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

Area of Science:

• Biochemistry
• Proteomics
• Chemical Biology

Background:

• ATP-competitive kinase inhibitors are vital tools but developing selective ones is difficult due to conserved active sites across >500 human kinases.
• Efficient methods are needed to profile kinase inhibitor targets in cell lysates for discovering selective compounds and understanding polypharmacology.

Purpose of the Study:

• To describe a microgram-scale chemoproteomic profiling protocol for ATP-competitive kinase inhibitors using kinobeads.
• To enable rapid and sensitive profiling of kinase expression and inhibitor selectivity in native proteomes.

Main Methods:

• A gel-free in situ digestion protocol coupled with nanoflow liquid chromatography-mass spectrometry (LC-MS).
• Utilized kinobeads for chemoproteomic profiling, analyzing ~200 kinases in single runs with minimal sample input (5 μL kinobeads, 300 μg protein).
• Employed competition-binding experiments with label-free quantification for inhibitor profiling.

Main Results:

• Achieved broad kinome coverage, monitoring 312 kinases across 11 cancer cell lines.
• Successfully profiled pyrrolopyrimidine- and pyrazolopyrimidine-based kinase inhibitors.
• Discovered a novel, selective, and potent inhibitor of protein kinase D (PKD) 1, 2, and 3.

Conclusions:

• The developed kinobeads protocol offers a rapid and sensitive method for profiling kinase expression levels.
• This approach is effective for assessing ATP-competitive kinase inhibitor selectivity within native proteomes.
• Facilitates the discovery of selective kinase inhibitors and elucidation of their mechanisms of action.