Cannabinoid Receptor CB2 Structure and CB2/Gi Signaling Mechanisms

Abstract

Cannabinoid receptor subtype 2 (CB2) is a class‐A family G protein‐coupled receptor (GPCR), located primarily in immune‐associated tissues and implicated in several immune and inflammatory diseases. Drugs targeting CB2 are predicted to be effective treatment alternatives for chronic neuropain and neuroinflammatory autoimmune diseases without risk of addiction or deleterious CB1‐associated psychotropic effects. However, development of potent CB2‐selective small molecules designed to “fit” the active CB2 protein structure has been hampered due to lack of a high‐resolution 3D structure, attributable primarily to its inherent structural heterogeneity, typical of many GPCRs. The objective of this research is to gain an unprecedented understanding of the structural and functional mechanisms inherent to CB2 using high‐end biophysical and biochemical approaches and purified functional CB2. To this end, we have performed a series of CB2 protein manipulations, including insertion of a novel proprietary peptide in the N‐terminus, to obtain high levels of receptor expression in Baculovirus‐infected Sf9 cells and good stability of functional CB2 monomer during purification. Mutagenesis studies were also conducted to examine CB2 structural and functional domains and the ligand binding pocket. Analysis of the active pharmacophore was performed utilizing our published CB2 homology model and cannabinoid cheminformatics, to discover structure‐based agonist/antagonist recognition sites, permitting the design and discovery of new CB2 ligands. The Aims of this study are: 1) to optimize design/synthesis of novel CB2 chemical probes to facilitate CB2 structural studies; this will be accomplished by computer‐aided development of a new series of CB2 ligands with high affinity and specificity, as well as those forming covalent bonds, to stabilize CB2 protein expression, purification and crystallization; and 2) to elucidate CB2 structure and CB2/Gi signaling mechanisms by X‐ray and cryo‐EM to guide structure‐based design of CB2 agonists and inverse agonists. We will determine the crystal structure of CB2 by X‐ray diffraction. Thermostable CB2 mutants, other rationally designed CB2 constructs with novel insertions, and ability of structurally diverse CB2 agonists/inverse agonists (from commercial and in‐house sources) to confer thermostability to CB2, will be tested to optimize crystallization conditions and obtain the best CB2 crystals for X‐ray diffraction. We will also determine the G protein coupling and conformational change of CB2 by cryo‐EM. Multiple approaches will be used to stabilize the CB2‐Gi complex (e.g., anti‐Gi Fab or PtdInsP2) to obtain promising negative staining and cryo‐EM data, which will be utilized to study the CB2 active signaling. Fulfillment of these Aims will facilitate our long‐term goal of utilizing new detailed structural information of CB2 to develop a new class of drugs that will benefit millions of patients suffering from the various immune‐related afflictions in which CB2 is implicated.Support or Funding Information1. NIH NIDA P30 PDA035778A (Xie, PI/Director) 07/01/2014–06/30/19 2.4 Calendar DOD AZ150100 (Xie, PI) 09/15/2016–09/14/19 1.2 CalendarThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.