Affinity-Based Probes Based on Type II Kinase Inhibitors

Protein kinases are key components of most mammalian
signal transduction
networks and are therapeutically relevant drug targets. Efforts to
study protein kinase function would benefit from new technologies
that are able to profile kinases in complex proteomes. Here, we describe
active site-directed probes for profiling kinases in whole cell extracts
and live cells. These probes contain general ligands that stabilize
a specific inactive conformation of the ATP-binding sites of protein
kinases, as well as trifluoromethylphenyl diazirine and alkyne moieties
that allow covalent modification and enrichment of kinases, respectively.
A diverse group of serine/threonine and tyrosine kinases were identified
as specific targets of these probes in whole cell extracts. In addition,
a number of kinase targets were selectively labeled in live cells.
Our chemical proteomics approach should be valuable for interrogating
protein kinase active sites in physiologically relevant environments.