Determining Cysteines Available for Covalent Inhibition Across the Human Kinome

Abstract

Covalently bound protein kinase inhibitors have been frequently designed to target noncatalytic cysteines at the ATP binding site. Thus, it is important to know if a given cysteine can form a covalent bond. Here we combine a function-site interaction fingerprint method and DFT calculations to determine the potential of cysteines to form a covalent interaction with an inhibitor. By harnessing the human structural kinome, a comprehensive structure-based binding site cysteine data set was assembled. The orientation of the cysteine thiol group indicates which cysteines can potentially form covalent bonds. These covalent inhibitor easy-available cysteines are located within five regions: P-loop, roof of pocket, front pocket, catalytic-2 of the catalytic loop, and DFG-3 close to the DFG peptide. In an independent test set these cysteines covered 95% of covalent kinase inhibitors. This study provides new insights into cysteine reactivity and preference which is important for the prospective development of covalent kinase inhibitors.

Figure 1

Model system and reaction schemes for cysteine-involved addition. d_i is the interatomic distance between two atoms.

Figure 2

(a) The locations of cysteines and the structure template from pdb id 3byu. (b) Statistics for the accessible cysteines, the details of the interactions between cysteines and ligands, and the existed covalent interactions between cysteines and ligands.

Figure 3

The detailed interactions of cysteines. (a) The six atoms of cysteine. (b) The distribution of different types of interactions for different atoms. (c) The rate of contributed interactions for different atoms at eachlocation.

Figure 4

Cysteines at different locations; sulfur atom in yellow, cysteine in green; and ligand in blue. (a) Cysteine at P-loop (pdb id 4r6v). (b) Cysteine at roof of the binding pocket (pdb id 4riy). (c) Cysteines at location Hinge-1 of the hinge region (representative pdb id 3m2w). (d) Cysteines at location Hinge-2 of the hinge region (representative pdb id 2ywp). (e) Cysteines at location Hinge-3 of the hinge region (representative pdb id 3lco). (f) Cysteines at location Catalytic-2 of the catalytic loop (representative pdb id 1t46). (g) Cysteines at location Catalytic-9 of the catalytic loop (representative pdb id 4a0j). (h) Cysteine at location DFG-4 close to the DFG peptide (representative pdb id 3wf7).

Figure 5

The potential energy surfaces(PESs). (a) For scheme 1 in different dielectric constants. (b) For scheme 2 in different dielectric constants. ΔE for energy barrier from the reactant to the transition state (TS) and the energy unit is kcal mol⁻¹. ε is the dielectric constant.

Figure 6

(a) Distribution of the cysteine dihedralangle χ₁at location Hinge -2. (b) PES for the conformation space of the cysteine dihedralangle χ₁;Y -axis for the interatomic distance d₁.

Figure 7

Reactive cysteines across the human kinome. (a) The reactive cysteines distributed at the five regions of binding sites marked in different colors. (b) The kinases with released 3D kinase structures. (c) The kinases without released kinase structures. (b) and (c) were generated using KinMap (http://kinhub.org/) and the high resolution figures are available in supporting information Figure S2–3.

Figure 8

(a) Distribution of the kinases with the released CKIs. (b) Distribution of cysteines of contributing to form covalent adducts.

Figure 9

Cysteines located at different locations. (a) Cysteine at Hinge-1 with the covalent ligand (pdb id 4xcu). (b) Remote cysteine from the binding site (pdb id 1ua2). (c) Cysteine at the activation loop (pdb id 4e3c). (d) Cysteine at the extended front pocket (pdb id 3itz).