Human protein kinase inhibitor screening by capillary electrophoresis using transverse diffusion of laminar flow profiles for reactant mixing.

摘要: A capillary electrophoresis (CE)-based enzyme assay method has been developed to screen protein kinase inhibitors. Four human kinases GSK3β, DYRK1A, CDK5/p25 and CDK1/cyclin B were chosen test this novel method. These enzymes have identified as very promising targets develop treatments against cancer neurodegenerative diseases. The efficiency of drugs these relevant biological never carried out by CE. For proposal, the was used a nanoreactor in which four reactants (the enzyme, its two substrates potential inhibitor) successively injected, mixed using transverse diffusion laminar flow profiles incubated. adenosine 5'-diphosphate (ADP) formed during enzymatic reaction detected UV quantified. CE validated determining IC50 values wide variety inhibitors covering large domain affinity toward containing representative chemically divergent skeletons. Excellent agreement found between results obtained those reported literature when conventional radiometric assays. Moreover, successfully determine inhibitory effect several that not yet assessed methods is crucial for structure activity relation studies. This simple, rapid, economic (few tens nanoliters per IC50) eco-friendly since no radioactivity required. It could be extended high-throughput screening inhibitors, great interest biomedical pharmaceutical research fields.