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


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.

Accession codes

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.


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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.

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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).

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