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.
Highlights
Cannabis sativa has been used to relieve pain and insomnia since prehistoric EurasiaAfrica [1]
To rapidly and efficiently identify drugs that may act as CB2 receptors (CB2R) agonists, we developed a double luciferase screening system to screen CB2R agonists from 69 botanical compounds; evaluated their agonistic activity on CB2R using cyclic adenosine monophosphate (cAMP) levels, molecular docking, and molecular dynamics simulations; and further verified their anti-osteoporotic activities on osteoblasts and osteoclasts, hoping that the results could provide novel clues for discovering anti-osteoporotic drugs
We evaluated the regulatory effect of the screened botanical compounds on osteoblasts and osteoclasts and found that dihydromethysticin, desmethoxyyangonin, flavokawain A, echinatin, mangiferin, and 11-keto-beta-boswellic acid could improve the bone formation of osteoblasts, as evidenced by the proliferation, alkaline phosphatase (ALP) activities, and mineralization of the bone matrix, and desmethoxyyangonin, flavokawain A, echinatin, mangiferin, and11keto-beta-boswellic acid suppressed the bone resorption of the osteoclasts, as revealed by the tartrate resistant acid phosphatase (TRAP) activities and morphologic characteristics of the F-actin ring of osteoclasts
Summary
Cannabis sativa has been used to relieve pain and insomnia since prehistoric EurasiaAfrica [1]. More than 120 cannabinoids have been isolated and identified from this plant and intensively studied for their utility in the treatment of chronic and acute pain, epilepsy and anxiety, and modulation of appetite [2,3,4]. The effects of these cannabinoids are thought to be mediated mainly by type 1 and 2 cannabinoid receptors (CB1R and CB2R), as well as other G protein-coupled receptors (GPCRs) [5]. Drugs that solely target CB2R may be able to abrogate these negative side effects [12]
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