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The CB1 and CB2 receptors are two subtypes of cannabinoid receptors.Both CB1 and CB2 belong to the family I rhodopsin-like G-protein coupled receptors (GPCRs).The CB 1 receptor is mainly located in the CNS system and is response for addiction and serious side psychoactive effect of cannabis.CB2 is mostly expressed in the immune system and plays a critical role in the cannabinoid-mediated immune response.CB2 has become one of important drug targets.Due to the general lack of experimental 3D structures, great efforts have been devoted for the computer-aided GPCR-targeted drug design using ligand-based computer modeling techniques based on the pharmacophore models derived from known bioactive ligands affecting GPCR.We applied a comprehensive approach of ligand-based molecular design and in vitro bioactivity testing to discover novel CB2 ligands.At first, we focused on the study of the different three-dimensional (3D) structural requirements for selective ligands of CB1 and CB2, respectively, by applying combined approaches of NMR and molecular modeling.It was production of the 3D quantitative structure-activity relationship (QSAR) models by performing the comparative molecular field analyses (CoMFA) on a set of CB1 and CB2 ligands using the NMR-determined molecular conformers as templates.Diverse pharmacophoric features of selective ligands (agonist or antagonist) for CB1 and CB2 were then respectively induced by comparison of CoMFA steric and potential contourmaps for differently binding affinity of ligands at these two subtypes.A pharmacophore model was derived based on the generated 3D-QSAR model.Pharmacophore-based virtual screening was then carried out to predict the potential CB2-selective ligands from compound database.Docking simulations were further completed to verify the in silico screened compound binding to the CB2 receptor on the basis of the generated CB2 homology model.The previously modeling results also led to serve as a basis for the structural optimization design of later-generation analogues.In vitro bioactive tests let us to identify several new CB2-selective ligands with nanomolar to micromolar binding affinity.The results would help us to better understand the pharmacophoric requirements of the CB2-selective ligand and serve as a basis for the future SAR analyses and structural modification to develop novel CB2-seletivce drug candidate with improved bioactivity.