Affinity Profiling of the Cellular Kinome for the Nucleotide Cofactors ATP, ADP, and GTP

Most kinase inhibitor drugs target the binding site of
the nucleotide
cosubstrate ATP. The high intracellular concentration of ATP can strongly
affect inhibitor potency and selectivity depending on the affinity
of the target kinase for ATP. Here we used a defined chemoproteomics
system based on competition-binding assays in cell extracts from Jurkat
and SK-MEL-28 cells with immobilized ATP mimetics (kinobeads). This
system enabled us to assess the affinities of more than 200 kinases
for the cellular nucleotide cofactors ATP, ADP, and GTP and the effects
of the divalent metal ions Mg2+ and Mn2+. The
affinity values determined in this system were largely consistent
across the two cell lines, indicating no major dependence on kinase
expression levels. Kinase-ATP affinities range from low micromolar
to millimolar, which has profound consequences for the prediction
of cellular effects from inhibitor selectivity profiles. Only a small
number of kinases including CK2, MEK, and BRAF exhibited affinity
for GTP. This extensive and consistent data set of kinase-nucleotide
affinities, determined for native enzymes under defined experimental
conditions, will represent a useful resource for kinase drug discovery.