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

Nature Chemical Biology volume 3, pages 229238 (2007) | Download Citation

Abstract

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.

  • Compound

    Wortmannin

  • Compound

    N-(4-(3-Bromophenylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(3-Ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine

  • Compound

    N-(4-(Phenylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-(4-tert-Butylphenylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-(4-Bromophenylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-((1,1'-Biphenyl)-4-ylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-(4-Isopropylphenylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-(Naphthalen-2-ylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-(Naphthalen-1-ylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-((Naphthalen-1-yl)methylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-(Phenethylamino)quinazolin-6-yl)acrylamide

  • Compound

    N-(4-(4-tert-Butylphenylamino)quinazolin-6-yl)propionamide

  • Compound

    N-(3-Chloro-4-fluoro-phenyl)-7-methoxy-6-(3-morpholin-4-ylpropoxy)quinazolin-4-amine

  • Compound

    AX7593

  • Compound

    N-(7-(2-(2-(2-(2-(4-Nitrobenzo[c][1,2,5]oxadiazol-7-ylamino)ethoxy)ethoxy)ethoxy)ethoxy)-4-(3-bromophenylamino)quinazolin-6-yl)acrylamide

  • Compound

    4-Chloro-7-nitro-2,1,3-benzoxadiazole

  • Compound

    N6-Benzyl-ADP

  • Compound

    N-(5-Chloro-1,3-benzodioxol-4-yl)-7-(2-(4-methylpiperazin-1-yl)ethoxy)-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine

  • Compound

    N-(3-Chloro-4-((3-fluorophenyl)methoxy)phenyl)-6-(5-((2-methylsulfonylethylamino)methyl)-2-furyl)quinazolin-4-amine

  • Compound

    N'-(2-Cyano-4-nitrophenyl)-N,N-dimethylformamidine

  • Compound

    N-(3-Bromophenyl)-6-nitroquinazolin-4-amine

  • Compound

    6-Nitro-N-phenylquinazolin-4-amine

  • Compound

    N-(4-tert-Butylphenyl)-6-nitroquinazolin-4-amine

  • Compound

    N-(4-Bromophenyl)-6-nitroquinazolin-4-amine

  • Compound

    N-((1,1'-Biphenyl)-4-yl)-6-nitroquinazolin-4-amine

  • Compound

    N-(4-Isopropylphenyl)-6-nitroquinazolin-4-amine

  • Compound

    N-(Naphthalen-2-yl)-6-nitroquinazolin-4-amine

  • Compound

    N-(Naphthalen-1-yl)-6-nitroquinazolin-4-amine

  • Compound

    N-((Naphthalen-1-yl)methyl)-6-nitroquinazolin-4-amine

  • Compound

    6-Nitro-N-phenethylquinazolin-4-amine

  • Compound

    N4-(3-Bromophenyl)quinazoline-4,6-diamine

  • Compound

    N4-Phenylquinazoline-4,6-diamine

  • Compound

    N4-(4-tert-Butylphenyl)quinazoline-4,6-diamine

  • Compound

    N4-(4-Bromophenyl)quinazoline-4,6-diamine

  • Compound

    N4-((1,1'-Biphenyl)-4-yl)-quinazoline-4,6-diamine

  • Compound

    N4-(4-Isopropylphenyl)quinazoline-4,6-diamine

  • Compound

    N4-(Naphthalen-2-yl)quinazoline-4,6-diamine

  • Compound

    N4-(Naphthalen-1-yl)quinazoline-4,6-diamine

  • Compound

    N4-((Naphthalen-1-yl)methyl)quinazoline-4,6-diamine

  • Compound

    N4-Phenethylquinazoline-4,6-diamine

  • Compound

    7-Fluoroquinazolin-4(3H)-one

  • Compound

    7-Fluoro-6-nitroquinazolin-4(3H)-one

  • Compound

    4-Chloro-7-fluoro-6-nitroquinazoline

  • Compound

    N-(3-Bromophenyl)-7-fluoro-6-nitroquinazolin-4-amine

  • Compound

    2-(2-(2-(2-(4-(3-Bromophenylamino)-6-nitroquinazolin-7-yloxy)ethoxy)ethoxy)ethoxy)ethanol

  • Compound

    2-(2-(2-(2-(4-(3-Bromophenylamino)-6-nitroquinazolin-7-yloxy)ethoxy)ethoxy)ethoxy)ethyl methanesulfonate

  • Compound

    7-(2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethoxy)-N-(3-bromophenyl)-6-nitroquinazolin-4-amine

  • Compound

    7-(2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethoxy)-N4-(3-bromophenyl)quinazoline-4,6-diamine

  • Compound

    7-(2-(2-(2-(2-(4-Nitrobenzo[c][1,2,5]oxadiazol-7-ylamino)ethoxy)ethoxy)ethoxy)ethoxy)-N4-(3-bromophenyl)quinazoline-4,6-diamine

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Acknowledgements

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

Author notes

    • Haridas Rode

    Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany. Present addresses: Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, D-44227 Dortmund, Germany (D.R.) and Novartis Institutes of Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA (C.K.).

Affiliations

  1. Department of Chemistry, University of California, Berkeley, Berkeley, California 94720, USA.

    • Jimmy A Blair
    •  & Kevan M Shokat
  2. Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 600 16th Street, MC 2280, San Francisco, California 94158, USA.

    • Jimmy A Blair
    • , Daniel Rauh
    • , Charles Kung
    • , Chao Zhang
    •  & Kevan M Shokat
  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Cai-Hong Yun
    •  & Michael J Eck
  4. Department of Cancer Biology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.

    • Cai-Hong Yun
    •  & Michael J Eck
  5. Departments of Neurology, Neurological Surgery and Pediatrics, and the Brain Tumor Research Center, University of California, San Francisco, San Francisco, California 94143, USA.

    • Qi-Wen Fan
    •  & William A Weiss

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Contributions

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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Kevan M Shokat.

Supplementary information

PDF files

  1. 1.

    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.

  2. 2.

    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.

  3. 3.

    Supplementary Table 1

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

  4. 4.

    Supplementary Table 2

    Data collection and refinement statistics for EGFR and c-Src-cys complex structures.

  5. 5.

    Supplementary Table 3

    Inhibition data of C4-derivatized PD 168393 analogs screened against EGFR kinase.

  6. 6.

    Supplementary Table 4

    Ramachandran statistics for EGFR and c-Src-cys complex structures.

  7. 7.

    Supplementary Methods

  8. 8.

    Supplementary Note

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DOI

https://doi.org/10.1038/nchembio866

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