Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

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

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Cohen, P. Protein kinases–the major drug targets of the twenty-first century? Nat. Rev. Drug Discov. 1, 309–315 (2002).

    Article  CAS  Google Scholar 

  2. Knight, Z.A. & Shokat, K.M. Features of selective kinase inhibitors. Chem. Biol. 12, 621–637 (2005).

    Article  CAS  Google Scholar 

  3. Kung, C., Kenski, D.M., Krukenberg, K., Madhani, H.D. & Shokat, K.M. Selective kinase inhibition by exploiting differential pathway sensitivity. Chem. Biol. 13, 399–407 (2006).

    Article  CAS  Google Scholar 

  4. Ventura, J.J. et al. Chemical genetic analysis of the time course of signal transduction by JNK. Mol. Cell 21, 701–710 (2006).

    Article  CAS  Google Scholar 

  5. Evans, M.J. & Cravatt, B.F. Mechanism-based profiling of enzyme families. Chem. Rev. 106, 3279–3301 (2006).

    Article  CAS  Google Scholar 

  6. Shreder, K.R., Wong, M.S., Nomanbhoy, T., Leventhal, P.S. & Fuller, S.R. Synthesis of AX7593, a quinazoline-derived photoaffinity probe for EGFR. Org. Lett. 6, 3715–3718 (2004).

    Article  CAS  Google Scholar 

  7. Yee, M.C., Fas, S.C., Stohlmeyer, M.M., Wandless, T.J. & Cimprich, K.A. A cell-permeable, activity-based probe for protein and lipid kinases. J. Biol. Chem. 280, 29053–29059 (2005).

    Article  CAS  Google Scholar 

  8. Yuan, H. et al. Synthesis and activity of C11-modified wortmannin probes for PI3 kinase. Bioconjug. Chem. 16, 669–675 (2005).

    Article  CAS  Google Scholar 

  9. Liu, Y. et al. Wortmannin, a widely used phosphoinositide 3-kinase inhibitor, also potently inhibits mammalian polo-like kinase. Chem. Biol. 12, 99–107 (2005).

    Article  CAS  Google Scholar 

  10. Liu, Y. et al. Structural basis for selective inhibition of Src family kinases by PP1. Chem. Biol. 6, 671–678 (1999).

    Article  CAS  Google Scholar 

  11. Buzko, O. & Shokat, K.M. A kinase sequence database: sequence alignments and family assignment. Bioinformatics 18, 1274–1275 (2002).

    Article  CAS  Google Scholar 

  12. Bishop, A.C. et al. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature 407, 395–401 (2000).

    Article  CAS  Google Scholar 

  13. Fry, D.W. et al. Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc. Natl. Acad. Sci. USA 95, 12022–12027 (1998).

    Article  CAS  Google Scholar 

  14. Singh, J. et al. Structure-based design of a potent, selective, and irreversible inhibitor of the catalytic domain of the erbB receptor subfamily of protein tyrosine kinases. J. Med. Chem. 40, 1130–1135 (1997).

    Article  CAS  Google Scholar 

  15. Cohen, M.S., Zhang, C., Shokat, K.M. & Taunton, J. Structural bioinformatics-based design of selective, irreversible kinase inhibitors. Science 308, 1318–1321 (2005).

    Article  CAS  Google Scholar 

  16. Stamos, J., Sliwkowski, M.X. & Eigenbrot, C. Structure of the epidermal growth factor receptor kinase domain alone and in complex with a 4-anilinoquinazoline inhibitor. J. Biol. Chem. 277, 46265–46272 (2002).

    Article  CAS  Google Scholar 

  17. Fan, Q.W., Zhang, C., Shokat, K.M. & Weiss, W.A. Chemical genetic blockade of transformation reveals dependence on aberrant oncogenic signaling. Curr. Biol. 12, 1386–1394 (2002).

    Article  CAS  Google Scholar 

  18. Tsou, H.R. et al. 6-Substituted-4-(3-bromophenylamino)quinazolines as putative irreversible inhibitors of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER-2) tyrosine kinases with enhanced antitumor activity. J. Med. Chem. 44, 2719–2734 (2001).

    Article  CAS  Google Scholar 

  19. Seeliger, M.A. et al. High yield bacterial expression of active c-Abl and c-Src tyrosine kinases. Protein Sci. 14, 3135–3139 (2005).

    Article  CAS  Google Scholar 

  20. Hennequin, L.F. et al. N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5- (tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine, a novel, highly selective, orally available, dual-specific c-Src/Abl kinase inhibitor. J. Med. Chem. 49, 6465–6488 (2006).

    Article  CAS  Google Scholar 

  21. Yun, C.-H. et al. Structures of lung cancer-derived EGFR mutants and inhibitor complexes: mechanism of activation and insights into differential inhibitor sensitivity. Cancer Cell (in the press).

  22. Heron, N.M. et al. SAR and inhibitor complex structure determination of a novel class of potent and specific Aurora kinase inhibitors. Bioorg. Med. Chem. Lett. 16, 1320–1323 (2006).

    Article  CAS  Google Scholar 

  23. Shewchuk, L. et al. Binding mode of the 4-anilinoquinazoline class of protein kinase inhibitor: X-ray crystallographic studies of 4-anilinoquinazolines bound to cyclin-dependent kinase 2 and p38 kinase. J. Med. Chem. 43, 133–138 (2000).

    Article  CAS  Google Scholar 

  24. Wood, E.R. et al. A unique structure for epidermal growth factor receptor bound to GW572016 (lapatinib): relationships among protein conformation, inhibitor off-rate, and receptor activity in tumor cells. Cancer Res. 64, 6652–6659 (2004).

    Article  CAS  Google Scholar 

  25. Fry, D.W. Mechanism of action of erbB tyrosine kinase inhibitors. Exp. Cell Res. 284, 131–139 (2003).

    Article  CAS  Google Scholar 

  26. Breitenlechner, C.B. et al. Crystal structures of active SRC kinase domain complexes. J. Mol. Biol. 353, 222–231 (2005).

    Article  CAS  Google Scholar 

  27. Cowan-Jacob, S.W. et al. The crystal structure of a c-Src complex in an active conformation suggests possible steps in c-Src activation. Structure 13, 861–871 (2005).

    Article  CAS  Google Scholar 

  28. Xu, W., Harrison, S.C. & Eck, M.J. Three-dimensional structure of the tyrosine kinase c-Src. Nature 385, 595–602 (1997).

    Article  CAS  Google Scholar 

  29. Leftheris, K. et al. The discovery of orally active triaminotriazine aniline amides as inhibitors of p38 MAP kinase. J. Med. Chem. 47, 6283–6291 (2004).

    Article  CAS  Google Scholar 

  30. Fry, D.W. et al. A specific inhibitor of the epidermal growth factor receptor tyrosine kinase. Science 265, 1093–1095 (1994).

    Article  CAS  Google Scholar 

  31. Pollack, V.A. et al. Inhibition of epidermal growth factor receptor-associated tyrosine phosphorylation in human carcinomas with CP-358,774: dynamics of receptor inhibition in situ and antitumor effects in athymic mice. J. Pharmacol. Exp. Ther. 291, 739–748 (1999).

    CAS  PubMed  Google Scholar 

  32. Wakeling, A.E. et al. ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res. 62, 5749–5754 (2002).

    CAS  PubMed  Google Scholar 

  33. Fan, Q.W. et al. Combinatorial efficacy achieved through two-point blockade within a signaling pathway-a chemical genetic approach. Cancer Res. 63, 8930–8938 (2003).

    CAS  PubMed  Google Scholar 

  34. Smaill, J.B. et al. Tyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2-d]pyrimidine-6-acrylamides bearing additional solubilizing functions. J. Med. Chem. 43, 1380–1397 (2000).

    Article  CAS  Google Scholar 

  35. Rasmussen, S.G. et al. Biophysical characterization of the cocaine binding pocket in the serotonin transporter using a fluorescent cocaine analogue as a molecular reporter. J. Biol. Chem. 276, 4717–4723 (2001).

    Article  CAS  Google Scholar 

  36. Uchiyama, S., Santa, T., Okiyama, N., Fukushima, T. & Imai, K. Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton. Biomed. Chromatogr. 15, 295–318 (2001).

    Article  CAS  Google Scholar 

  37. Jorissen, R.N. et al. Epidermal growth factor receptor: mechanisms of activation and signalling. Exp. Cell Res. 284, 31–53 (2003).

    Article  CAS  Google Scholar 

  38. Witucki, L.A. et al. Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-acceptor specificity of the wild-type enzyme. Chem. Biol. 9, 25–33 (2002).

    Article  CAS  Google Scholar 

  39. Wissner, A. et al. 4-Anilino-6,7-dialkoxyquinoline-3-carbonitrile inhibitors of epidermal growth factor receptor kinase and their bioisosteric relationship to the 4-anilino-6,7-dialkoxyquinazoline inhibitors. J. Med. Chem. 43, 3244–3256 (2000).

    Article  CAS  Google Scholar 

  40. Lynch, T.J. et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 350, 2129–2139 (2004).

    Article  CAS  Google Scholar 

  41. Paez, J.G. et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304, 1497–1500 (2004).

    Article  CAS  Google Scholar 

  42. Kobayashi, S. et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N. Engl. J. Med. 352, 786–792 (2005).

    Article  CAS  Google Scholar 

  43. Pao, W. et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2, e73 (2005).

    Article  Google Scholar 

  44. Engelman, J.A. et al. Allelic dilution obscures detection of a biologically significant resistance mutation in EGFR-amplified lung cancer. J. Clin. Invest. 116, 2695–2706 (2006).

    Article  CAS  Google Scholar 

  45. Inukai, M. et al. Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer. Cancer Res. 66, 7854–7858 (2006).

    Article  CAS  Google Scholar 

  46. Carter, T.A. et al. Inhibition of drug-resistant mutants of ABL, KIT, and EGF receptor kinases. Proc. Natl. Acad. Sci. USA 102, 11011–11016 (2005).

    Article  CAS  Google Scholar 

  47. Kobayashi, S. et al. An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor. Cancer Res. 65, 7096–7101 (2005).

    Article  CAS  Google Scholar 

  48. Kwak, E.L. et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc. Natl. Acad. Sci. USA 102, 7665–7670 (2005).

    Article  CAS  Google Scholar 

  49. Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  50. DeLano, W.L. The PyMOL Molecular Graphics System (DeLano Scientific, San Carlos, California, USA, 2002).

    Google Scholar 

Download references

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

Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Kevan M Shokat.

Ethics declarations

Competing interests

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio866

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing