Identification of raloxifene as a novel CB2 inverse agonist

• A cell-based, high-throughput assay was validated for the CB2 cannabinoid receptor.
• Using the validated assay a library of FDA-approved drugs was screened against CB2.
• Anti-osteoporosis drug raloxifene was discovered to be a novel CB2 inverse agonist.
• Our discovery provides new insights into the possibility of repurposing raloxifene.
• Our data suggest novel mechanisms for the known therapeutic effects of raloxifene.

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 [3H]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.