Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity

Kinases
are involved in the regulation of many cellular processes
and aberrant kinase signaling has been implicated in human disease.
As a consequence, kinases are attractive drug targets. Assessing kinase
function and drug selectivity in a more physiological context is challenging
and often hampered by the generally low expression level of kinases
and the extensive post-translation modification in vivo. Kinase drug selectivity screens by chemical proteomics have gained
attention because they allow the profiling of hundreds of kinases
against one drug at the same time. Here, we directly compared two
such methods, notably, immobilized broad spectrum kinase inhibitors
(kinobeads) and active site labeling using desthiobiotin-ATP and -ADP
probes. Affinity purification of ∼100 kinases by either kinobeads
or ATP/ADP probes was readily achieved using 1 mg of cellular protein.
Bioinformatic analysis revealed a high degree of complementarity of
the two techniques. Kinobeads covered the Tyrosine Kinase family particularly
well and ATP probes enriched higher numbers of STE family kinases.
A consecutive combination of both enrichment strategies therefore
allowed for the coverage of a larger part of the kinome than any one
technique alone. While kinobeads are very selective for kinases, the
ATP/ADP probes also enriched a large number of other nucleotide binding
proteins. Both methods were applied to the selectivity profiling of
the small molecular Aurora kinase inhibitor tozasertib in K562 cells.
Our data confirmed Aurora A, B, and BCR-ABL as the main targets of
tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar
off targets.