Broad-Spectrum Kinase Profiling in Live Cells with Lysine-Targeted Sulfonyl Fluoride Probes

Protein
kinases comprise a large family of structurally related
enzymes. A major goal in kinase-inhibitor development is to selectively
engage the desired kinase while avoiding myriad off-target kinases.
However, quantifying inhibitor interactions with multiple endogenous
kinases in live cells remains an unmet challenge. Here, we report
the design of sulfonyl fluoride probes that covalently label a broad
swath of the intracellular kinome with high efficiency. Protein crystallography
and mass spectrometry confirmed a chemoselective reaction between
the sulfonyl fluoride and a conserved lysine in the ATP binding site.
Optimized probe 2 (XO44) covalently modified up to 133
endogenous kinases, efficiently competing with high intracellular
concentrations of ATP. We employed probe 2 and label-free
mass spectrometry to quantify intracellular kinase engagement by the
approved drug, dasatinib. The data revealed saturable dasatinib binding
to a small subset of kinase targets at clinically relevant concentrations,
highlighting the utility of lysine-targeted sulfonyl fluoride probes
in demanding chemoproteomic applications.