A probe-based target engagement assay for kinases in live cells

Protein kinases are an important class of drug targets due to their crucial roles in almost every aspect of cellular function, such as cell growth, metabolism, proliferation, differentiation, migration, effector functions and cell death. Starting from the previously described covalent promiscuous kinase-targeting scaffold XO44, we evaluated alterations of the kinase binding core and on the enrichment moiety for kinase promiscuity and capturing in lysate and in live cells. By exchanging the alkyne click handle to a trans-cyclooctene, the more efficient Diels-Alder-Click reaction enabled the specific and displaceable enrichment of 95 kinases and 31 non-kinases from live cells. This improved tool enables a chemoproteomics assay to probe dose-dependent intracellular target engagement for probe-captured kinases for the kinase inhibitors dasatinib and dinaciclib. Further, we demonstrate that sepiapterin reductase and the multidrug resistance protein ABCC1 can be captured by this probe, suggesting these proteins as off-targets of kinase inhibitor scaffolds.