Structural basis of selective cannabinoid CB2 receptor activation

Abstract

Cannabinoid CB2 receptor (CB2R) agonists are investigated as therapeutic agents in the clinic. However, their molecular mode-of-action is not fully understood. Here, we report the discovery of LEI-102, a CB2R agonist, used in conjunction with three other CBR ligands (APD371, HU308, and CP55,940) to investigate the selective CB2R activation by binding kinetics, site-directed mutagenesis, and cryo-EM studies. We identify key residues for CB2R activation. Highly lipophilic HU308 and the endocannabinoids, but not the more polar LEI-102, APD371, and CP55,940, reach the binding pocket through a membrane channel in TM1-TM7. Favorable physico-chemical properties of LEI-102 enable oral efficacy in a chemotherapy-induced nephropathy model. This study delineates the molecular mechanism of CB2R activation by selective agonists and highlights the role of lipophilicity in CB2R engagement. This may have implications for GPCR drug design and sheds light on their activation by endogenous ligands.

Platform Reagents Enable Synthesis of Ligand-Directed Covalent Probes: Study of Cannabinoid Receptor 2 in Live Cells

“…The drastic drop in affinity observed toward CB 1 R cannot be fully explained by the CB 2 R and CB 1 R amino acid residue substitutions alone. Thus, we speculate that significant contributions arise from the differences in ligand accessibility to the CB 2 R versus CB 1 R binding sites, as reported for the parent ligand of 1a , HU-308. , …”

“…Thus, we speculate that significant contributions arise from the differences in ligand accessibility to the CB 2 R versus CB 1 R binding sites, as reported for the parent ligand of 1a, HU-308. 73,74 TR-FRET-Based Assay to Assess Covalent NBD Transfer to CB 2 R. The binding data for 2a−e suggest that conjugation of 1a with the electrophilic O-NBD motif is well tolerated. In previous work on ligand-directed chemistry, covalent labeling was validated by Western blotting, 53 mass spectrometry, 46,75,76 UV−vis spectroscopy, 63 or time-resolved Forster resonance energy transfer (TR-FRET).…”

Discovery of a Photoaffinity Probe that Captures the Active Conformation of the Cannabinoid CB2 Receptor

“…[9] The CB 2 R-G i structure was reported in complex with CB 2 R selective agonist LEI-102 with a resolution 2.9 Å. [9] Considering previous structure activity relationship (SAR) reports [8] , we substituted the thiomorpholine 1,1-dioxide (1) of LEI-121 for a diaziridinepiperidine (2), a difluoropiperidine (3) or a piperidine (4) to investigate their effect on the potency and functionality of the probe. [8] Furthermore, compound 2 was designed to more closely resemble the structure of LEI-102, in an attempt to create an agonist probe.…”

“…The construction of probe 2 was similar to the synthesis of LEI-102 (Scheme 2). [9] To start, reductive amination of 4-bromobenzaldehyde and 2-aminoacetamide led to compound 16. After cyclization the formed…”

“…[9] LEI-102 is a high-affinity CB 2 R ligand (pK i of 8.0 � 0.1) with over 1000-fold selectivity over CB 1 R. LEI-102 activates the CB 2 receptor as a partial agonist (pEC 50 6.9 � 0.2, E max 76 � 1 %). [9]…”