NMR and computer modeling studies of G-protein coupled cannabinoid receptors and bioactive cannabinoid ligands

The research objectives are to build/examine a three-dimensional (3D) structural model of the second cannabinoid receptor subtype (CB2), and to study the preferred conformation properties and 3D-QSAR models of key cannabinoid ligands by using combined NMR and computer modeling approaches for pharmacophore-based drug design. This is very important information to assist rational drug design. ^ A CB2 3D structural model was first constructed by using homology method, and further employed to analyze the CB2 structural information and to predict possible CB2 binding cavity. Subsequently, NMR studies were performed to reveal the amino acid ends of CB2 seven transmembrane domains. Such studies provided valuable experimental data that were then integrated into the homology model for further structural refinement of the 3D model of the CB2 receptor. ^ In the efforts to study and understand the biological importance of the helix VIII domains in G-protein activation of the CB1 and CB2 receptors, the comparative NMR structural analyses were carried out for the segments of both receptors. The NMR-determined 3D structures of both peptides show well-defined amphipathic α-helix in the membrane-mimicking environment. It was hypothesized that the salt bridge features may play significant roles in the function and regulation of the C-terminal juxtamembrane domains of both receptors. ^ In addition, we did the pharmacophoric studies of the key cannabinoid ligands. The combined NMR and CoMFA analyses were performed to develop 3D-QSAR models, which distinguished the different steric and electronic requirements of the antagonist for the selective efficacy at the CB1 and CB2 receptors. The results provided some clues for designing more potent and selective antagonists. Furthermore, solution/solid-state NMR and molecular modeling were applied to investigate conformational features of anandamide, which encode important pharmacophoric features regarding the activation of the cannabinoid receptors. ^ The present dissertation research work has contributed to define the 3D CB2 structure from which a potential binding pocket has been predicted in correlating with the site-directed mutagenesis. Moreover, the conformational and 3D-QSAR studies of key cannabinoid ligands helped to derive reasonable pharmacophore models. The work accomplished is essential for the future in-silico design and virtual screening as planned.