Differential Kinobeads Profiling for Target Identification of Irreversible Kinase Inhibitors

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.