Computational analysis of the binding specificity of Gleevec to Abl, c-Kit, Lck, and c-Src tyrosine kinases

Abstract

Gleevec, a well-known cancer therapeutic agent, is an effective inhibitor of several tyrosine kinases, including Abl and c-Kit, but displays less potency to inhibit closely homologous tyrosine kinases, such as Lck and c-Src. Because many structural features of the binding site are highly conserved in these homologous kinases, the molecular determinants responsible for the binding specificity of Gleevec remain poorly understood. To address this issue, free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent was used to compute the binding affinity of Gleevec to Abl, c-Kit, Lck, and c-Src. The results of the FEP/MD calculations are in good agreement with experiments, enabling a detailed and quantitative dissection of the absolute binding free energy in terms of various thermodynamic contributions affecting the binding specificity of Gleevec to the kinases. Dominant binding free energy contributions arises from the van der Waals dispersive interaction, compensating about two-thirds of the unfavorable free energy penalty associated with the loss of translational, rotational, and conformational freedom of the ligand upon binding. In contrast, the contributions from electrostatic and repulsive interactions nearly cancel out due to solvent effects. Furthermore, the calculations show the importance of the conformation of the kinase activation loop. Among the kinases examined, Abl provides the most favorable binding environment for Gleevec via optimal protein-ligand interactions and a small free energy cost for loss of the translational, rotational, and conformational freedom upon ligand binding. The FEP/MD calculations additionally reveal that Lck and c-Src provide similar nonbinding interactions with the bound-Gleevec, but the former pays less entropic penalty for the ligand losing its translational, rotational, and conformational motions to bind, examining the empirically observed differential binding affinities of Gleevec between the two Src-family kinases.

Figure 1

(A) Schematic diagram of Gleevec. (B) Pairwise percentage of sequence identity of Abl, c-Kit, Lck, and c-Src based on multiple sequence alignment of Clustalw2 program. (C) Superimposing the conformation of Gleevec bound to the kinase domains of Abl (colored in red), c-Kit (in orange), Lck (in green), and c-Src (in blue). Gleevec is represented by thick sticks.

Figure 2

Progression of the number of water molecules in the Gleevec-binding pockets of Abl, c-Kit, Lck, and c-Src in response to binding during the FEP calculations. The simulations are divided into 31 stages, starting from repulsive stage 1 and progressing to fully interacting ligand in stage 31. In stages 1–9, the ligand repulsion is gradually switched on. In stages 10–20, the ligand dispersion is turned on. In the stages 21–31, the charges of the ligand are added progressively.

Figure 3

Differential van der Waals (ΔE_vdW) interaction energies (in kcal/mol) between each individual kinase residue and Gleevec in c-Kit (in color of orange), Lck (in color of green), and c-Src(c) (in color of blue) relative to the corresponding residue in Abl. In each text box, Abl residue is at the bottom and the corresponding c-Kit/Lck/c-Src(c) residue is on the top.

Figure 4

(A) Superposition of P-loop in the equilibrated complexes of Abl (in red), c-Kit (in orange), Lck (in green), and c-Src (in blue). P-loops are represented by cartoon and Gleevec is shown in thick sticks. (B) Snapshot of the interactions of the P-loop with surrounding residues in Gleevec-bound Abl kinase.

Figure 5

Differential electrostatic (E_elec) interaction energies (in kcal/mol) between each individual kinase residue and Gleevec in c-Kit (in color of orange), Lck (in color of green), and c-Src (in color of blue) relative to the corresponding residue in Abl. In each text box, Abl residue is at the bottom and the corresponding c-Kit/Lck/c-Src residue is on the top. The actual ΔE_elec of water in Lck, which is 16.4 kcal/mol, is truncated for the consideration of figure resolution.

Figure 6

Structurally defined A-loop conformations in c-Src kinase domain. Blue: closed confirmation of A-loop in c-Src(c); magenta: open-form A-loop in c-Src(o). Gleevec is represented by thick sticks.