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A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors

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Abstract

Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target–independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.

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Figure 1: Characterization of the proteome reactivities of covalent kinase inhibitors in human cancer cells as determined by gel-based ABPP.
Figure 2: Characterization of the proteomic reactivities of covalent kinase inhibitors in cancer cells by ABPP-SILAC.
Figure 3: Analysis of protein targets of covalent kinase inhibitors.
Figure 4: Minimizing the off-target reactivity of covalent kinase inhibitors by modification of the Michael acceptor reactive group.
Figure 5: The in situ proteomic reactivity of covalent kinase inhibitors is altered by modification of the Michael acceptor.
Figure 6: Cytotoxicity and proteomic reactivity of DMAM-modified EGFR inhibitors.

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Acknowledgements

We thank G. Simon for assistance with proteomic data analysis. This work was supported by the National Institutes of Health (CA087660), Pfizer and the Skaggs Institute for Chemical Biology.

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Author notes

  1. Bryan R Lanning and Landon R Whitby: These authors contributed equally to this work.

Authors and Affiliations

  1. The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA

    Bryan R Lanning, Landon R Whitby, Melissa M Dix, Chris Joslyn, Chu Wang, Jonathan J Hulce & Benjamin F Cravatt

  2. Pfizer Worldwide Research and Development, Cambridge, Massachusetts, USA

    John Douhan, Erik C Hett, Lee R Roberts & Mark E Schnute

  3. Pfizer Worldwide Research and Development, Groton, Connecticut, USA

    Adam M Gilbert, Laurence O Whiteley & Matthew M Hayward

  4. Pfizer Worldwide Research and Development, San Diego, California, USA

    Theodore O Johnson, John C Kath & Sherry Niessen

  5. Pfizer Worldwide Research and Development, Andover, Massachusetts, USA

    Baoxian Wei

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Contributions

B.R.L., L.R.W., M.M.D. and B.F.C. designed the experiments; B.R.L., L.R.W. and M.M.D. performed the experiments and analyzed data; C.J. assisted with experiments; C.W. and J.J.H. assisted with data analysis; J.D., A.M.G., E.C.H., T.O.J., J.C.K., S.N., L.R.R., M.E.S., B.W., L.O.W. and M.M.H. provided intellectual input for probe design, experimental strategy and interpretation of proteomic data. M.M.H. and B.F.C. provided inter-institutional leadership and coordination; and B.R.L., L.R.W., M.M.H. and B.F.C. wrote the manuscript.

Corresponding authors

Correspondence to Matthew M Hayward or Benjamin F Cravatt.

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Competing interests

The authors declare competing financial interests. J.D., A.M.G., E.C.H., T.O.J., J.C.K., S.N., L.R.R., M.E.S., B.W., L.O.W. and M.M.H. are employees of Pfizer. The research was partially funded by Pfizer.

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Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3, Supplementary Figures 1–12 and Supplementary Note. (PDF 29097 kb)

Supplementary Table 3

Full proteomic data sets for SILAC-ABPP studies (XLSX 15605 kb)

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Lanning, B., Whitby, L., Dix, M. et al. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. Nat Chem Biol 10, 760–767 (2014). https://doi.org/10.1038/nchembio.1582

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