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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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.


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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.


  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


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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.

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    Supplementary Information

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

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