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. 2022 Jan 11;27(2):453.
doi: 10.3390/molecules27020453.

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Jiayi Yuan  1   2 , Chen Jiang  1   2 , Junmei Wang  1   2 , Chih-Jung Chen  1   2 , Yixuan Hao  1   2 , Guangyi Zhao  1   2 , Zhiwei Feng  1   2 , Xiang-Qun Xie  1   2
Affiliations

In Silico Prediction and Validation of CB2 Allosteric Binding Sites to Aid the Design of Allosteric Modulators

Jiayi Yuan et al. Molecules. .

Abstract

Although the 3D structures of active and inactive cannabinoid receptors type 2 (CB2) are available, neither the X-ray crystal nor the cryo-EM structure of CB2-orthosteric ligand-modulator has been resolved, prohibiting the drug discovery and development of CB2 allosteric modulators (AMs). In the present work, we mainly focused on investigating the potential allosteric binding site(s) of CB2. We applied different algorithms or tools to predict the potential allosteric binding sites of CB2 with the existing agonists. Seven potential allosteric sites can be observed for either CB2-CP55940 or CB2-WIN 55,212-2 complex, among which sites B, C, G and K are supported by the reported 3D structures of Class A GPCRs coupled with AMs. Applying our novel algorithm toolset-MCCS, we docked three known AMs of CB2 including Ec2la (C-2), trans-β-caryophyllene (TBC) and cannabidiol (CBD) to each site for further comparisons and quantified the potential binding residues in each allosteric binding site. Sequentially, we selected the most promising binding pose of C-2 in five allosteric sites to conduct the molecular dynamics (MD) simulations. Based on the results of docking studies and MD simulations, we suggest that site H is the most promising allosteric binding site. We plan to conduct bio-assay validations in the future.

Keywords: allosteric binding site; cannabinoid receptor 2; negative allosteric modulators; positive allosteric modulators.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Predicted allosteric binding sites of CB2 by CavityPlus, Protein Allosteric and Regulatory Sites (PARS) and Sybyl-X. The orthosteric binding site was highlighted in yellow surface, while predicted allosteric sites are highlighted in dots.
Figure 2
Figure 2
The detailed interactions and residue energy contribution of C-2, TBC and CBD in sites B, C and K. (a) The detailed binding pose of C-2 in site B. (b) The detailed binding pose of TBC in site C. (c) The detailed binding pose of CBD in site C. (d) The detailed binding pose of C-2 in site K. (e) The detailed binding pose of TBC in site K. (f) The detailed binding pose of CBD in site K. (g) The residue energy contribution of key residues for C-2 in site B. (h) The comparisons of residue energy contribution of key residues for TBC and CBD in site C. (i) The comparisons of residue energy contribution of key residues for C-2, TBC and CBD in site K.
Figure 3
Figure 3
The detailed interactions and residue energy contribution of C-2, TBC and CBD in site G. (a) The detailed binding pose of C-2 in site G. (b) The detailed binding pose of TBC in site G. (c) The detailed binding pose of CBD in site G. (d) The comparisons of residue energy contribution of the key residues for these AMs in site G.
Figure 4
Figure 4
The detailed interactions and residue energy contribution of C-2, TBC and CBD in site H. (a) The detailed binding pose of C-2 in site H. (b) The detailed binding pose of TBC in site H. (c) The detailed binding pose of CBD in site H. (d) The comparisons of residue energy contribution of these AMs in site H.
Figure 5
Figure 5
The detailed interactions and residue energy contribution of C-2, TBC and CBD in site I. (a) The detailed binding pose of C-2 in site I. (b) The detailed binding pose of TBC in site I. (c) The detailed binding pose of CBD in site I. (d) The comparisons of residue energy contribution of binding residues for these AMs in site I.
Figure 6
Figure 6
The detailed interactions and residue energy contribution of C-2, TBC and CBD in site J. (a) The detailed binding pose of C-2 in site J. (b) The detailed binding pose of TBC in site J. (c) The detailed binding pose of CBD in site J. (d) The comparisons of residue energy contribution of the binding residues for these AMs in site J.
Figure 7
Figure 7
The time course of root–mean–square deviations (RMSD) of mainchain atoms of the transmembrane domains (7TM, black), heavy atoms of CP55940 after least-square (LS) fitting (9GF, red) and heavy atoms of CP55940 without fitting (9GF, green). To calculate the No-Fit RMSDs for CP55940, the mainchain atoms of 7TM were first aligned and the resulting translation–rotation matrix were then applied to the ligand. After the coordinate transformation, the RMSDs were calculated directly. (A) CP55940/CB2 without C-2 binding, (B) site B, (C) site G, (D) site H, (E) site I and (F) site J.
Figure 8
Figure 8
The time course of root–mean–square deviations of mainchain atoms of the transmembrane domains (7TM, black), heavy atoms of C-2 after least-square (LS) fitting (LIG, red) and heavy atoms of C-2 without fitting (LIG, green). To calculate the No-Fit RMSDs for C-2, the mainchain atoms of 7TM were first aligned and the resulting translation–rotation matrix were then applied to the ligand. After the coordinate transformation, the RMSDs were calculated directly. (A) site B, (B) site G, (C) site H, (D) site I and (E) site J.
Figure 9
Figure 9
The detailed interactions between C-2, CP55940 and CB2 receptor where C-2 resides in site H. (A) The crystal structure of CP55940/CB2 with C-2, the PAM binding at site H. (B) The CB2 residues surrounding CP55940 and C-2. (C) The representative MD structure (the one that is structurally most similar to the average structure). (D) The residues surrounding CP55940 and C-2 in the representative MD structure. Two hydrogen bonds are formed between C-2 and CB2 residues (H952.65 and S2857.38), and one hydrogen bond is formed between CP55940 and Y25. The hydrogen bonds are shown as magenta dashed lines.
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