As key components in nearly every signal transduction pathway, protein kinases are attractive targets for the regulation of cellular signaling by small-molecule inhibitors. We report the structure-guided development of 6-acrylamido-4-anilinoquinazoline irreversible kinase inhibitors that potently and selectively target rationally designed kinases bearing two selectivity elements that are not found together in any wild-type kinase: an electrophile-targeted cysteine residue and a glycine gatekeeper residue. Cocrystal structures of two irreversible quinazoline inhibitors bound to either epidermal growth factor receptor (EGFR) or engineered c-Src show covalent inhibitor binding to the targeted cysteine (Cys797 in EGFR and Cys345 in engineered c-Src). To accommodate the new covalent bond, the quinazoline core adopts positions that are different from those seen in kinase structures with reversible quinazoline inhibitors. Based on these structures, we developed a fluorescent 6-acrylamido-4-anilinoquinazoline affinity probe to report the fraction of kinase necessary for cellular signaling, and we used these reagents to quantitate the relationship between EGFR stimulation by EGF and its downstream outputs—Akt, Erk1 and Erk2.
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We thank E. Garner and R.D. Mullins (University of California, San Francisco) and the J.A. Wells lab (University of California, San Francisco) for reagents and use of instrumentation. We thank M. Seeliger and J. Kuriyan (University of California, Berkeley) for the plasmid containing the chicken c-Src gene, the plasmid containing tyrosine phosphatase YopH, and the purified c-Src kinase domain. We thank G. Montelione (Rutgers) for the plasmid containing GroEL and trigger factor. We thank J. Taunton, T. Hirano, D. Maly and R. Bateman for assistance with organic synthesis and data collection, and Q. Justman, M. Feldman, A. Dar and B. Olson for helpful comments on the manuscript. We thank the staff and funding agencies of beamlines 8.2.1 and 8.2.2 (Advanced Light Source) and beamline ID24 (Argonne National Laboratory Advanced Photon Source) for their assistance with X-ray diffraction data collection. This work was supported in part by US National Institutes of Health grants AI44009 (K.M.S.), CA080942 (M.J.E.), CA116020 (M.J.E.), NCRR RR015804 and NCRR RR001614 (NIH Resource to University of California, San Francisco) and by the Sandler Program in Basic Sciences (K.M.S. and W.A.W.) and the Burroughs Wellcome Fund (W.A.W.). M.J.E. is the recipient of a Scholar Award from the Leukemia and Lymphoma Society.
The authors declare no competing financial interests.
Stereodiagrams for irreversible 6-acrylamido-4-anilinoquinazoline inhibitor 4 covalently bound to the ATP site of both EGFR and c-Src-cys show different binding modes for each kinase. (PDF 1019 kb)
Allele-selective inhibitor 5 modeled into the ATP binding pocket of c-Src with different selectivity elements illustrates a potential selectivity mechanism afforded by the gatekeeper residue. (PDF 1069 kb)
Inhibition data of C4-derivatized PD 168393 analogs screened against four Fyn kinase variants reveal potent, selective inhibitors for Fyn-dm. (PDF 46 kb)
Data collection and refinement statistics for EGFR and c-Src-cys complex structures. (PDF 31 kb)
Inhibition data of C4-derivatized PD 168393 analogs screened against EGFR kinase. (PDF 27 kb)
Ramachandran statistics for EGFR and c-Src-cys complex structures. (PDF 28 kb)
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Blair, J., Rauh, D., Kung, C. et al. Structure-guided development of affinity probes for tyrosine kinases using chemical genetics. Nat Chem Biol 3, 229–238 (2007). https://doi.org/10.1038/nchembio866
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