Kinobead and Single-Shot LC-MS Profiling Identifies Selective PKD Inhibitors

ATP-competitive
protein kinase inhibitors are important research
tools and therapeutic agents. Because there are >500 human kinases
that contain highly conserved active sites, the development of selective
inhibitors is extremely challenging. Methods to rapidly and efficiently
profile kinase inhibitor targets in cell lysates are urgently needed
to discover selective compounds and to elucidate the mechanisms of
action for polypharmacological inhibitors. Here, we describe a protocol
for microgram-scale chemoproteomic profiling of ATP-competitive kinase
inhibitors using kinobeads. We employed a gel-free in situ digestion protocol coupled to nanoflow liquid chromatography–mass
spectrometry to profile ∼200 kinases in single analytical runs
using as little as 5 μL of kinobeads and 300 μg of protein.
With our kinobead reagents, we obtained broad coverage of the kinome,
monitoring the relative expression levels of 312 kinases in a diverse
panel of 11 cancer cell lines. Further, we profiled a set of pyrrolopyrimidine-
and pyrazolopyrimidine-based kinase inhibitors in competition-binding
experiments with label-free quantification, leading to the discovery
of a novel selective and potent inhibitor of protein kinase D (PKD)
1, 2, and 3. Our protocol is useful for rapid and sensitive profiling
of kinase expression levels and ATP-competitive kinase inhibitor selectivity
in native proteomes.