Global Kinase Screening. Applications of Frontal Affinity Chromatography Coupled to Mass Spectrometry in Drug Discovery

• Published in: Analytical chemistry (Washington) 2005-03, Vol.77 (5), p.1268-1274
• Main Authors: Slon-Usakiewicz, Jacek J, Dai, Jin-Rui, Ng, William, Foster, J. Estelle, Deretey, Eugen, Toledo-Sherman, Leticia, Redden, Peter R, Pasternak, Andrew, Reid, Neil
• Format: Article
• Language: English
• Subjects: Alkaloids - chemistry; Alkaloids - metabolism; Analytical chemistry; Animals; Benzophenanthridines - chemistry; Benzophenanthridines - metabolism; Binding Sites; Binding, Competitive; Catalytic Domain; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, Affinity - methods; Drug Evaluation, Preclinical - methods; Drugs; Enzymes; Exact sciences and technology; Humans; Imidazoles - chemistry; Imidazoles - metabolism; Mass spectrometry; Mass Spectrometry - methods; Mice; Molecular Structure; Molecules; Other chromatographic methods; Peptides - chemistry; Peptides - metabolism; Phosphorylation; Protein Binding; Protein Kinase C-alpha - antagonists & inhibitors; Protein Kinase C-alpha - chemistry; Protein Kinase C-alpha - metabolism; Protein Kinase Inhibitors - chemistry; Protein Kinase Inhibitors - metabolism; Protein Kinase Inhibitors - pharmacology; Protein Kinases - chemistry; Protein Kinases - metabolism; Pyridines - chemistry; Pyridines - metabolism; Quinazolines - chemistry; Quinazolines - metabolism; Receptor, EphB2 - antagonists & inhibitors; Receptor, EphB2 - chemistry; Receptor, EphB2 - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Spectrometric and optical methods
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac048716q

Utilizing frontal affinity chromatography with mass spectrometry detection (FAC-MS), we have identified novel applications in the discovery of small-molecule hits to protein targets that are difficult if not impossible to accomplish using traditional assays. We demonstrate for the first time an ability to distinguish between competitive ligands for the ATP and substrate sites of protein kinase C independently in the same experiment and show that ATP competitive ligands using a functionally inactive receptor tyrosine kinase can be identified. This ability of FAC-MS to simultaneously monitor binding at the ATP and substrate binding sites, as well as measure ligand binding to both active and inactive kinases, suggests that FAC-MS can be used as a “global kinase binding assay”.