Chemical genetic strategy for targeting protein kinases based on covalent complementarity

Abstract

The conserved nature of the ATP-binding site of the > 500 human kinases renders the development of specific inhibitors a challenging task. A widely used chemical genetic strategy to overcome the specificity challenge exploits a large-to-small mutation of the gatekeeper residue (a conserved hydrophobic amino acid) and the use of a bulky inhibitor to achieve specificity via shape complementarity. However, in a number of cases, introduction of a glycine or alanine gatekeeper results in diminished kinase activity and ATP affinity. A new chemical genetic approach based on covalent complementarity between an engineered gatekeeper cysteine and an electrophilic inhibitor was developed to address these challenges. This strategy was evaluated with Src, a proto-oncogenic tyrosine kinase known to lose some enzymatic activity using the shape complementarity chemical genetic strategy. We found that Src with a cysteine gatekeeper recapitulates wild type activity and can be irreversibly inhibited both in vitro and in cells. A cocrystal structure of T338C c-Src with a vinylsulfonamide-derivatized pyrazolopyrimidine inhibitor was solved to elucidate the inhibitor binding mode. A panel of electrophilic inhibitors was analyzed against 307 kinases and MOK (MAPK/MAK/MRK overlapping kinase), one of only two human kinases known to have an endogenous cysteine gatekeeper. This analysis revealed remarkably few off-targets, making these compounds the most selective chemical genetic inhibitors reported to date. Protein engineering studies demonstrated that it is possible to increase inhibitor potency through secondary-site mutations. These results suggest that chemical genetic strategies based on covalent complementarity should be widely applicable to the study of protein kinases.

Fig. 1.

Chemical genetic strategies for inhibiting protein kinases. Kinases are depicted in red and inhibitors types are represented by gray shapes. WT kinases generally harbor hydrophobic gatekeeper residues and are difficult to inhibit selectively. An AS protein kinase has an engineered glycine or alanine gatekeeper and may be selectively inhibited by a bulky inhibitor. An ES protein kinase contains an engineered cysteine gatekeeper and may be selectively inhibited by an electrophilic inhibitor.

Fig. 2.

Crystal structure of compound 9 bound covalently to c-Src-ES1. The experimental electron density of c-Src-ES1 at 2.20 Å resolution is shown (2F₀-F_c map at 1σ). (A) The pyrazolopyrimidine portion of compound 9 (green) interacts with the hinge region of c-Src (Met-341 and Glu-339), while the sulfonamide group makes a hydrogen bond with Glu-310 of the αC helix. (B) Electron density reveals a covalent linkage between Cys-338 and compound 9. The oxygen atoms of the sulfonamide interact with the backbone of Asp-404 and via a water molecule with Phe-405, both of which are part of the DFG-motif of the kinase. (C) Comparison of structural features of compound 9 (green) bound to c-Src-ES1 (gray) and a published Type II pyrazolopyrimidine compound bound to WT c-Src (pink, pdb code: 3el7) (29). Both compounds engage the hinge region in a similar fashion and bind the αC helix in the “in” conformation. Furthermore, both compounds participate in hydrogen bonding interactions with Glu-310 and backbone amides of the DFG-motif. However while the Type II inhibitor binds in the “DFG-out” conformation, compound 9 engages the “DFG-in” orientation. In addition, while the positioning of the gatekeeper main chain is almost identical, the sulfhydryl of the Cys-338 points in the opposite direction relative to the hydroxyl group of Thr-338 in order to facilitate a covalent bond with compound 9.

Fig. 3.

Assay for MOK inhibition by cysteine gatekeeper-targeting compounds. (Top) FLAG-MOK expressed in COS7 cells was immunoprecipitated and assayed in vitro with a myelin basic protein (MBP) substrate and inhibitors at a concentration of 1 μM. Autoradiography is shown. (Center) Quantification of the percent MBP phosphorylated from three independent experiments with associated standard errors. All values are normalized relative to the MOK + DMSO lane. (Bottom) Western blot of loading controls for FLAG-MOK are shown.

Fig. 4.

Cellular dose response analysis for inhibition of v-Src-ES1 (I338C) with electrophilic inhibitors. NIH-3T3 cells stably transduced with either v-Src-ES1 or I338T v-Src were treated with electrophilic inhibitors or nonreactive analogs for one hour. Kinase activity was monitored by blotting for global phosphotyrosine levels. Actin blots were included to control for protein content.