Inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the primary hydrolytic enzymes for the respective endocannabinoids N-arachidonoylethanolamine (AEA) and ...2-arachidonylglycerol (2-AG), produces antinociception but with minimal cannabimimetic side effects. Although selective inhibitors of either enzyme often show partial efficacy in various nociceptive models, their combined blockade elicits augmented antinociceptive effects, but side effects emerge. Moreover, complete and prolonged MAGL blockade leads to cannabinoid receptor type 1 (CB1) receptor functional tolerance, which represents another challenge in this potential therapeutic strategy. Therefore, the present study tested whether full FAAH inhibition combined with partial MAGL inhibition would produce sustained antinociceptive effects with minimal cannabimimetic side effects. Accordingly, we tested a high dose of the FAAH inhibitor PF-3845 (N-3-pyridinyl-4-3-5-(trifluoromethyl)-2-pyridinyloxyphenylmethyl-1-piperidinecarboxamide; 10 mg/kg) given in combination with a low dose of the MAGL inhibitor JZL184 4-nitrophenyl 4-(dibenzod1,3dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (4 mg/kg) in mouse models of inflammatory and neuropathic pain. This combination of inhibitors elicited profound increases in brain AEA levels (>10-fold) but only 2- to 3-fold increases in brain 2-AG levels. This combination produced significantly greater antinociceptive effects than single enzyme inhibition and did not elicit common cannabimimetic effects (e.g., catalepsy, hypomotility, hypothermia, and substitution for Δ(9)-tetrahydrocannabinol in the drug-discrimination assay), although these side effects emerged with high-dose JZL184 (i.e., 100 mg/kg). Finally, repeated administration of this combination did not lead to tolerance to its antiallodynic actions in the carrageenan assay or CB1 receptor functional tolerance. Thus, full FAAH inhibition combined with partial MAGL inhibition reduces neuropathic and inflammatory pain states with minimal cannabimimetic effects.
Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB
receptor (CB
R) distal C-terminal-associated protein that alters CB
R interactions with G-proteins. We tested the hypothesis that CRIP1a ...is capable of also altering CB
R interactions with β-arrestin proteins that interact with the CB
R at the C-terminus. Coimmunoprecipitation studies indicated that CB
R associates in complexes with either CRIP1a or β-arrestin, but CRIP1a and β-arrestin fail to coimmunoprecipitate with each other. This suggests a competition for CRIP1a and β-arrestin binding to the CB
R, which we hypothesized could attenuate the action of β-arrestin to mediate CB
R internalization. We determined that agonist-mediated reduction of the density of cell surface endogenously expressed CB
Rs was clathrin and dynamin dependent and could be modeled as agonist-induced aggregation of transiently expressed GFP-CB
R. CRIP1a overexpression attenuated CP55940-mediated GFP-CB
R as well as endogenous β-arrestin redistribution to punctae, and conversely, CRIP1a knockdown augmented β-arrestin redistribution to punctae. Peptides mimicking the CB
R C-terminus could bind to both CRIP1a in cell extracts as well as purified recombinant CRIP1a. Affinity pull-down studies revealed that phosphorylation at threonine-468 of a CB
R distal C-terminus 14-mer peptide reduced CB
R-CRIP1a association. Coimmunoprecipitation of CB
R protein complexes demonstrated that central or distal C-terminal peptides competed for the CB
R association with CRIP1a, but that a phosphorylated central C-terminal peptide competed for association with β-arrestin 1, and phosphorylated central or distal C-terminal peptides competed for association with β-arrestin 2. Thus, CRIP1a can compete with β-arrestins for interaction with C-terminal CB
R domains that could affect agonist-driven, β-arrestin-mediated internalization of the CB
R.
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•The crosstalk between opioid and chemokine receptors play a role in chronic pain.•The putative MOR-CXCR4 heterodimers may be involved in pain modulation.•Bivalent ligands are ...powerful to investigate GPCR dimerization and relevant diseases.•Bivalent ligand 1a showed reasonable recognition to both the MOR and CXCR4.•Bivalent ligand 1a showed potential antinociception in lactic acid-induced pain model.
The functional interactions between opioid and chemokine receptors have been implicated in the pathological process of chronic pain. Mounting studies have indicated the possibility that a MOR-CXCR4 heterodimer may be involved in nociception and related pharmacologic effects. Herein we have synthesized a series of bivalent ligands containing both MOR agonist and CXCR4 antagonist pharmacophores with an aim to investigate the functional interactions between these two receptors. In vitro studies demonstrated reasonable recognition of designed ligands at both respective receptors. Further antinociceptive testing in mice revealed compound 1a to be the most promising member of this series. Additional molecular modeling studies corroborated the findings observed. Taken together, we identified the first bivalent ligand 1a showing promising antinociceptive effect by targeting putative MOR-CXCR4 heterodimers, which may serve as a novel chemical probe to further develop more potent bivalent ligands with potential application in analgesic therapies for chronic pain management.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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•15 Low efficacy partial agonists of the mu-opioid receptor were synthesized.•15 Compounds displayed high binding affinity to molecular target.•Compound 12 significantly blocked ...antinociceptive effects of morphine.•Compound 12 precipitated significantly less withdrawal than naloxone.•Substitutions at 5′-position of thiophene may be a solution to improve safety.
The number of opioid-related overdose deaths and individuals that have suffered from opioid use disorders have significantly increased over the last 30 years. FDA approved maintenance therapies to treat opioid use disorder may successfully curb drug craving and prevent relapse but harbor adverse effects that reduce patient compliance. This has created a need for new chemical entities with improved patient experience. Previously our group reported a novel lead compound, NAT, a mu-opioid receptor antagonist that potently antagonized the antinociception of morphine and showed significant blood–brain barrier permeability. However, NAT belongs to thiophene containing compounds which are known structural alerts for potential oxidative metabolism. To overcome this, 15 NAT derivatives with various substituents at the 5′-position of the thiophene ring were designed and their structure–activity relationships were studied. These derivatives were characterized for their binding affinity, selectivity, and functional activity at the mu opioid receptor and assessed for their ability to antagonize the antinociceptive effects of morphine in vivo. Compound 12 showed retention of the basic pharmacological attributes of NAT while improving the withdrawal effects that were experienced in opioid-dependent mice. Further studies will be conducted to fully characterize compound 12 to examine whether it would serve as a new lead for opioid use disorder treatment and management.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Modern antiretroviral therapies have provided HIV-1 infected patients longer lifespans and better quality of life. However, several neurological complications are now being seen in these patients due ...to HIV-1 associated injury of neurons by infected microglia and astrocytes. In addition, these effects can be further exacerbated with opiate use and abuse. One possible mechanism for such potentiation effects of opiates is the interaction of the mu opioid receptor (MOR) with the chemokine receptor CCR5 (CCR5), a known HIV-1 co-receptor, to form MOR-CCR5 heterodimer. In an attempt to understand this putative interaction and its relevance to neuroAIDS, we designed and synthesized a series of bivalent ligands targeting the putative CCR5-MOR heterodimer. To understand how these bivalent ligands may interact with the heterodimer, biological studies including calcium mobilization inhibition, binding affinity, HIV-1 invasion, and cell fusion assays were applied. In particular, HIV-1 infection assays using human peripheral blood mononuclear cells, macrophages, and astrocytes revealed a notable synergy in activity for one particular bivalent ligand. Further, a molecular model of the putative CCR5-MOR heterodimer was constructed, docked with the bivalent ligand, and molecular dynamics simulations of the complex was performed in a membrane-water system to help understand the biological observation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Discovery of analgesics void of abuse liability is critical to battle the opioid crisis in the United States. Among many strategies to achieve this goal, targeting more than one opioid receptor seems ...promising to minimize this unwanted side effect while achieving a reasonable therapeutic profile. In the process of understanding the structure–activity relationship of nalfurafine, we identified a potential analgesic agent, NMF, as a dual kappa opioid receptor/delta opioid receptor agonist with minimum abuse liability. Further characterizations, including primary in vitro ADMET studies (hERG toxicity, plasma protein binding, permeability, and hepatic metabolism), and in vivo pharmacodynamic and toxicity profiling (time course, abuse liability, tolerance, withdrawal, respiratory depression, body weight, and locomotor activity) further confirmed NMF as a promising drug candidate for future development.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid ...receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated (35)SGTPγS (guanylyl-5'-O-thio-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated (35)SGTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist-stimulated CB1R signaling and downregulation of CB1Rs. Thus, CRIP1a appears to act as a broad negative regulator of CB1R function.
Rationale
Low-efficacy mu opioid receptor agonists may be useful for some clinical indications, but clinically available low-efficacy mu agonists also have low selectivity for mu vs. kappa opioid ...receptors. NAQ (17-cyclopropylmethyl-3,14ß-dihydroxy-4,5α-epoxy-6α-(3′-isoquinolyl)acetamidomorphinan) is a novel opioid receptor ligand with low-efficacy at mu receptors and greater mu-receptor selectivity than existing low-efficacy agonists.
Objectives
This study examined behavioral effects of NAQ in rats using an intracranial self-stimulation (ICSS) procedure that has been used previously to examine other opioids. NAQ effects were examined before, during, and after chronic morphine treatment, and effects of NAQ were compared to effects of nalbuphine and naltrexone.
Methods
Adult male Sprague–Dawley rats were trained to respond for electrical brain stimulation delivered via electrodes implanted in the medial forebrain bundle. A range of brain stimulation frequencies maintained a wide range of baseline ICSS rates. Effects of NAQ (0.32–10 mg/kg), nalbuphine (1.0 mg/kg), and naltrexone (0.1 mg/kg) were determined before morphine treatment and during treatment with 3.2 and 18 mg/kg/day morphine. NAQ effects were also redetermined beginning 2 weeks after termination of morphine treatment.
Results
NAQ produced weak ICSS facilitation in morphine-naïve rats but more robust ICSS facilitation during and after morphine treatment and also reversed morphine withdrawal-associated depression of ICSS. These effects were similar to effects of nalbuphine.
Conclusions
These results agree with the in vitro characterization of NAQ as a low-efficacy mu agonist. Opioid exposure may enhance abuse-related effects of NAQ, but NAQ may also serve as a low-efficacy and relatively safe option for treatment of opioid withdrawal or dependence.
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DOBA, EMUNI, FIS, FSPLJ, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, VSZLJ, ZAGLJ
Cannabinoid receptor interacting protein 1a (CRIP1a) is a CB1 receptor (CB1R) distal C‐terminus‐associated protein that modulates CB1R signaling via G proteins, and CB1R down‐regulation but not ...desensitization (Blume et al. 2015 Cell Signal., 27, 716–726; Smith et al. 2015 Mol. Pharmacol., 87, 747–765). In this study, we determined the involvement of CRIP1a in CB1R plasma membrane trafficking. To follow the effects of agonists and antagonists on cell surface CB1Rs, we utilized the genetically homogeneous cloned neuronal cell line N18TG2, which endogenously expresses both CB1R and CRIP1a, and exhibits a well‐characterized endocannabinoid signaling system. We developed stable CRIP1a‐over‐expressing and CRIP1a‐siRNA‐silenced knockdown clones to investigate gene dose effects of CRIP1a on CB1R plasma membrane expression. Results indicate that CP55940 or WIN55212‐2 (10 nM, 5 min) reduced cell surface CB1R by a dynamin‐ and clathrin‐dependent process, and this was attenuated by CRIP1a over‐expression. CP55940‐mediated cell surface CB1R loss was followed by a cycloheximide‐sensitive recovery of surface receptors (30–120 min), suggesting the requirement for new protein synthesis. In contrast, WIN55212‐2‐mediated cell surface CB1Rs recovered only in CRIP1a knockdown cells. Changes in CRIP1a expression levels did not affect a transient rimonabant (10 nM)‐mediated increase in cell surface CB1Rs, which is postulated to be as a result of rimonabant effects on ‘non‐agonist‐driven’ internalization. These studies demonstrate a novel role for CRIP1a in agonist‐driven CB1R cell surface regulation postulated to occur by two mechanisms: 1) attenuating internalization that is agonist‐mediated, but not that in the absence of exogenous agonists, and 2) biased agonist‐dependent trafficking of de novo synthesized receptor to the cell surface.
The CB1R‐interacting protein CRIP1a functions to differentially modulate agonist‐promoted versus non‐agonist‐mediated CB1R internalization and cell surface equilibrium. Association of CRIP1a with the CB1R precludes agonist‐driven internalization via a mechanism involving β‐arrestin, clathrin and dynamin. CRIP1a also functions to fine‐tune CB1R cell surface levels through delivery of newly synthesized CB1R receptors to the plasma membrane.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Repeated administration of Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive constituent of Cannabis sativa, induces profound tolerance that correlates with desensitization and ...downregulation of CB(1) cannabinoid receptors in the CNS. However, the consequences of repeated administration of the endocannabinoid N-arachidonoyl ethanolamine (anandamide, AEA) on cannabinoid receptor regulation are unclear because of its rapid metabolism by fatty acid amide hydrolase (FAAH). FAAH(-/-) mice dosed subchronically with equi-active maximally effective doses of AEA or THC displayed greater rightward shifts in THC dose-effect curves for antinociception, catalepsy, and hypothermia than in AEA dose-effect curves. Subchronic THC significantly attenuated agonist-stimulated (35)SGTP gamma S binding in brain and spinal cord, and reduced (3)HWIN55,212-2 binding in brain. Interestingly, AEA-treated FAAH(-/-) mice showed less CB(1) receptor downregulation and desensitization than THC-treated mice. Experiments examining tolerance and cross-tolerance indicated that the behavioral effects of THC, a low efficacy CB(1) receptor agonist, were more sensitive to receptor loss than those of AEA, a higher efficacy agonist, suggesting that the expression of tolerance was more affected by the intrinsic activity of the ligand at testing than during subchronic treatment. In addition, the CB(1) receptor antagonist, rimonabant, precipitated a markedly reduced magnitude of withdrawal in FAAH(-/-) mice treated subchronically with AEA compared with mice treated repeatedly with THC. The findings that repeated AEA administration produces lesser adaptive changes at the CB(1) receptor and has reduced dependence liability compared with THC suggest that pharmacotherapies targeting endocannabinoid catabolic enzymes are less likely to promote tolerance and dependence than direct acting CB(1) receptor agonists.
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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
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