Structure-guided development of affinity probes for tyrosine kinases using chemical genetics


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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Figure 1: A chemical genetic strategy sensitizes kinases to irreversible inhibitors that do not inhibit wild-type kinases.
Figure 2: The screening of a panel of C4-derivatized PD 168393 analogs reveals potent, selective inhibitors for an engineered double mutant of c-Src kinase.
Figure 3: Deconvoluted mass spectra of c-Src variants treated with irreversible inhibitors suggest covalent inhibitor binding to kinase active sites bearing a properly positioned cysteine.
Figure 4: Stereodiagrams for irreversible 6-acrylamido-4-anilinoquinazoline inhibitor 2 covalently bound to the ATP site of both EGFR and c-Src-cys show different binding modes for each kinase.
Figure 5: Comparison of the binding modes of 2 and 4 in EGFR and c-Src-cys with a canonical kinase-quinazoline binding mode reveals inhibitor movements necessary to accommodate covalent attachment.
Figure 6: Reversal of tyrosine phosphorylation in cells demonstrates both the cell permeability and allele selectivity of two rationally designed 6-acrylamido-4-anilinoquinazoline inhibitors.
Figure 7: EGFR-as3 activity in intact cells, as reported by an affinity probe for protein kinases, correlates with the downstream signals of EGFR-as3.

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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.

Author information




J.A.B. and C.K. synthesized the panel of inhibitors, expressed the Fyn variants and measured the Fyn in vitro IC50 values. J.A.B. and D.R. expressed the c-Src variants, crystallized and measured the c-Src-cys cocrystals and measured the EGFR-as3 cellular activity. J.A.B. conducted the protein mass spectrometry and measured the c-Src and EGFR in vitro IC50 values. D.R. and H.R. synthesized and characterized probe 16. D.R. solved the c-Src-cys complex structures. Q.W.F. established the 3T3:EGFR cell lines and conducted the cellular inhibition experiments with 2 and 5. C.H.Y. expressed, crystallized and solved the EGFR complex structures. J.A.B. prepared the manuscript, with help from and editing by all the co-authors. C.Z. conceptualized the initial chemical genetic design. W.A.W., M.J.E. and K.M.S. helped conceive of experiments.

Corresponding author

Correspondence to Kevan M Shokat.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

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)

Supplementary Fig. 2

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)

Supplementary Table 1

Inhibition data of C4-derivatized PD 168393 analogs screened against four Fyn kinase variants reveal potent, selective inhibitors for Fyn-dm. (PDF 46 kb)

Supplementary Table 2

Data collection and refinement statistics for EGFR and c-Src-cys complex structures. (PDF 31 kb)

Supplementary Table 3

Inhibition data of C4-derivatized PD 168393 analogs screened against EGFR kinase. (PDF 27 kb)

Supplementary Table 4

Ramachandran statistics for EGFR and c-Src-cys complex structures. (PDF 28 kb)

Supplementary Methods (PDF 366 kb)

Supplementary Note (PDF 48 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).

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