be regulated may present an opportunity to refine therapeutics for the treatment of pain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, ...and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role.
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•3D structures of CB2-AM12033-Gi, CB1-AM841-Gi, and CB2-AM12033 are determined•Structural evidence of G protein selectivity by CB1 and CB2 is identified•MD simulations reveal the distinct binding behavior of HU308 in CB2 and CB1•Cholesterol molecule as an endogenous allosteric modulator of CB1 is uncovered
Structure and simulations of cannabinoid receptors CB2 and CB1 in their inactive, active-like, and activated signaling states reveal residue differences that may provide G protein selectivity, the distinct binding behavior of CB2 agonists in CB2 and CB1, as well as evidence for modulation of CB1 by cholesterol binding.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
The cannabinoid CB
2
receptor (CB
2
R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed ...and widely used to target CB
2
R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB
2
R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB
2
R agonists to study the role of CB
2
R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.
The ability of a ligand to preferentially promote engagement of one signaling pathway over another downstream of GPCR activation has been referred to as signaling bias, functional selectivity, and ...biased agonism. The presentation of ligand bias reflects selectivity between active states of the receptor, which may result in the display of preferential engagement with one signaling pathway over another. In this study, we provide evidence that the G protein-biased mu opioid receptor (MOR) agonists SR-17018 and SR-14968 stabilize the MOR in a wash-resistant yet antagonist-reversible G protein-signaling state. Furthermore, we demonstrate that these structurally related biased agonists are noncompetitive for radiolabeled MOR antagonist binding, and while they stimulate G protein signaling in mouse brains, partial agonists of this class do not compete with full agonist activation. Importantly, opioid antagonists can readily reverse their effects in vivo. Given that chronic treatment with SR-17018 does not lead to tolerance in several mouse pain models, this feature may be desirable for the development of long-lasting opioid analgesics that remain sensitive to antagonist reversal of respiratory suppression.
It has been demonstrated that opioid agonists that preferentially act at μ-opioid receptors to activate G protein signaling over βarrestin2 recruitment produce antinociception with less respiratory ...suppression. However, most of the adverse effects associated with opioid therapeutics are realized after extended dosing. Therefore, we tested the onset of tolerance and dependence, and assessed for neurochemical changes associated with prolonged treatment with the biased agonist SR-17018. When chronically administered to mice, SR-17018 does not lead to hot plate antinociceptive tolerance, receptor desensitization in periaqueductal gray, nor a super-sensitization of adenylyl cyclase in the striatum, which are hallmarks of opioid neuronal adaptations that are seen with morphine. Interestingly, substitution with SR-17018 in morphine-tolerant mice restores morphine potency and efficacy, whereas the onset of opioid withdrawal is prevented. This is in contrast to buprenorphine, which can suppress withdrawal, but produces and maintains morphine antinociceptive tolerance. Biased agonists of this nature may therefore be useful for the treatment of opioid dependence while restoring opioid antinociceptive sensitivity.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
The mu opioid receptor-selective agonist, SR-17018, preferentially activates GTPγS binding over βarrestin2 recruitment in cellular assays, thereby demonstrating signaling bias. In mice, SR-17018 ...stimulates GTPγS binding in brainstem and produces antinociception with potencies similar to morphine. However, it produces much less respiratory suppression and mice do not develop antinociceptive tolerance in the hot plate assay upon repeated dosing. Herein we evaluate the effects of acute and repeated dosing of SR-17018, oxycodone and morphine in additional models of pain-related behaviors. In the mouse warm water tail immersion assay, an assessment of spinal reflex to thermal nociception, repeated administration of SR-17018 produces tolerance as does morphine and oxycodone. SR-17018 retains efficacy in a formalin-induced inflammatory pain model upon repeated dosing, while oxycodone does not. In a chemotherapeutic-induced neuropathy pain model SR-17018 is more potent and efficacious than morphine or oxycodone, moreover, this efficacy is retained upon repeated dosing of SR-17018. These findings demonstrate that, with the exception of the tail flick test, SR-17018 retains efficacy upon chronic treatment across several pain models.
•SR-17018 retains efficacy after repeated dosing in a mouse formalin assay.•SR-17018 retains efficacy after repeated dosing in a chemotherapeutic neuropathy model.•SR-17018 produces tolerance in a mouse tail flick model upon chronic treatment.•Oxycodone and morphine produce tolerance in these models.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
One emerging strategy to address the opioid crisis is the development of mu opioid receptor (MOR) ligands that preferentially signal the G-protein vs. β-arrestin pathway. The present study compared ...the relative potency and effectiveness of two G-protein biased (GPB)-MOR ligands TRV130 and SR-14968 to five unbiased MOR ligands (NAQ, nalbuphine, buprenorphine, morphine, and methadone) on therapeutic-related (e.g. antinociception) and abuse-related (e.g. discriminative stimulus effects) endpoints. Male and female rats were tested in a warm water tail-withdrawal procedure (50 °C) or trained to discriminate fentanyl (0.04 mg/kg, SC) from saline in a two-lever food-reinforced discrimination procedure. TRV130 and SR-14968 were approximately two-fold more potent to produce fentanyl stimulus effects vs. antinociception. Morphine, fentanyl, and methadone were significantly more potent in the fentanyl discrimination vs. tail withdrawal procedure. In addition, maximum antinociceptive and discriminative stimulus effects of fixed-proportion fentanyl/naltrexone mixtures (1:0.018, 1:0.054, 1:0.18, 1:0.3, and 1:0.54) were used to quantify 1) the relative in vivo efficacy of the two GPB-MOR agonists and five unbiased MOR ligands, and 2) potential species differences in MOR ligand effects between rats and monkeys. The efficacy-effect function generated from the fentanyl/naltrexone mixtures stratified the five unbiased ligands consistent with agonist-stimulated GTPγS binding (NAQ = nalbuphine < buprenorphine < morphine < methadone). However, TRV130 and SR-14968 produced greater antinociception and less fentanyl-like stimulus effects than was predicted. Furthermore, there was a significant positive correlation between rat and monkey antinociceptive effects. Overall, these results demonstrate GPB-MOR agonists produce undesirable abuse-related effects, albeit with slightly better potency and efficacy ratios compared to unbiased agonists.
This article is part of the Special Issue entitled ‘New Vistas in Opioid Pharmacology’.
•G-protein biased mu agonists produced fentanyl-like discriminative stimulus effects.•GPB-MOR agonists produced better potency and efficacy ratios than existing opioids.•Fixed-proportion fentanyl/naltrexone mixtures manipulate in vivo drug efficacy.•Mu agonist antinociceptive effects in rats and monkeys were positively correlated.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Serious clinical liabilities associated with the prescription of opiates for pain control include constipation, respiratory depression, pruritus, tolerance, abuse, and addiction. A recognized ...strategy to circumvent these side effects is to combine opioids with other antinociceptive agents. The combination of opiates with the primary active constituent of cannabis (Δ(9)-tetrahydrocannabinol) produces enhanced antinociceptive actions, suggesting that cannabinoid receptor agonists can be opioid sparing. Here, we tested whether elevating the endogenous cannabinoid 2-arachidonoylglycerol through the inhibition of its primary hydrolytic enzyme monoacylglycerol lipase (MAGL), will produce opioid-sparing effects in the mouse chronic constriction injury (CCI) of the sciatic nerve model of neuropathic pain. The dose-response relationships of i.p. administration of morphine and the selective MAGL inhibitor 2,5-dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate (MJN110) were tested alone and in combination at equieffective doses for reversal of CCI-induced mechanical allodynia and thermal hyperalgesia. The respective ED50 doses (95% confidence interval) of morphine and MJN110 were 2.4 (1.9-3.0) mg/kg and 0.43 (0.23-0.79) mg/kg. Isobolographic analysis of these drugs in combination revealed synergistic antiallodynic effects. Acute antinociceptive effects of the combination of morphine and MJN110 required μ-opioid, CB1, and CB2 receptors. This combination did not reduce gastric motility or produce subjective cannabimimetic effects in the drug discrimination assay. Importantly, combinations of MJN110 and morphine given repeatedly (i.e., twice a day for 6 days) continued to produce antiallodynic effects with no evidence of tolerance. Taken together, these findings suggest that MAGL inhibition produces opiate-sparing events with diminished tolerance, constipation, and cannabimimetic side effects.
Common pharmacological treatments of neuropathic and chronic inflammatory pain conditions generally lack efficacy and/or are associated with significant untoward side effects. However, recent ...preclinical data indicate that combined inhibition of cyclooxygenase (COX) and fatty acid amide hydrolase (FAAH), the primary catabolic enzyme of the endocannabinoid N-arachidonoylethanolamine (anandamide; AEA), produces enhanced antinociceptive effects in a variety of murine models of pain. Accordingly, the primary objective of the present study was to investigate the consequences of co-administration of the COX inhibitor diclofenac and the highly selective FAAH inhibitor PF-3845 in models of neuropathic pain (i.e., chronic constrictive injury of the sciatic nerve (CCI)) and inflammatory pain induced by an intraplantar injection of carrageenan. Here, we report that combined administration of subthreshold doses of these drugs produced enhanced antinociceptive effects in CCI and carrageenan pain models, the latter of which was demonstrated to require both CB1 and CB2 receptors. The combined administration of subthreshold doses of these drugs also increased AEA levels and decreased prostaglandin levels in whole brain. Together, these data add to the growing research that dual blockade of FAAH and COX represents a potential therapeutic strategy for the treatment of neuropathic and inflammatory pain states.
Tandem inhibition of FAAH and COX attenuates inflammatory and neuropathic pain states, which may avoid potentially harmful side effects of other therapeutic options, such as NSAIDs or opioids.
•Combination of diclofenac and PF-3845 attenuates both neuropathic and inflammatory pain.•CB1 and CB2 antagonists block the anti-inflammatory effects of inhibition of FAAH and COX enzymes.•PF-3845 elevates prostaglandins in whole brain, which is abolished the NSAID diclofenac.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Diacylglycerol lipase (DAGL)
and
, the major biosynthetic enzymes of the endogenous cannabinoid (endocannabinoid) 2-arachidonylglycerol (2-AG), are highly expressed in the nervous system and immune ...system, respectively. Genetic deletion or pharmacological inhibition of DAGL-
protects against lipopolysaccharide (LPS)-induced inflammatory responses in mouse peritoneal macrophages and reverses LPS-induced allodynia in mice. To gain insight into the contribution of DAGL-
in LPS-induced allodynia, we tested global knockout mice as well as DO34, a dual DAGL-
/
inhibitor. Intraperitoneal administration of DO34 (30 mg/kg) significantly decreased whole-brain levels of 2-AG (∼83%), anandamide (∼42%), and arachidonic acid (∼58%). DO34 dose-dependently reversed mechanical and cold allodynia, and these antinociceptive effects did not undergo tolerance after 6 days of repeated administration. In contrast, DO34 lacked acute thermal antinociceptive, motor, and hypothermal pharmacological effects in naive mice. As previously reported, DAGL-
(-/-) mice displayed a protective phenotype from LPS-induced allodynia. However, DAGL-
(-/-) mice showed full allodynic responses, similar to their wild-type littermates. Interestingly, DO34 (30 mg/kg) fully reversed LPS-induced allodynia in DAGL-
(+/+) and (-/-) mice, but did not affect the antinociceptive phenotype of DAGL-
(-/-) mice in this model, indicating a DAGL-
-independent site of action. These findings suggest that DAGL-
and DAGL-
play distinct roles in LPS-induced nociception. Whereas DAGL-
appears to be dispensable for the development and expression of LPS-induced nociception, DAGL-
inhibition represents a promising strategy to treat inflammatory pain.
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