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Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells

Abstract

The interaction between the HSP90 chaperone and its client kinases is sensitive to the conformational status of the kinase, and stabilization of the kinase fold by small molecules strongly decreases chaperone interaction. Here we exploit this observation and assay small-molecule binding to kinases in living cells, using chaperones as 'thermodynamic sensors'. The method allows determination of target specificities of both ATP-competitive and allosteric inhibitors in the kinases' native cellular context in high throughput. We profile target specificities of 30 diverse kinase inhibitors against >300 kinases. Demonstrating the value of the assay, we identify ETV6-NTRK3 as a target of the FDA-approved drug crizotinib (Xalkori). Crizotinib inhibits proliferation of ETV6-NTRK3-dependent tumor cells with nanomolar potency and induces the regression of established tumor xenografts in mice. Finally, we show that our approach is applicable to other chaperone and target classes by assaying HSP70/steroid hormone receptor and CDC37/kinase interactions, suggesting that chaperone interactions will have broad application in detecting drug-target interactions in vivo.

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Figure 1: Principle of the chaperone assay.
Figure 2: Characterization of ATP-competitive ABL inhibitor potencies with the chaperone interaction assay.
Figure 3: Characterizing allosteric ABL modulators with the chaperone interaction assay.
Figure 4: Expanding the scope of the chaperone interaction assay.
Figure 5: Profiling kinase inhibitor specificities with the chaperone interaction assay.
Figure 6: Crizotinib inhibits the ETV6-NTRK3 translocation fusion kinase.

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Acknowledgements

We thank the Lindquist laboratory members for valuable discussions and comments on the manuscript. We also thank M. Azam (Cincinnati Children's Hospital Medical Center) for providing mutant BCR-ABL clones and P. Thiru, I. Barrasa and G. Bell (Whitehead Institute) for help with primer design and statistical analysis. M.T. was supported by Human Frontier Science Programme long-term fellowship. S.L. is a Howard Hughes Medical Institute investigator. Support for this study was also provided by the US National Institutes of Health (NIH) Genomics Based Drug Discovery-Driving Medical Projects grant UL1-DE019585, administratively linked to NIH grants RL1-GM084437, RL1-CA133834 and RL1-HG004671.

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Contributions

M.T. and S.L. planned the project. M.T. designed the experiments, developed the assay, performed the experiments together with I.K., and analyzed the data. Xenograft experiments were done by L.W., and S.S. performed immunohistochemistry on tumor samples. J.Z. tested BCR-ABL variants for GNF-2 sensitivity in BaF/3 cells. J.Z., Q.L. and N.S.G. synthesized and provided kinase inhibitors and helped design experiments. M.T. and S.L. wrote the paper with input from all co-authors.

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Correspondence to Susan Lindquist.

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Competing interests

M.T. and S.L. are named inventors on a patent application on the technology described in this manuscript.

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Taipale, M., Krykbaeva, I., Whitesell, L. et al. Chaperones as thermodynamic sensors of drug-target interactions reveal kinase inhibitor specificities in living cells. Nat Biotechnol 31, 630–637 (2013). https://doi.org/10.1038/nbt.2620

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