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Targeting Bcr–Abl by combining allosteric with ATP-binding-site inhibitors

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.

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Figure 1: NMR spectroscopy provides evidence for GNF-2 binding to the C-terminal myristate pocket of Abl.
Figure 2: Crystal structure of GNF-2 bound to the Abl myristoyl pocket.
Figure 3: Location and cellular IC 50 of Bcr–Abl GNF-2 resistance mutations.
Figure 4: Cellular and enzymatic inhibition of wild-type and mutants by combination treatments.
Figure 5: Hydrogen-exchange mass spectrometry on binding of GNF-5 to Abl.
Figure 6: In vivo efficacy studies with GNF-5 on wild-type and T315I Bcr–Abl dependent proliferation in xenograft and bone-marrow transplantation models.

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Primary accessions

Protein Data Bank

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

    CAS  PubMed  Article  Google Scholar 

  2. Quintas-Cardama, A., Kantarjian, H. & Cortes, J. Flying under the radar: the new wave of BCR-ABL inhibitors. Nature Rev. Drug Discov. 6, 834–848 (2007)

    CAS  Article  Google Scholar 

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

    CAS  PubMed  Article  ADS  Google Scholar 

  4. 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)

    CAS  PubMed  Article  Google Scholar 

  5. 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)

    CAS  PubMed  Google Scholar 

  6. Bradeen, H. A. 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)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  8. Jahnke, W. & Widmer, H. Protein NMR in biomedical research. Cell. Mol. Life Sci. 61, 580–599 (2004)

    CAS  PubMed  Article  Google Scholar 

  9. Vajpai, N. 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)

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  12. Ray, A. 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)

    CAS  PubMed  Article  Google Scholar 

  13. von Bubnoff, N. 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)

    CAS  PubMed  Article  Google Scholar 

  14. 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)

    CAS  PubMed  Article  Google Scholar 

  15. Azam, M. 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)

    CAS  PubMed  Article  ADS  Google Scholar 

  16. 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)

    CAS  PubMed  Article  Google Scholar 

  17. 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)

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  20. Kornberg, A. & Pricer, W. E. Di- and triphosphopyridine nucleotide isocitric dehydrogenases in yeast. J. Biol. Chem. 189, 123–136 (1951)

    CAS  PubMed  Google Scholar 

  21. Choi, Y. 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)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  22. Wales, T. E. & Engen, J. R. Hydrogen exchange mass spectrometry for the analysis of protein dynamics. Mass Spectrom. Rev. 25, 158–170 (2006)

    CAS  PubMed  Article  ADS  Google Scholar 

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

    CAS  PubMed  Article  ADS  Google Scholar 

  24. van Etten, R. A. Disease progression in a murine model of bcr/abl leukemogenesis. Leuk. Lymphoma 11 (suppl. 1). 239–242 (1993)

    PubMed  Article  Google Scholar 

  25. Joensuu, H. 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)

    CAS  PubMed  Article  Google Scholar 

  26. 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)

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  ADS  Google Scholar 

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

    CAS  PubMed  Article  ADS  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. Burke, J. R. 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)

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

  35. Tokumitsu, H. 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)

    CAS  PubMed  Google Scholar 

  36. Lee, T. S. 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)

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. Oppi, C., Shore, S. K. & Reddy, E. P. 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)

    CAS  PubMed  Article  ADS  Google Scholar 

  40. Wales, T. E., Fadgen, K. E., Gerhardt, G. C. & Engen, J. R. High-speed and high-resolution UPLC separation at zero degrees Celsius. Anal. Chem. 80, 6815–6820 (2008)

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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

    CAS  PubMed  Article  Google Scholar 

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.

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

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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.

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Zhang, J., Adrián, F., Jahnke, W. et al. Targeting Bcr–Abl by combining allosteric with ATP-binding-site inhibitors. Nature 463, 501–506 (2010). https://doi.org/10.1038/nature08675

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