Abstract

In an effort to find new pharmacological modalities to overcome resistance to ATP-binding-site inhibitors of Bcr–Abl, we recently reported the discovery of GNF-2, a selective allosteric Bcr–Abl inhibitor. Here, using solution NMR, X-ray crystallography, mutagenesis and hydrogen exchange mass spectrometry, we show that GNF-2 binds to the myristate-binding site of Abl, leading to changes in the structural dynamics of the ATP-binding site. GNF-5, an analogue of GNF-2 with improved pharmacokinetic properties, when used in combination with the ATP-competitive inhibitors imatinib or nilotinib, suppressed the emergence of resistance mutations in vitro, displayed additive inhibitory activity in biochemical and cellular assays against T315I mutant human Bcr–Abl and displayed in vivo efficacy against this recalcitrant mutant in a murine bone-marrow transplantation model. These results show that therapeutically relevant inhibition of Bcr–Abl activity can be achieved with inhibitors that bind to the myristate-binding site and that combining allosteric and ATP-competitive inhibitors can overcome resistance to either agent alone.

  • Subscribe to Nature for full access:

    $199

    Subscribe

Additional access options:

Already a subscriber?  Log in  now or  Register  for online access.

Accessions

Primary accessions

Data deposits

The coordinates and structure factors of the complete Abl/imatinib/GNF-2 complex crystal structure are deposited in the Protein Data Bank under accession 3K5V.

References

  1. 1.

    et al. Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl. Cancer Cell 7, 129–141 (2005)

  2. 2.

    , & Flying under the radar: the new wave of BCR-ABL inhibitors. Nature Rev. Drug Discov. 6, 834–848 (2007)

  3. 3.

    et al. Overriding imatinib resistance with a novel ABL kinase inhibitor. Science 305, 399–401 (2004)

  4. 4.

    et al. Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification. Science 293, 876–880 (2001)

  5. 5.

    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)

  6. 6.

    et al. Comparison of imatinib mesylate, dasatinib (BMS-354825), and nilotinib (AMN107) in an N-ethyl-N-nitrosourea (ENU)-based mutagenesis screen: high efficacy of drug combinations. Blood 108, 2332–2338 (2006)

  7. 7.

    et al. Allosteric inhibitors of Bcr-abl-dependent cell proliferation. Nature Chem. Biol. 2, 95–102 (2006)

  8. 8.

    & Protein NMR in biomedical research. Cell. Mol. Life Sci. 61, 580–599 (2004)

  9. 9.

    et al. Solution conformations and dynamics of ABL kinase-inhibitor complexes determined by NMR substantiate the different binding modes of imatinib/nilotinib and dasatinib. J. Biol. Chem. 283, 18292–18302 (2008)

  10. 10.

    et al. Structural basis for the autoinhibition of c-Abl tyrosine kinase. Cell 112, 859–871 (2003)

  11. 11.

    et al. A myristoyl/phosphotyrosine switch regulates c-Abl. Cell 112, 845–857 (2003)

  12. 12.

    et al. Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study. Blood 109, 5011–5015 (2007)

  13. 13.

    et al. A cell-based screen for resistance of Bcr-Abl-positive leukemia identifies the mutation pattern for PD166326, an alternative Abl kinase inhibitor. Blood 105, 1652–1659 (2005)

  14. 14.

    , & Mechanisms of autoinhibition and STI-571/imatinib resistance revealed by mutagenesis of BCR-ABL. Cell 112, 831–843 (2003)

  15. 15.

    et al. Activity of dual SRC-ABL inhibitors highlights the role of BCR/ABL kinase dynamics in drug resistance. Proc. Natl Acad. Sci. USA 103, 9244–9249 (2006)

  16. 16.

    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)

  17. 17.

    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)

  18. 18.

    & Quantitative analysis of dose–effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv. Enzyme Regul. 22, 27–55 (1984)

  19. 19.

    et al. Resistance to imatinib of bcr/abl p190 lymphoblastic leukemia cells. Cancer Res. 66, 5387–5393 (2006)

  20. 20.

    & Di- and triphosphopyridine nucleotide isocitric dehydrogenases in yeast. J. Biol. Chem. 189, 123–136 (1951)

  21. 21.

    et al. N-myristoylated c-Abl tyrosine kinase localizes to the endoplasmic reticulum upon binding to an allosteric inhibitor. J. Biol. Chem. 284, 29005–29014 (2009)

  22. 22.

    & Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom. Rev. 25, 158–170 (2006)

  23. 23.

    et al. Conformational disturbance in Abl kinase upon mutation and deregulation. Proc. Natl Acad. Sci. USA 106, 1386–1391 (2009)

  24. 24.

    Disease progression in a murine model of bcr/abl leukemogenesis. Leuk. Lymphoma 11 (suppl. 1). 239–242 (1993)

  25. 25.

    et al. Effect of the tyrosine kinase inhibitor STI571 in a patient with a metastatic gastrointestinal stromal tumor. N. Engl. J. Med. 344, 1052–1056 (2001)

  26. 26.

    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)

  27. 27.

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

  28. 28.

    et al. Prediction of resistance to small molecule FLT3 inhibitors: implications for molecularly targeted therapy of acute leukemia. Cancer Res. 64, 6385–6389 (2004)

  29. 29.

    et al. Characterization of a conserved structural determinant controlling protein kinase sensitivity to selective inhibitors. Chem. Biol. 11, 691–701 (2004)

  30. 30.

    et al. A mammalian protein targeted by G1-arresting rapamycin-receptor complex. Nature 369, 756–758 (1994)

  31. 31.

    et al. A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl Acad. Sci. USA 92, 7686–7689 (1995)

  32. 32.

    et al. Identification and characterization of pleckstrin-homology-domain-dependent and isoenzyme-specific Akt inhibitors. Biochem. J. 385, 399–408 (2005)

  33. 33.

    et al. BMS-345541 is a highly selective inhibitor of IκB kinase that binds at an allosteric site of the enzyme and blocks NF-κB-dependent transcription in mice. J. Biol. Chem. 278, 1450–1456 (2003)

  34. 34.

    et al. Development of thioquinazolinones, allosteric Chk1 kinase inhibitors. Bioorg. Med. Chem. Lett. 19, 1240–1244 (2009)

  35. 35.

    et al. KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II. J. Biol. Chem. 265, 4315–4320 (1990)

  36. 36.

    et al. Molecular basis explanation for imatinib resistance of BCR-ABL due to T315I and P-loop mutations from molecular dynamics simulations. Cancer 112, 1744–1753 (2008)

  37. 37.

    et al. Efficient uniform isotope labeling of Abl kinase expressed in Baculovirus-infected insect cells. J. Biomol. NMR 31, 343–349 (2005)

  38. 38.

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

  39. 39.

    , & Nucleotide sequence of testis-derived c-abl cDNAs: implications for testis-specific transcription and abl oncogene activation. Proc. Natl Acad. Sci. USA 84, 8200–8204 (1987)

  40. 40.

    , , & High-speed and high-resolution UPLC separation at zero degrees Celsius. Anal. Chem. 80, 6815–6820 (2008)

  41. 41.

    , & Semi-automated data processing of hydrogen exchange mass spectra using HX-Express. J. Am. Soc. Mass Spectrom. 17, 1700–1703 (2006)

Download references

Acknowledgements

We thank C. Henry and G. Rummel for technical assistance; R. Beigi for help with the bone-marrow transplantation studies; A. Velentza for performing the DSC experiments; and J. Kuriyan, M. Seeliger, C. Yun, M. Eck, E. Weisberg, D. Fabbro, P. L. Yang, G. Superti-Furga and A. Kung for helpful discussions. We also acknowledge the support of staff at beamline PXII of the Swiss Light Source, Villigen, Switzerland, during X-ray data collection, the ICCB-Longwood Screening facility at Harvard Medical School for the cell proliferation and enzyme assay, and Barnet Institute for hydrogen-exchange experiments.

Author Contributions F.J.A., J.Z., J.P., Y.C., G.L., M.A. and G.D. designed and performed cellular and biochemical experiments. J.Z. performed bacterial Abl expression and enzyme assays. W.J., N.V. and S.G. designed and performed the NMR experiments. S.W.C.-J. designed and performed the crystallographic experiments. G.F. and A.S. produced the protein for the NMR and X-ray experiments. T.S., Q.D., B.O., A.W. and X.D. designed and synthesized the compounds. A.G.L., C.D., F.S., G.-R.G. and T.T. conducted the in vivo studies. Y.L. and B.B. contributed to the design of the compounds. R.E.I. and J.R.E. performed and designed the hydrogen-exchange experiments. M.W. contributed to the design of the in vivo experiments. F.J.A., M.W. and P.M. provided critical input to the overall research direction. N.S.G. directed the research and wrote the paper with input from all co-authors.

Author information

Author notes

    • Jianming Zhang
    •  & Francisco J. Adrián

    These authors contributed equally to this work.

Affiliations

  1. Dana-Farber Cancer Institute, Harvard Medical School, Department of Cancer Biology and Department of Biological Chemistry and Molecular Pharmacology, 250 Longwood Avenue, Seeley G. Mudd Building 628, Boston, Massachusetts 02115, USA

    • Jianming Zhang
    • , Taebo Sim
    • , Yongmun Choi
    • , Amy Wojciechowski
    • , Xianming Deng
    •  & Nathanael S. Gray
  2. Genomics Institute of the Novartis Research Foundation, Department of Chemistry, 10675 John Jay Hopkins Drive, San Diego, California 92121, USA

    • Francisco J. Adrián
    • , Allen G. Li
    • , Christine Dierks
    • , Fangxian Sun
    • , Gui-Rong Guo
    • , Qiang Ding
    • , Guoxun Liu
    • , Tove Tuntland
    • , Yi Liu
    •  & Badry Bursulaya
  3. Novartis Institutes for Biomedical Research, CH-4056 Basel, Switzerland

    • Wolfgang Jahnke
    • , Sandra W. Cowan-Jacob
    • , Gabriele Fendrich
    • , André Strauss
    •  & Paul W. Manley
  4. The Barnett Institute of Chemical & Biological Analysis and Department of Chemistry & Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA

    • Roxana E. Iacob
    •  & John R. Engen
  5. Life Sciences Research Division, Korea Institute of Science and Technology 39-1, Hawolgok-dong, Seongbuk-gu, Seoul, 136-791, Korea

    • Taebo Sim
  6. Division of Pediatric Hematology/Oncology, Children’s Hospital and Dana-Farber Cancer Institute; Division of Hematology, Brigham and Women’s Hospital; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School; Howard Hughes Medical Institute; Boston, Massachusetts 02115, USA

    • John Powers
    • , Mohammad Azam
    •  & George Q. Daley
  7. Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA

    • Barun Okram
  8. Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

    • Navratna Vajpai
    •  & Stephan Grzesiek
  9. Novartis Institutes for BioMedical Research, Inc., 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA

    • Markus Warmuth

Authors

  1. Search for Jianming Zhang in:

  2. Search for Francisco J. Adrián in:

  3. Search for Wolfgang Jahnke in:

  4. Search for Sandra W. Cowan-Jacob in:

  5. Search for Allen G. Li in:

  6. Search for Roxana E. Iacob in:

  7. Search for Taebo Sim in:

  8. Search for John Powers in:

  9. Search for Christine Dierks in:

  10. Search for Fangxian Sun in:

  11. Search for Gui-Rong Guo in:

  12. Search for Qiang Ding in:

  13. Search for Barun Okram in:

  14. Search for Yongmun Choi in:

  15. Search for Amy Wojciechowski in:

  16. Search for Xianming Deng in:

  17. Search for Guoxun Liu in:

  18. Search for Gabriele Fendrich in:

  19. Search for André Strauss in:

  20. Search for Navratna Vajpai in:

  21. Search for Stephan Grzesiek in:

  22. Search for Tove Tuntland in:

  23. Search for Yi Liu in:

  24. Search for Badry Bursulaya in:

  25. Search for Mohammad Azam in:

  26. Search for Paul W. Manley in:

  27. Search for John R. Engen in:

  28. Search for George Q. Daley in:

  29. Search for Markus Warmuth in:

  30. Search for Nathanael S. Gray in:

Competing interests

F.J.A., W.J., S.W.C.-J., A.G.L., F.S., G.-R.G., Q.D., B.O., G.L., G.F., T.T., B.B., P.W.M. and M.W. are employed by Novartis Pharmaceuticals or the Genomics Institute of the Novartis Research Foundation. N.G. received research funding for this project from Novartis Pharmaceuticals.

Corresponding authors

Correspondence to Francisco J. Adrián or Markus Warmuth or Nathanael S. Gray.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains Supplementary Methods, Supplementary Tables S1-S3, Supplementary Figures1-15 and Supplementary References.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature08675

Rights and permissions

To obtain permission to re-use content from this article visit RightsLink.

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.