Crystal Structure of the Human Cannabinoid Receptor CB2

Abstract

The cannabinoid receptor CB2 is predominately expressed in the immune system, and selective modulation of CB2 without the psychoactivity of CB1 has therapeutic potential in inflammatory, fibrotic, and neurodegenerative diseases. Here, we report the crystal structure of human CB2 in complex with a rationally designed antagonist, AM10257, at 2.8 Å resolution. The CB2-AM10257 structure reveals a distinctly different binding pose compared with CB1. However, the extracellular portion of the antagonist-bound CB2 shares a high degree of conformational similarity with the agonist-bound CB1, which led to the discovery of AM10257's unexpected opposing functional profile of CB2 antagonism versus CB1 agonism. Further structural analysis using mutagenesis studies and molecular docking revealed the molecular basis of their function and selectivity for CB2 and CB1. Additional analyses of our designed antagonist and agonist pairs provide important insight into the activation mechanism of CB2. The present findings should facilitate rational drug design toward precise modulation of the endocannabinoid system.

Keywords: G-protein coupled receptor; cannabinoid receptor CB2; crystal structure; ligand design; subtype selectivity.

Figure 1.. Synthesis and Characterization of AM10257.

(A) Systematic lead optimization of AM10257, a CB2 antagonist, and radioligand binding affinity against [³H]CP55,940, Ki, inhibition constant. (B) AM10257 acts as a competitive antagonist/inverse agonist at CB2 against CP55,940, a potent agonist, in the forskolin-stimulated adenylyl cyclase assay as presented by the Schild plot where logK_B = −8.30 ± 0.05; (EC₅₀ for CP55,940 = 8.1 ± 0.6 nM). (C) AM10257 acts in a manner consistent with competitive antagonist/inverse agonist at CB2 in β-arrestin2 recruitment assay. CP55,940: EC₅₀: 3.4 ± 0.6 nM, E_max (fold): 2.5 ± 0.2; AM10257: EC₅₀: 0.53 ± 0.29 nM, E_max (fold): 0.70 ± 0.05. Data are presented as the mean ± s.e.m. of 3 experiments performed in duplicate. See also Figures S1 and S2 and Table S1.

Figure 2.. Overall structure of AM10257-CB2 Complex

(A) Side view of the CB2-AM10257 complex. CB2 is shown in green with ligand AM10257 in magenta. (B) Overall structure comparison of antagonist-bound CB2 and CB1. CB2 color scheme as in (A). CB1 is shown in light blue with antagonist AM6538 (cyan sticks). See also Figures S1 and S5.

Figure 3.. Interactions between AM10257 and CB2.

(A) The ‘three-arm’ scaffold of AM10257. The benzene ring, 5-hydroxypentyl group, and adamantyl group in red, blue and green squares are termed arm 1, arm 2 and arm 3, respectively. The key residues, which form binding interactions with the arms, are also shown. (B) Key residues (green sticks) involved in AM10257 (magenta sticks) binding. The water is shown as a red sphere and the hydrogen bonds are shown as dashed lines. (C) Dose response studies of CP55,940 activity for each mutant compared to wild-type CB2. As determined in parallel studies: WT EC₅₀: 1.6 ± 0.1 nM, E_max (fold): 2.2 ± 0.1; F87A EC₅₀: 664.9 ± 57.4 nM, E_max (fold): 2.4 ± 0.1; F91A EC₅₀: 56.6 ± 5.5 nM, E_max (fold): 2.4 ± 0.1; F94A EC₅₀: 125.6 ± 11.6 nM, E_max (fold): 2.3 ± 0.1; H95A EC₅₀: 349.9 ± 29.5, E_max (fold): 2.2 ± 0.1. Data are presented as mean ± s.e.m. of >3 experiments performed in duplicate. See also Figures S3 and S5.

Figure 4.. Comparison of ligand-binding modes and binding pockets for cannabinoid receptors.

(A) Superposition of the CB2-AM10257 and CB1-AM6538 ligand binding pockets. The residues Phe^2.57 and Phe^2.61 are shown as spheres. AM10257 (magenta sticks)-bound CB2, and AM6538 (cyan sticks)-bound CB1 are shown in green and light blue cartoon, respectively. (B) Conformational difference of Phe^3.36 and Trp^6.48 in antagonist-bound CB2 and CB1. Color scheme as in (A). (C–E) Surface representations of ligand binding pocket for: CB2 with AM10257 (C), CB1 with AM6538 (D), and CB1 with AM11542 (E). See also Figures S4 and S5.

Figure 5.. Structural comparison of antagonist-bound CB2 and agonist-bound CB1.

(A) Overall structural comparison of AM10257 (magenta sticks)-bound CB2 and AM11542 (yellow sticks)-bound CB1. CB2 and CB1 receptors are shown in green and orange, respectively. (B) Binding pose comparison of AM10257 in CB2 and AM11542 in CB1. The residues involved in the ligand binding are shown as sticks. (C) AM10257 (K_i = 13 nM for CB1, as determined by radioligand competition assays against [³H]-CP55,940) and functions as a partial agonist of CB1 as determined by β-arrestin2 recruitment assay. CP55,940 EC₅₀: 17.9 ± 1.4 nM, E_max (fold): 20.6 ± 0.9; AM10257 EC₅₀: 51.5 ± 7.4 nM, E_max (fold): 2.4 ± 0.1. Data are presented as mean ± s.e.m. of >4 experiments performed in duplicate. See also Figures S4 and S6.

Figure 6.. Molecular docking and pharmacology of designed CB2 agonist/antagonist pair.

(A) Chemical structures of rationally designed agonist MRI2594 and antagonist/inverse agonist MRI2687. (B) The docking poses of MRI2594 (orange sticks) and MRI2687 (blue sticks) in AM10257-bound CB2 (AM10257 is removed). (C) MRI2594 performs as an agonist of CB2 and MRI2687 performs as an inverse agonist of CB2 as determined by β-arrestin2 recruitment assay. CP55,940 EC₅₀: 2.3 ± 0.6 nM, E_max (fold): 2.3 ± 0.1; MRI2594 EC₅₀: 0.17 ± 0.03 nM, E_max (fold): 2.0 ± 0.2; MRI2687 EC₅₀: 0.24 ± 0.06 nM, E_max (fold): 0.75 ± 0.04; AM10257 EC₅₀: 0.20 ± 0.04 nM, E_max (fold): 0.70 ± 0.05. (D) MRI2594 and MRI2687 perform as an agonist and a partial agonist of CB1 as determined by β-arrestin2 recruitment assay. CP55,940 EC₅₀: 17.9 ± 1.4 nM, E_max (fold): 20.6 ± 0.9 (from Figure 5C); MRI2594 EC₅₀: 310.0 ± 54.0 nM, E_max (fold): 28.0 ± 2.0; MRI2687 EC₅₀: 114.0 ± 15.0 nM, E_max (fold): 6.0 ± 0.3. Data are presented as mean ± s.e.m. of > 4 experiments performed in duplicate. See also Figures S2 and S6.