Identification of Novel Cannabinoid CB2 Receptor Agonists from Botanical Compounds and Preliminary Evaluation of Their Anti-Osteoporotic Effects

Abstract

As cannabinoid CB2 receptors (CB2R) possess various pharmacological effects-including anti-epilepsy, analgesia, anti-inflammation, anti-fibrosis, and regulation of bone metabolism-without the psychoactive side effects induced by cannabinoid CB1R activation, they have become the focus of research and development of new target drugs in recent years. The present study was intended to (1) establish a double luciferase screening system for a CB2R modulator; (2) validate the agonistic activities of the screened compounds on CB2R by determining cAMP accumulation using HEK293 cells that are stably expressing CB2R; (3) predict the binding affinity between ligands and CB2 receptors and characterize the binding modes using molecular docking; (4) analyze the CB2 receptors-ligand complex stability, conformational behavior, and interaction using molecular dynamics; and (5) evaluate the regulatory effects of the screened compounds on bone metabolism in osteoblasts and osteoclasts. The results demonstrated that the screening system had good stability and was able to screen cannabinoid CB2R modulators from botanical compounds. Altogether, nine CB2R agonists were identified by screening from 69 botanical compounds, and these CB2R agonists exhibited remarkable inhibitory effects on cAMP accumulation and good affinity to CB2R, as evidenced by the molecular docking and molecular dynamics. Five of the nine CB2R agonists could stimulate osteoblastic bone formation and inhibit osteoclastic bone resorption. All these findings may provide useful clues for the development of novel anti-osteoporotic drugs and help elucidate the mechanism underlying the biological activities of CB2R agonists identified from the botanical materials.

Keywords: cannabinoid CB2 receptor agonists; double luciferase screening system; molecular docking and dynamics; osteoblast; osteoclast.

Figure 1

Establishment of the double luciferase screening system for screening CB2R modulators. (a) HEK293 with or without transfected CB2R (×100, Scale bar: 50 μm). (b) CB2 mRNA expression in HEK293 with or without transfected CB2R. The data shown are from six independent experiments and expressed as mean ± SD (n = 6). ### p < 0.001 compared with untransferred cell lines. (c) CB2 protein expression in HEK293 with or without transfected CB2R, using GAPDH as loading control. (d) The agonistic effect of HU308 on CB2R in HEK293-CB2 cells. HEK293-CB2 cells were treated with HU308 at concentrations of 10 nM~100 µM for 4–8 h and relative luminescence values were used to indicate the agonistic effect of HU308 on CB2R. (e) The reversal effect of AM630 on the agonistic activity of HU308. (f) The agonistic effect of HU308 on HEK293-CB2 cells with different passages. The data shown are from six independent experiments and expressed as mean ± SD (n = 6). ### p < 0.001 compared with the control. ** p < 0.01 and *** p < 0.001 compared with HU308 treatment group. GFP, green fluorescent protein; CB2, type 2 cannabinoid; EGFP, enhanced green fluorescent protein; HEK293-EGFP, the HEK293 cells only transferred into the empty vector(pIRES2-EGFP); HEK293-CB2, the HEK293 cells transferred into pIRES2-EGFP and CB2 gene; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PA, passage.

Figure 2

The agonistic effect of botanical compounds on CB2R based on double luciferase reporter assay. (a–c) Effects of botanical compounds on CB2R activation in HEK293-CB2 cells. HEK293-CB2 cells were treated with botanical compounds for 6 h and relative luminescence values were used to indicate the agonistic effect of the botanical compounds on CB2R. (d) Effects of the botanical compounds on CB2R activation in HEK293-EGFP cells. (e) The reversal effect of AM630 on the agonistic activity of CB2R in HU308 and screened compounds in HEK293-CB2 cells. The data shown are from three independent experiments and expressed as mean ± SD (n = 3). # p < 0.05, ## p < 0.01 and ### p < 0.001 compared with the control. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared with the corresponding compounds treatment group. EGFP, enhance green fluorescent protein; CB2, type 2 cannabinoid.

Figure 3

Effects of the screened compounds on intracellular cAMP accumulation in HEK293-CB2 cells. HEK293-CB2 cells were pretreated with forskolin for 30 min and then treated with the screened compounds for 6 h. cAMP level in HEK293-CB2 cells was determined with the cAMP assay kit according to the manufacturer’s instructions. (a) Flavokawain C. (b) Dihydromethysticin. (c) Desmethoxyyangonin. (d) Flavokawain A. (e) Echinatin. (f) Mangiferin. (g) orientin. (h) Asperuloside. (i) 11-keto-beta-boswellic acid. The data shown are from three independent experiments and expressed as the mean ± SD (n = 3). cAMP, cyclic adenosine monophosphate.

Figure 4

Docking poses of HU308 and screened CB2R agonist in CB2R protein. (a) Overall structure of the 5ZTY-HU308 complex. Protein 5ZTY is shown in cyan, with the ligand HU308 in firebrick. (b) Binding pose of HU308 with CB2 protein 5ZTY. Key residues, cyan sticks; involved in HU308, firebrick sticks; binding in CB2-Gi complex structure, cyan cartoon. (c) The binding poses of the screened compounds in CB2 protein 5ZTY, among which, HU308, firebrick sticks; flavokawain A, green sticks; echinatin, deepblue sticks; mangiferin, orange sticks; orientin, cyan sticks; asperuloside, pink sticks; hydrogen bonded residues, yellow sticks; residues with π-π interactions, skyblue sticks; conservative residues, gray sticks.

Figure 5

Molecular dynamics analysis of HU308 and representative screened compounds with CB2 protein. (a) RMSD analysis and (b) RMSF analysis.

Figure 6

Effects of the screened compounds on the function of osteoblasts derived from bone marrow mesenchymal stem cells (BMSCs). (a) Proliferation of BMSCs. After 48-h treatment of BMSCs with HU308 and screened compounds, cell viability was detected by CCK8. (b) ALP activity. BMSCs were treated for seven days in the presence or absence of 1 μM AM630, ALP activities were measured by p-nitrophenyl sodium phosphate method. (c) ALP staining. The BMSCs cells were treated for five days in the presence or absence of 1 μM AM630, ALP activity was stained with BCIP/NBT assay kit. (d) Bone mineralization nodule staining. BMSCs were treated with screened compounds for 14 days in the presence or absence of 1 μM AM630, the bone mineralization nodules were detected by alizarin red staining. The data shown are from three independent experiments and expressed as mean ± SD (n = 3). # p < 0.05 and ## p < 0.01 compared with the control. ** p < 0.01 compared with the corresponding compounds treatment group.

Figure 7

Inhibitory effects of HU308 and screened compounds on the activity of osteoclasts derived from RAW264.7 cells. (a) Cell viability. RAW264.7 cells were treated with 1 μM of HU308 or the screened compounds for three days, and the cell viability was detected by CCK8 method. (b) The tartrate resistant acid phosphatase (TRAP) activity. After three-day treatment of osteoclasts derived from RAW264.7 cells in the presence or absence of 1 μM AM630, TRAP activity was measured by p-nitrophenyl sodium phosphate method. (c) TRAP staining. After three-day treatment of osteoclasts derived from RAW264.7 cells in the presence or absence of 1 μM AM630, TRAP activity was detected with the assay kit. (d) F-Actin ring staining. After five-day treatment of osteoclasts derived from RAW264.7 cells in the presence or absence of 1 μM AM630, F-Actin ring was stained with phalloidin and DAPI and then imaged with a fluorescence microscope. The data shown are from three independent experiments and expressed as mean ± SD (n = 3). # p < 0.05 compared with the control. * p < 0.05 compared with corresponding compounds treatment group. TRAP, tartrate resistant acid phosphatase.

Figure 8

The schematic diagram shows the plant monomers that can further promote osteogenesis (blue circle) and inhibit osteoclast differentiation (red circle) through the CB2 pathway in natural compounds that activate CB2R.