title: High-throughput screening of kinase inhibitors by multiplex capillary electrophoresis with UV absorption detection.
abstract: Protein kinases play a major role in the transformation of cells and are often used as molecular targets for the new generation of anticancer drugs. We present a novel technique for high-throughput screening of inhibitors of protein kinases. The technique involves the use of multiplexed capillary electrophoresis (CE) for the rapid separation of the peptides, phosphopeptides, and various inhibitors. By means of UV detection, diversified peptides with native amino acid sequences and their phosphorylated counterparts can be directly analyzed without the need for radioactive or fluorescence labeling. The effects of different inhibitors and their IC(50) value were determined using three different situations involving the use of a single purified kinase, two purified kinases, and crude cell extracts, respectively. The results suggest that multiplexed CE/UV may prove to be a straightforward and general approach for high-throughput screening of compound libraries to find potent and selective inhibitors of the various protein kinases.
authors.affiliation: Ames Laboratory-USDOE and Department of Chemistry, Iowa State University, Ames, IA, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.forename: Edward S
authors.initials: ES
authors.lastname: Yeung
mesh_terms: D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000302:isolation & purification; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D047428:Protein Kinase Inhibitors; D013056:Spectrophotometry, Ultraviolet
chemical_list: D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D017868:Cyclic AMP-Dependent Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases