Zhao-Hui Song

Involvement of the 90-kDa heat shock protein (Hsp-90) in CB2 cannabinoid receptor-mediated cell migration: a new role of Hsp-90 in migration signaling of a G protein-coupled receptor.

The endocannabinoid 2-arachidonoylglycerol (2-AG) enhances cell migration through the CB2 cannabinoid receptor. In this study, using an immunoprecipitation and mass spectrometry-based proteomic approach, we first identified the 90-kDa heat shock protein (Hsp90), a chaperone protein with novel signaling functions, as a CB2-interacting protein. The CB2/Hsp90 interaction was confirmed in human embryonic kidney 293 cells expressing transfected CB2 and in differentiated HL-60 cells expressing endogenous CB2, by coimmunoprecipitation and Western blot experiments, as well as by treatment with geldanamycin (GA), a specific Hsp90 inhibitor. Disruption of the CB2/Hsp90 interaction by treatment with GA or reducing Hsp90 levels with specific short interfering RNAs markedly inhibited 2-AG-induced cell migration, demonstrating that Hsp90 is crucial for 2-AG-induced cell migration. 2-AG treatment resulted in a CB2-mediated stimulation of Rac1 activity, and treatment with GA blocked 2AG-induced activation of Rac1. It is noteworthy that expression of the dominant-negative form of Rac1 reduced 2-AG-induced cell migration. These data demonstrate that 2-AG-induced activation of Rac1 is essential for 2-AG-induced cell migration, and the CB2/Hsp90 interaction is needed for 2-AG-induced activation of Rac1. Furthermore, 2-AG-induced Rac1 activation was sensitive to pertussis toxin treatment, hence involving Gi proteins. In addition, treatment with GA significantly inhibited the CB2/Gαi2 interaction. As a whole, our data indicate that Hsp90 may serve as scaffold to keep the CB2 receptor and its signaling components, including Gαi2, in proximity, thus facilitating CB2-mediated signaling to cell migration through the Gi-Rac1 pathway. By demonstrating that Hsp90 is essential for CB2-mediated signaling to cell migration, this study reveals a novel role of Hsp90 in the signaling events mediated by a G protein-coupled receptor.

Residues accessible in the binding-site crevice of transmembrane helix 6 of the CB2 cannabinoid receptor.

We have used the substituted-cysteine accessibility method (SCAM) to map the residues in the sixth membrane-spanning segment of the CB2 cannabinoid receptor that contribute to the surface of the water-accessible binding-site crevice. Using a background of the mutant C2.59S which is relatively insensitive to the methanethiosulfonate (MTS) reagents, we mutated to cysteine, one at a time, 34 consecutive residues in TMH6 of the CB2 receptor. These mutant receptors were then expressed in HEK293 cells. By incubating HEK293 cells stably transfected with CB2 receptors with the small, charged, hydrophilic, thiol-specific reagent methanethiosulfonate ethylammonium (MTSEA), [(3)H]CP55940 binding was significantly inhibited for six mutant receptors. All six of the mutants that reacted with MTSEA were protected from the reaction when pretreated with the cannabinoid agonist WIN55212-2, suggesting that MTSEA modification occurred within the binding crevice. Therefore, the side chains of the residues at these reactive loci (V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62) are on the water-accessible surface of the binding-site crevice. These residues are extracellular to the TMH6 CWXP hinge motif. The pattern of accessibility is consistent with a alpha-helical conformation for this segment of TMH6. Molecular modeling studies performed in the context of the CB2 model show that V6.51, L6.52, L6.54, M6.55, L6.59, and T6.62 face into the CB2 binding pocket, further confirming our SCAM results. These results are similar to the accessibility patterns determined by SCAM studies of TMH6 in the opioid and dopamine D2 receptors.

Effects of palmitoylethanolamide on aqueous humor outflow.

PURPOSE To study the effects of palmitoylethanolamide (PEA), a fatty acid ethanolamide, on aqueous humor outflow facility. METHODS The effects of PEA on outflow facility were measured using a porcine anterior segment-perfused organ culture model. The involvements of different receptors in PEA-induced changes were investigated using receptor antagonists and adenovirus delivered small hairpin RNAs (shRNAs). PEA-induced activation of p42/44 mitogen-activated protein kinase (MAPK) was determined by Western blot analysis using an antiphospho p42/44 MAPK antibody. RESULTS PEA caused a concentration-dependent enhancement of outflow facility, with the maximum effect (151.08 ± 11.12% of basal outflow facility) achieved at 30 nM of PEA. Pretreatment of anterior segments with 1 μM cannabinoid receptor 2 antagonist SR144528 and 1 μM PPARα antagonist GW6471, but not 1 μM cannabinoid receptor 1 antagonist SR141716A, produced a partial antagonism on the PEA-induced increase of outflow facility. Treatment of TM cells with PEA for 10 minutes activated phosphorylation of p42/44 MAPK, which was blocked by pretreatment with SR1444528 and GW6471, but not SR141716A. Knocking down the expression of either GPR55 or PPARα receptors with specific shRNAs for these receptors partially blocked PEA-induced increase in outflow facility and abolished PEA-induced phosphorylation of p42/44 MAPK. PD98059, an inhibitor of the p42/44 MAPK pathway, blocked both PEA-induced enhancement of aqueous humor outflow facility and PEA-induced phosphorylation of p42/44 MAPK. CONCLUSIONS Our results demonstrate that PEA increases aqueous humor outflow through the TM pathway and these effects are mediated by GPR55 and PPARα receptors through activation of p42/44 MAPK.

Identification of raloxifene as a novel CB2 inverse agonist.

The purpose of the current study was to apply a high throughput assay to systematically screen a library of food and drug administration (FDA)-approved drugs as potential ligands for the cannabinoid receptor 2 (CB2). A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring changes in intracellular cAMP levels was validated and found to be suitable for testing ligands that may act on CB2. Among the 640 FDA-approved drugs screened, raloxifene, a drug used to treat/prevent post-menopausal osteoporosis, was identified for the first time to be a novel CB2 inverse agonist. Our results demonstrated that by acting on CB2, raloxifene enhances forskolin-stimulated cAMP accumulation in a concentration-dependant manner. Furthermore, our data showed that raloxifene competes concentration-dependently for specific [(3)H]CP-55,940 binding to CB2. In addition, raloxifene pretreatment caused a rightward shift of the concentration-response curves of the cannabinoid agonists CP-55,940, HU-210, and WIN55,212-2. Raloxifene antagonism is most likely competitive in nature, as these rightward shifts were parallel and were not associated with any changes in the efficacy of cannabinoid agonists on CB2. Our discovery that raloxfiene is an inverse agonist for CB2 suggests that it might be possible to repurpose this FDA-approved drug for novel therapeutic indications for which CB2 is a target. Furthermore, identifying raloxifene as a CB2 inverse agonist also provides important novel mechanisms of actions to explain the known therapeutic effects of raloxifene.

The importance of hydrogen bonding and aromatic stacking to the affinity and efficacy of cannabinoid receptor CB2 antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528).

Despite the therapeutic promise of the subnanomolar affinity cannabinoid CB2 antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528, 1), little is known about its binding site interactions and no primary interaction site for 1 at CB2 has been identified. We report here the results of Glide docking studies in our cannabinoid CB2 inactive state model that were then tested via compound synthesis, binding, and functional assays. Our results show that the amide functional group of 1 is critical to its CB2 affinity and efficacy and that aromatic stacking interactions in the TMH5/6 aromatic cluster of CB2 are also important. Molecular modifications that increased the positive electrostatic potential in the region between the fenchyl and aromatic rings led to more efficacious compounds. This result is consistent with the EC-3 loop negatively charged amino acid, D275 (identified via Glide docking studies) acting as the primary interaction site for 1 and its analogues.

GPR3 and GPR6, novel molecular targets for cannabidiol.

GPR3 and GPR6 are members of a family of constitutively active, Gs protein-coupled receptors. Previously, it has been reported that GPR3 is involved in Alzheimers disease whereas GPR6 plays potential roles in Parkinsons disease. GPR3 and GPR6 are considered orphan receptors because there are no confirmed endogenous agonists for them. However, GPR3 and GPR6 are phylogenetically related to the cannabinoid receptors. In this study, the activities of endocannabinoids and phytocannabinoids were tested on GPR3 and GPR6 using a β-arrestin2 recruitment assay. Among the variety of cannabinoids tested, cannabidiol (CBD), the major non-psychoactive component of marijuana, significantly reduced β-arrestin2 recruitment to both GPR3 and GPR6. In addition, the inhibitory effects of CBD on β-arrestin2 recruitment were concentration-dependent for both GPR3 and GPR6, with a higher potency for GPR6. These data show that CBD acts as an inverse agonist at both GPR3 and GPR6 receptors. These results demonstrate for the first time that both GPR3 and GPR6 are novel molecular targets for CBD. Our discovery that CBD acts as a novel inverse agonist on both GPR3 and GPR6 indicates that some of the potential therapeutic effects of CBD (e.g. treatment of Alzheimers disease and Parkinsons disease) may be mediated through these important receptors.

Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119

The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor.

Involvement of a non-CB1/CB2 cannabinoid receptor in the aqueous humor outflow-enhancing effects of abnormal-cannabidiol

The purpose of this study was to investigate the effects of abnormal-cannabidiol (abn-cbd), a non-psychoactive cannabinoid agonist, on aqueous humor outflow via the trabecular meshwork (TM) of porcine eye, and to examine the involvement of a non-CB1/CB2 cannabinoid receptor and the p42/44 mitogen-activated protein kinase (p42/44 MAPK) pathway. The effects of abn-cbd on aqueous humor outflow were measured using a porcine anterior segment perfused organ culture model. The activation of p42/44 MAPK by abn-cbd was determined in cultured TM cells with western blot analysis using an anti-phospho-p42/44 MAPK antibody. Administration of abn-cbd caused a concentration-dependent enhancement of aqueous humor outflow facility with a maximum effect (155.0 ± 11.7% of basal outflow facility) after administration of 30 nM abn-cbd. Pretreatment with 1 μM of O-1918, a cannabidiol analog that acts as a selective antagonist at the non-CB1/CB2 receptor, produced a full antagonism of 30 nM abn-cbd induced increase of aqueous humor outflow facility. Pretreatment with 1 μM of CB1 antagonist SR141716A partially blocked, whereas pretreatment with either 1 μM of CB1 antagonist AM251 or 1 μM of CB2 antagonist SR144528 had no effect on abn-cbd induced enhancement of outflow facility. Treatment of TM cells with 30 nM of abn-cbd activated p42/44 MAPK, which was blocked completely by pretreatment with O-1918, and partially by pretreatment with SR141716A, but not by either AM251 or SR144528. In addition, PD98059, an inhibitor of p42/44 MAPK pathway, blocked completely the abn-cbd induced p42/44 MAPK activation and blocked partially the abn-cbd induced enhancement of outflow facility. In conclusion, the results from this study demonstrate that abn-cbd increases aqueous humor outflow through the TM pathway of the eye, and this effect is mediated by a non-CB1/CB2 cannabinoid receptor, with an involvement of p42/44 MAPK signaling pathway.

Cannabidiol, a novel inverse agonist for GPR12

GPR12 is a constitutively active, Gs protein-coupled receptor that currently has no confirmed endogenous ligands. GPR12 may be involved in physiological processes such as maintenance of oocyte meiotic arrest and brain development, as well as pathological conditions such as metastatic cancer. In this study, the potential effects of various classes of cannabinoids on GPR12 were tested using a cAMP accumulation assay. Our data demonstrate that cannabidiol (CBD), a major non-psychoactive phytocannabinoid, acted as an inverse agonist to inhibit cAMP accumulation stimulated by the constitutively active GPR12. Thus, GPR12 is a novel molecular target for CBD. The structure-activity relationship studies of CBD indicate that both the free hydroxyl and the pentyl side chain are crucial for the effects of CBD on GPR12. Furthermore, studies using cholera toxin, which blocks Gs protein and pertussis toxin, which blocks Gi protein, revealed that Gs, but not Gi is involved in the inverse agonism of CBD on GPR12. CBD is a promising novel therapeutic agent for cancer, and GPR12 has been shown to alter viscoelasticity of metastatic cancer cells. Since we have demonstrated that CBD is an inverse agonist for GPR12, this provides novel mechanism of action for CBD, and an initial chemical scaffold upon which highly potent and efficacious agents acting on GPR12 may be developed with the ultimate goal of blocking cancer metastasis.

CB2 cannabinoid receptor is a novel target for third-generation selective estrogen receptor modulators bazedoxifene and lasofoxifene.

The purpose of the current study was to investigate the ability of the third-generation selective estrogen receptor modulators (SERMs) bazedoxifene and lasofoxifene to bind and act on CB2 cannabinoid receptor. We have identified, for the first time, that CB2 is a novel target for bazedoxifene and lasofoxifene. Our results showed that bazedoxifene and lasofoxifene were able to compete for specific [(3)H]CP-55,940 binding to CB2 in a concentration-dependent manner. Our data also demonstrated that by acting on CB2, bazedoxifene and lasofoxifene concentration-dependently enhanced forskolin-stimulated cAMP accumulation. Furthermore, bazedoxifene and lasofoxifene caused parallel, rightward shifts of the CP-55,940, HU-210, and WIN55,212-2 concentration-response curves without altering the efficacy of these cannabinoid agonists on CB2, which indicates that bazedoxifene- and lasofoxifene-induced CB2 antagonism is most likely competitive in nature. Our discovery that CB2 is a novel target for bazedoxifene and lasofoxifene suggests that these third-generation SERMs can potentially be repurposed for novel therapeutic indications for which CB2 is a target. In addition, identifying bazedoxifene and lasofoxifene as CB2 inverse agonists also provides important novel mechanisms of actions to explain the known therapeutic effects of these SERMs.