Kinase inhibitors show great promise as a new class of therapeutics. Here we describe an efficient way to determine kinase inhibitor specificity by measuring binding of small molecules to the ATP site of kinases. We have profiled 20 kinase inhibitors, including 16 that are approved drugs or in clinical development, against a panel of 119 protein kinases. We find that specificity varies widely and is not strongly correlated with chemical structure or the identity of the intended target. Many novel interactions were identified, including tight binding of the p38 inhibitor BIRB-796 to an imatinib-resistant variant of the ABL kinase, and binding of imatinib to the SRC-family kinase LCK. We also show that mutations in the epidermal growth factor receptor (EGFR) found in gefitinib-responsive patients do not affect the binding affinity of gefitinib or erlotinib. Our results represent a systematic small molecule-protein interaction map for clinical compounds across a large number of related proteins.
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Dancey, J. & Sausville, E.A. Issues and progress with protein kinase inhibitors for cancer treatment. Nat. Rev. Drug Discov. 2, 296–313 (2003).
Cohen, P. Protein kinases–the major drug targets of the twenty-first century? Nat. Rev. Drug Discov. 1, 309–315 (2002).
Manning, G., Whyte, D.B., Martinez, R., Hunter, T. & Sudarsanam, S. The protein kinase complement of the human genome. Science 298, 1912–1934 (2002).
Davies, S.P., Reddy, H., Caivano, M. & Cohen, P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95–105 (2000).
Bain, J., McLauchlan, H., Elliott, M. & Cohen, P. The specificities of protein kinase inhibitors: an update. Biochem. J. 371, 199–204 (2003).
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).
Paez, J.G. et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science 304, 1497–1500 (2004).
Sche, P.P., McKenzie, K.M., White, J.D. & Austin, D.J. Display cloning: functional identification of natural product receptors using cDNA-phage display. Chem. Biol. 6, 707–716 (1999).
Kumar, S., Boehm, J. & Lee, J.C. p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat. Rev. Drug Discov. 2, 717–726 (2003).
Lee, J.C. et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372, 739–746 (1994).
Godl, K. et al. An efficient proteomics method to identify the cellular targets of protein kinase inhibitors. Proc. Natl. Acad. Sci. USA 100, 15434–15439 (2003).
Tong, L. et al. A highly specific inhibitor of human p38 MAP kinase binds in the ATP pocket. Nat. Struct. Biol. 4, 311–316 (1997).
Pargellis, C. et al. Inhibition of p38 MAP kinase by utilizing a novel allosteric binding site. Nat. Struct. Biol. 9, 268–272 (2002).
Fitzgerald, C.E. et al. Structural basis for p38alpha MAP kinase quinazolinone and pyridol-pyrimidine inhibitor specificity. Nat. Struct. Biol. 10, 764–769 (2003).
Gray, N.S. et al. Exploiting chemical libraries, structure, and genomics in the search for kinase inhibitors. Science 281, 533–538 (1998).
Meggio, F. et al. Different susceptibility of protein kinases to staurosporine inhibition. Kinetic studies and molecular bases for the resistance of protein kinase CK2. Eur. J. Biochem. 234, 317–322 (1995).
Bennett, B.L. et al. SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc. Natl. Acad. Sci. USA 98, 13681–13686 (2001).
Buchdunger, E., Matter, A. & Druker, B.J. Bcr-Abl inhibition as a modality of CML therapeutics. Biochim. Biophys. Acta 1551, M11–M18 (2001).
Wakeling, A.E. et al. ZD1839 (gefitinib): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy. Cancer Res. 62, 5749–5754 (2002).
Moyer, J.D. et al. Induction of apoptosis and cell cycle arrest by CP-358,774, an inhibitor of epidermal growth factor receptor tyrosine kinase. Cancer Res. 57, 4838–4848 (1997).
Allen, L.F., Lenehan, P.F., Eiseman, I.A., Elliott, W.L. & Fry, D.W. Potential benefits of the irreversible pan-erbB inhibitor, CI-1033, in the treatment of breast cancer. Semin. Oncol. 29, 11–21 (2002).
Rusnak, D.W. et al. The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol. Cancer Ther. 1, 85–94 (2001).
Torrance, C.J. et al. Combinatorial chemoprevention of intestinal neoplasia. Nat. Med. 6, 1024–1028 (2000).
Wedge, S.R. et al. ZD6474 inhibits vascular endothelial growth factor signaling, angiogenesis, and tumor growth following oral administration. Cancer Res. 62, 4645–4655 (2002).
Wood, J.M. et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res. 60, 2178–2189 (2000).
Kelly, L.M. et al. CT53518, a novel selective FLT3 antagonist for the treatment of acute myelogenous leukemia (AML). Cancer Cell 1, 421–432 (2002).
Vieth, M. et al. Kinomics-structural biology and chemogenomics of kinase inhibitors and targets. Biochim. Biophys. Acta 1697, 243–257 (2004).
Druker, B.J. Perspectives on the development of a molecularly targeted agent. Cancer Cell 1, 31–36 (2002).
Gorre, M.E. et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876–880 (2001).
von Bubnoff, N., Schneller, F., Peschel, C. & Duyster, J. BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: a prospective study. Lancet 359, 487–491 (2002).
Shah, N.P. et al. Multiple BCR-ABL kinase domain mutations confer polyclonal resistance to the tyrosine kinase inhibitor imatinib (STI571) in chronic phase and blast crisis chronic myeloid leukemia. Cancer Cell 2, 117–125 (2002).
Gambacorti-Passerini, C.B. et al. Molecular mechanisms of resistance to imatinib in Philadelphia-chromosome-positive leukaemias. Lancet Oncol. 4, 75–85 (2003).
Shah, N.P. et al. Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 305, 399–401 (2004).
Branford, S. et al. High frequency of point mutations clustered within the adenosine triphosphate-binding region of BCR/ABL in patients with chronic myeloid leukemia or Ph-positive acute lymphoblastic leukemia who develop imatinib (STI571) resistance. Blood 99, 3472–3475 (2002).
Azam, M., Latek, R.R. & Daley, G.Q. Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 112, 831–843 (2003).
Roumiantsev, S. et al. Clinical resistance to the kinase inhibitor STI-571 in chronic myeloid leukemia by mutation of Tyr-253 in the Abl kinase domain P-loop. Proc. Natl. Acad. Sci. USA 99, 10700–10705 (2002).
Warmuth, M. et al. Dual-specific Src and Abl kinase inhibitors, PP1 and CGP76030, inhibit growth and survival of cells expressing imatinib mesylate-resistant Bcr-Abl kinases. Blood 101, 664–672 (2003).
La Rosee, P., Corbin, A.S., Stoffregen, E.P., Deininger, M.W. & Druker, B.J. Activity of the Bcr-Abl kinase inhibitor PD180970 against clinically relevant Bcr-Abl isoforms that cause resistance to imatinib mesylate (imatinib, STI571). Cancer Res. 62, 7149–7153 (2002).
Huron, D.R. et al. A novel pyridopyrimidine inhibitor of abl kinase is a picomolar inhibitor of Bcr-abl-driven K562 cells and is effective against STI571-resistant Bcr-abl mutants. Clin. Cancer Res. 9, 1267–1273 (2003).
Druker, B.J. et al. Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat. Med. 2, 561–566 (1996).
Nagar, B. et al. Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res. 62, 4236–4243 (2002).
Goldberg, D.R. et al. Optimization of 2-phenylaminoimidazo[4,5-h]isoquinolin-9-ones: orally active inhibitors of lck kinase. J. Med. Chem. 46, 1337–1349 (2003).
Mattiuzzi, G.N. et al. Development of Varicella-Zoster virus infection in patients with chronic myelogenous leukemia treated with imatinib mesylate. Clin. Cancer Res. 9, 976–980 (2003).
Dietz, A.B. et al. imatinib mesylate inhibits T-cell proliferation in vitro and delayed-type hypersensitivity in vivo. Blood 104, 1094–1099 (2004).
Ma, Y. et al. The c-KIT mutation causing human mastocytosis is resistant to STI571 and other KIT kinase inhibitors; kinases with enzymatic site mutations show different inhibitor sensitivity profiles than wild-type kinases and those with regulatory-type mutations. Blood 99, 1741–1744 (2002).
Lynch, T.J. et al. Novel agents in the treatment of lung cancer: conference summary statement. Clin. Cancer Res. 10, 4199s–4204s (2004).
Pao, W. et al. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc. Natl. Acad. Sci. USA 101, 13306–13311 (2004).
We thank Tony Hunter, Nicholas Lydon and Webster Cavenee for a critical reading of the manuscript and helpful discussions, Dan Lockhart for writing software tools to facilitate data analysis, David Austin for helpful suggestions regarding compound synthesis and Nicholas Olney and Victor Perez for expert technical assistance.
All authors are current or former employees of Ambit Biosciences.
Kinase inhibitors for which specificity profiles were determined (PDF 32 kb)
Comparison of binding constants measured in the competition binding assays to published results. (PDF 19 kb)
Comparison of the results of cell-based assays and binding assays for FLT3 and EGFR inhibitors. (PDF 9 kb)
Complete quantitative results of screening twenty kinase inhibitors against 119 protein kinases. (PDF 28 kb)
Binding constants for eight small molecules binding to ten EGFR variants. Binding constant values are in nanomolar. (PDF 10 kb)
List of clone type used for each kinase assay. Domain clones include the complete kinase catalytic domain along with flanking sequences. (PDF 18 kb)
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