Negative allosteric modulators of cannabinoid receptor 2: protein modeling, binding site identification and molecular dynamics simulations in the presence of an orthosteric agonist

Abstract

Selective activation of the cannabinoid receptor subtype 2 (CB2) shows promise for treating pain, inflammation, multiple sclerosis, cancer, ischemic/reperfusion injury and osteoporosis. Target selectivity and off-target side effects are two major limiting factors for orthosteric ligands, and therefore, the search for allosteric modulators (AMs) is a widely used drug discovery approach. To date, only a limited number of negative CB2 AMs have been identified, possessing only micromolar activity at best, and the CB2 receptor's allosteric site(s) are not well characterized. Herein, we used computational approaches including receptor modeling, site mapping, docking, molecular dynamics (MD) simulations and binding free energy calculations to predict, characterize and validate allosteric sites within the complex of the CB2 receptor with bound orthosteric agonist CP55,940. After docking of known negative CB2 allosteric modulators (NAMs), dihydro-gambogic acid (DHGA) and trans-β-caryophyllene (TBC) (note that TBC also shows agonist activity), at the predicted allosteric sites, the best total complex with CB2, CP55,940 and NAM was embedded into a hydrated lipid bilayer and subjected to a 200 ns MD simulation. The presence of an AM affected the CB2-CP55,940 complex, altering the relative positioning of the toggle switch residues and promoting a strong π-π interaction between Phe117^3.36 and Trp258^6.48. Binding of either TBC or DHGA to a putative allosteric pocket directly adjacent to the orthosteric ligand reduced the binding free energy of CP55,940, which is consistent with the expected effect of a negative AM. The identified allosteric sites present immense scope for the discovery of novel classes of CB2 AMs.

Keywords: CB2 allosteric modulator; CB2 receptor; G-protein-coupled receptor; docking; molecular dynamics.

Figure 1.

Chemical structures of selected CB compounds, including CP55,940, trans-β-caryophyllene (TBC), and two diastereomers of dihydro-gambogic acid (DHGA) which differ at the 4-hydroxy position.

Figure 2.

Multiple sequence alignment of human CB2 (hCB2) with turkey β1 adrenergic receptor (β1-AR) (PDB-ID: 2Y02), human β2 adrenergic receptor (β2-AR) (PDB-IDs: 3SN6); and bovine rhodopsin (Rho) (PDB-ID: 3PQR). Identical residues are colored red while similar residues are colored blue. Transmembrane (TM) domains are highlighted with yellow background.

Figure 3.

(A) Cα-atom based overlay of the 100 generated models of the hCB2 receptor. Helices and loops are shown in magenta and blue, respectively. (B) Plots of DOPE (top) and molpdf (bottom) scores of the 100 generated models of the hCB2 receptor.

Figure 4.

(A) Overall cartoon view of the selected hCB2 receptor model, with helices labeled with Roman numerals and loops labeled as e (extracellular) or i (intracellular). (B) An alternative view of the hCB2 receptor model from the extracellular side depicting some key residues of hCB2 and a disulfide bridge in the second extracellular loop (EC2). (C). Ramachandran plot of the CB2 3D model showing the backbone angles (phi and psi) of the amino acids. Most-favorable region in red and favored region in yellow. Triangles represent glycines and squares represent prolines, which are not expected to match the typical phi/psi ratio of the other amino acids.

Figure 5.

The interaction profile of CP55,940 with the hCB2 receptor, from the best docking pose. (A) 3D representation, with CP55,940 (cyan carbons) and key residues of CB2 (gray carbons); H-bonds are shown as yellow dashed lines. (B) 2D representation.

Figure 6.

SiteMap-detected allosteric binding pockets. (A) Five potential allosteric binding pockets (represented as spheres with unique color for each pocket) found for the complex of the CB2 receptor (gray ribbon representation) and CP55,940 (cyan carbon CPK representation): Site 1 (gold); Site 2 (magenta); Site 3 (blue); Site 4 (yellow); and Site 5 (red). (B) Binding map of Site 1 within the complex of the CB2 receptor (multi-colored ribbon representation) and CP55,940 (blue carbon CPK representation); the appearance of the map types is as follows: Hydrophobic map (yellow mesh), Hydrophilic map (green mesh), Hydrogen-bond donor map (blue mesh), and Hydrogen-bond acceptor map (red mesh).

Figure 7.

The 3D representation of putative binding modes of the allosteric inhibitors into the CB2–CP55,940 (magenta carbon) complex: (A) 4R-DHGA (yellow carbon); (B) TBC (green carbon).

Figure 8.

RMSD for protein backbone atoms (gray line; running average in a window of 200 ps is indicated in black), CP55,940 heavy atoms (gold line; running average in a window of 200 ps is indicated in red), and NAM heavy atoms (cyan line; running average in a window of 200 ps is indicated in blue) for MD simulations of the CB2 receptor and the orthosteric ligand (CP55,940), without or with NAM: (A) CP55,940 only; (B) CP55,940 and TBC; and (C) CP55,940 and DHGA.

Fig 9.

MD simulation of the CB2 receptor with: (A) CP55,940 (the orthosteric ligand) only; (B) CP55,940 and DHGA; and (C) CP55,940 and TBC. An overlay of the first (yellow), middle (50000^th) (gray), and last (blue) frames of each of the three MD simulations is presented. The protein is represented in cartoon form, while the ligands are represented in stick form with matching colors.

Figure 10.

(A) 3D view of a representative structure of TBC (plum carbon) with the CB2 receptor in the presence of CP55,940 (green carbon) (nearby waters are also shown as stick model) and (B) the corresponding 2D interaction diagram of TBC interacting with the CB2 receptor in the presence of CP55,940.

Figure 11.

(A) 3D view of a representative structure of DHGA (yellow carbon) with the CB2 receptor in the presence of CP55,940 (green carbon) (nearby waters are also shown as stick model) and (B) the corresponding 2D interaction diagram of DHGA interacting with the CB2 receptor.

Figure 12.

The distance between Trp172^4.64 and CP55,940 (southern hydroxyl group) in: CB2–CP55,940–TBC complex (gray line; running average in a window of 200 ps is indicated in black); CB2–CP55,940 complex (gold line; running average in a window of 200 ps is indicated in red); and CB2–CP55,940–DHGA complex (cyan line; running average in a window of 200 ps is indicated in blue).

Figure 13.

The distance between Trp258^6.48 and CP55,940 (terminal carbon of dimethyl heptyl side chain) in: CB2–CP55,940–TBC complex (gray line; running average in a window of 200 ps is indicated in black); CB2–CP55,940 complex (gold line; running average in a window of 200 ps is indicated in red); and CB2–CP55,940–DHGA complex (cyan line; running average in a window of 200 ps is indicated in blue).

Fig 14.

(A) The distance between Ser285^7.39 (OH) and CP55,940 (phenolic OH) in the CB2–CP55,940 complex (gray line; running average in a window of 200 ps is indicated in black). (B) The putative binding mode of CP55,940 with the CB2 receptor after MD simulation.

Figure 15.

The putative binding mode after MD simulations of: (A) CP55,940 with CB2–TBC complex and (B) CP55,940 with CB2–DHGA complex.