Optimized Chemical Proteomics Assay for Kinase Inhibitor Profiling

Solid supported probes
have proven to be an efficient tool for
chemical proteomics. The kinobeads technology features kinase inhibitors
covalently attached to Sepharose for affinity enrichment of kinomes
from cell or tissue lysates. This technology, combined with quantitative
mass spectrometry, is of particular interest for the profiling of
kinase inhibitors. It often leads to the identification of new targets
for medicinal chemistry campaigns where it allows a two-in-one binding
and selectivity assay. The assay can also uncover resistance mechanisms
and molecular sources of toxicity. Here we report on the optimization
of the kinobead assay resulting in the combination of five chemical
probes and four cell lines to cover half the human kinome in a single
assay (∼260 kinases). We show the utility and large-scale applicability
of the new version of kinobeads by reprofiling the small molecule
kinase inhibitors Alvocidib, Crizotinib, Dasatinib, Fasudil, Hydroxyfasudil,
Nilotinib, Ibrutinib, Imatinib, and Sunitinib.