Kinase Inhibitors

Volume 795 of the series Methods in Molecular Biology pp 119-134


Chemoproteomic Characterization of Protein Kinase Inhibitors Using Immobilized ATP

  • James S. DuncanAffiliated withDepartment of Biochemistry, Schulich School of Medicine & Dentistry, University of Western OntarioDepartment of Pharmacology, University of North Carolina, School of Medicine
  • , Timothy A. J. HaysteadAffiliated withDepartment of Pharmacology & Cancer Biology, Duke University Medical Centre
  • , David W. LitchfieldAffiliated withDepartment of Biochemistry, Schulich School of Medicine & Dentistry, University of Western Ontario Email author 

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Protein kinase inhibitors have emerged as indispensable tools for the elucidation of the biological functions of specific signal transduction pathways and as promising candidates for molecular-targeted therapy. However, because many protein kinase inhibitors are ATP-competitive inhibitors targeting the catalytic site of specific protein kinases, the large number of protein kinases that are encoded within eukaryotic genomes and the existence of many other cellular proteins that bind ATP result in the prospect of off-target effects for many of these compounds. Many of the potential off-target effects remain unrecognized because protein kinase inhibitors are often developed and tested primarily on the basis of in vitro assays using purified components. To overcome this limitation, we describe a systematic approach to characterize ATP-competitive protein kinase inhibitors employing ATP-sepharose to capture the purine-binding proteome from cell extracts. Protein kinase inhibitors can be used in competition experiments to prevent binding of specific cellular proteins to ATP-sepharose or to elute bound proteins from ATP-sepharose. Collectively, these strategies can enable validation of interactions between a specific protein kinase and an inhibitor in complex mixtures and can yield the identification of inhibitor targets.

Key words

Protein kinase inhibitors Chemoproteomics ATP-sepharose Protein kinase CK2 Mass spectrometry 2D Gel electrophoresis