cb7b00617_si_006.xlsx (1.39 MB)

Differential Kinobeads Profiling for Target Identification of Irreversible Kinase Inhibitors

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posted on 06.09.2017, 00:00 by Lars Dittus, Thilo Werner, Marcel Muelbaier, Marcus Bantscheff
Chemoproteomics profiling of kinase inhibitors with kinobeads enables the assessment of inhibitor potency and selectivity for endogenously expressed protein kinases in cell lines and tissues. Using a small panel of targeted covalent inhibitors, we demonstrate the importance of measuring covalent target binding in live cells. We present a differential kinobeads profiling strategy for covalent kinase inhibitors where a compound is added either to live cells or to a cell extract that enables the comprehensive assessment of inhibitor selectivity for covalent and noncovalent targets. We found that Acalabrutinib, CC-292, and Ibrutinib potently and covalently bind TEC family kinases, but only Ibrutinib also potently binds to BLK. ZAK was identified as a submicromolar affinity Ibrutinib off-target due to covalent modification of Cys22. In contrast to Ibrutinib, 5Z-7-Oxozeaenol reacted with Cys150 next to the DFG loop, demonstrating an alternative route to covalent inactivation of this kinase, e.g., to inhibit canonical TGF-β dependent processes.