A Probe-Based Target Engagement Assay for Kinases in Live Cells

The efficacy and safety of kinase inhibitor drugs are largely influenced by their selectivity. Available profiling technologies are primarily based on overexpressed or endogenously expressed kinases in cell extracts. We compared kinase capture with the cell penetrant covalent probe XO44 to three derivatives and found that replacing the alkyne handle with a trans-cyclooctene group allowed the development of a more robust kinase capture and enrichment protocol. An intracellular chemoproteomics target profiling and engagement assay was devised by optimizing probe concentration and incubation time and using an isobaric mass tag-based strategy for relative quantification. Comparing intracellular kinase profiles of the marketed drug dasatinib and the tool compound dinaciclib with the lysate-based kinobeads assay revealed excellent agreement in rank-order of binding. Dinaciclib showed a systematic shift to higher IC₅₀s, suggesting that intracellular cosubstrate concentrations, cell penetration of the compound, as well as kinase localization and complexes in live cells influence target profiles. Further, we show that sepiapterin reductase SPR and multidrug resistance protein 1 ABCC1 are off-targets of kinase inhibitor scaffolds with potential implications on efficacy and safety.