Download MendeleyChemical genetic strategy for targeting protein kinases based on covalent complementarity.
Garske, A.L., Peters, U., Cortesi, A.T., Perez, J.L., Shokat, K.M.(2011) Proc Natl Acad Sci U S A 108: 15046-15052
- PubMed: 21852571
- DOI: https://doi.org/10.1073/pnas.1111239108
- Primary Citation of Related Structures:
3SVV - PubMed 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.
Organizational 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.
