title: 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.
authors.affiliation: Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, 600 16th Street, MC 2280, San Francisco, CA 94158, USA.
authors.forename: Adam L
authors.initials: AL
authors.lastname: Garske
authors.forename: Ulf
authors.initials: U
authors.lastname: Peters
authors.forename: Arianna T
authors.initials: AT
authors.lastname: Cortesi
authors.forename: Jenny L
authors.initials: JL
authors.lastname: Perez
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: D000818:Animals; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D005821:Genetic Techniques; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D041681:NIH 3T3 Cells; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D047428:Protein Kinase Inhibitors; D011494:Protein Kinases; D019061:src-Family Kinases; D003545:Cysteine