Difference and Influence of Inactive and Active States of Cannabinoid Receptor Subtype CB2: From Conformation to Drug Discovery.

Cannabinoid receptor 2 (CB2), a G protein-coupled receptor (GPCR), is a promising target for the treatment of neuropathic pain, osteoporosis, immune system, cancer, and drug abuse. The lack of experimental 3D CB2 structure hindered not only the development of conformational differences study between the inactive and active CB2 but also the rationale discovery of novel functional compounds targeting CB2. In this work, we constructed both the inactive and active CB2 model by homology modeling. Then we conducted two comparative 100 ns molecular dynamics (MD) simulations on the two systems — the active CB2 bound with both the agonist and G-proteins, and the inactive CB2 bound with inverse agonist — to analyze the conformational difference of CB2 proteins and the key residues involved in molecular recognition. Our results showed that the inactive CB2 and the inverse agonist kept stable during the MD simulation. However, during the MD simulations, we observed the dynamics details about the breakdown of the “ionic lock” between R1313.50 and D2406.30, as well as the outward/inward movements of trans-membrane domains (TM5, TM6 and TM7) at the active CB2 that bound with G proteins and agonist. All these results are congruent with the experimental data and recent reports. Moreover, our results indicated that W2586.48 in TM6 and residues in TM4 (V1644.56~L1694.61) contributed greatly to the binding of agonist based on the binding energy decomposition. While residues (S180-F183) in extracellular loop 2 (ECL2) may be importance of the recognition of inverse agonist. Furthermore, pharmacophore model and virtual screening were carried out for the inactive and active CB2 models in parallel. Among all twelve hits, two compounds exhibited the novel scaffolds and can be used as the novel chemical probes for future studies of CB2. Importantly, our studies showed that the hits obtained from the inactive CB2 model mainly acted as inverse agonist or neutral antagonist at low concentration. Moreover, the hits from the active CB2 model also behaved as a neutral antagonist at low concentration. Our studies provided new insight into the understanding of structural and conformational differences between two states of CB2 and illuminated the structural effects for virtual screening and drug design.