Pathway and mechanism of drug binding to chemokine receptors revealed by accelerated molecular simulations

Abstract

Background: Chemokine GPCRs play key roles in biology and medicine. Particularly, CXCR4 promotes cancer metastasis and facilitate HIV entry into host cells. Plerixafor (PLX) is a CXCR4 drug, but the pathway and binding site of PLX in CXCR4 remain unknown. Results & methodology: We have performed molecular docking and all-atom simulations using Gaussian accelerated molecular dynamics (GaMD), which are consistent with previous mutation experiments, suggesting that PLX binds to the orthosteric site of CXCR4 as an antagonist. The GaMD simulations further revealed an intermediate allosteric binding site at the extracellular mouth of CXCR4. Conclusion: The newly identified allosteric site can be targeted for novel drug design targeting CXCR4 and other chemokine receptors.

Keywords: GPCRs; Gaussian accelerated molecular dynamics; HIV; chemokine receptors; drug binding; plerixafor.

Figure 1.. Gaussian accelerated molecular dynamics simulation (Sim2 in Table 1) successfully captured spontaneous drug binding to the CXCR4 chemokine receptor.

(A) Computational model used for simulation of the CXCR4 receptor (blue ribbons) with ten PLX drug molecules (red spheres) placed away in the solvent. The receptor was inserted in a POPC lipid bilayer (cyan sticks) and solvated in an aqueous solution (cyan) of 0.15 M NaCl, (B) structure of PLX with numbered nitrogen atoms and the symmetry-corrected RMSD of PLX relative to its bound conformation plotted as a function of simulation time, (C) the distance between the C_γ atom of receptor residue D262^6.58 of the binding pocket and the N7 atom of PLX, (D) the distance between the Cγ atom of receptor residue D97^2.63 of the binding pocket and the N3 atom of PLX, (E) the distance between the C_δ atom of receptor residue E288^7.39 of the binding pocket and the N4 atom of PLX and (F) GaMD predicted binding pose of PLX (red sticks) at the orthosteric site of CXCR4 (blue ribbons) with their interacting residues labeled and highlighted in green sticks. Antagonist IT1t (orange) and docking conformation of PLX (yellow) are shown for reference. The seven TM helices I–VII and three extracellular loops (ECL) 1–3 are labeled in the CXCR4 receptor. GaMD: Gaussian accelerated molecular dynamics; PLX: Plerixafor; RMSD: Root-mean-square deviation; TM: Transmembrane.

Figure 2.. 2D potential of mean force free energy profiles of the CXCR4–plerixafor interactions.

(A) The distance between the C_γ atom of CXCR4 residue D262^6.58 of the binding pocket and the N7 atom of PLX as plotted in Figure 1D, (B) the distance between the C_γ atom of CXCR4 residue D97^2.63 of the binding pocket and the N3 atom of PLX as plotted in Figure 1E, (C) the distance between the C_δ atom of CXCR4 residue E288^7.39 of the binding pocket and the N4 atom of PLX as plotted in Figure 1F. (D) A 2D PMF of the distance between receptor residue D262^6.58 and PLX atom N7 versus the distance between receptor residue E288^7.39 and PLX atom N4. The I1 and I2 states at energy minima were identified at (D262:CG-PLX:N7, E288:CD-PLX:N4) distances of (13.01 Å, 8.60 Å) and (7.38 Å, 21.49 Å), respectively. (E) A 2D PMF of the distance between receptor residue D262^6.58 and PLX atom N7 versus the distance between receptor residue D97^2.63 and PLX atom N3. (F) Zoomed view of the 2D PMF as plotted in Figure 2E. The I1 and I2 states at energy minima (∼0.47 kcal/mol and 0.0 kcal/mol) were identified at (D262:CG-PLX:N7 and D97:CG-PLX:N3) distances of (13.01 Å and 16.40 Å) and (7.38 Å and 19.54 Å), respectively. Low-energy unbound, intermediate I1 and I2 and bound conformational states are labeled in the PMF profiles. GaMD: Gaussian accelerated molecular dynamic; PLX: Plerixafor; PMF: Potential of mean force; RMSD: Root-mean-square deviation.

Figure 3.. Novel intermediate drug binding site in the CXCR4 receptor. Intermediate I1 and I2 conformational states of PLX (red sticks) bound to the CXCR4 receptor (blue ribbons).

The charged nitrogen atoms of PLX are numbered as 1, 3, 4 and 7. (A) Intermediate I1 state with interacting residues highlighted in green sticks, including D187^ECL2 and D262^6.58 that formed ionic interactions with PLX atoms N4 and N3, respectively. (B) Intermediate I2 state with interacting residues highlighted in green sticks, including D187^ECL2, D193^5.32 and D262^6.58 that formed salt bridges with positively charged N7, N4 and N1 of PLX, respectively. GaMD: Gaussian accelerated molecular dynamic; PLX: Plerixafor.

Figure 4.. Binding pathway of the plerixafor drug to the CXCR4 chemokine receptor revealed from Gaussian accelerated molecular dynamic simulations.

Starting from diffusion in the solvent, PLX bound to the target site of the CXCR4 receptor via an intermediate site located between ECL2 and TM V–VI helices. The CXCR4 is shown in blue ribbons. The PLX structural clusters (sticks) are colored by the reweighted PMF free energy values in a green (0 kcal/mol)-white-red (2.5 kcal/mol) color scale. PLX: Plerixafor; PMF: Potential of mean force; TM: Transmembrane.