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.
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ABSTRACT: We report a capillary electrophoresis method in conjunction with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening of protein kinase inhibitors (PKIs) in natural extracts. Protein kinase A (PKA), substrate 5-carboxyfluorescein-labeled kemptide (CLK) and inhibitor H-89 were employed for the method development and validation. Enzymatic inhibition assay was performed with electrophoretically mediated microanalysis technique. Once the bioactivity of a natural extract was confirmed, an assay-guided isolation and structure elucidation using LC-MS/MS were accomplished to identify the compounds which are responsible for the observed bioactivity. Totally 33 natural extracts were screened with the method, and baicalin in the extract of Radix Scutellariae was identified to be a new PKI of PKA. This result demonstrated the practical applicability of our method in screening of PKIs from natural products.Journal of Chromatography A 02/2014; · 4.61 Impact Factor
Dataset: HTF paper 2007
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ABSTRACT: 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 to test this novel method. These enzymes have been identified as very promising targets to develop treatments against cancer and neurodegenerative diseases. The efficiency of drugs against these relevant biological targets has never been carried out by CE. For this proposal, the capillary was used as a nanoreactor in which four reactants (the enzyme, its two substrates and its potential inhibitor) were successively injected, mixed by using transverse diffusion of laminar flow profiles and incubated. The adenosine 5'-diphosphate (ADP) formed during the enzymatic reaction was detected by UV and quantified. The efficiency of the developed CE method was validated by determining the IC50 values of a wide variety of inhibitors covering a large domain of affinity toward kinases and containing representative and chemically divergent skeletons. Excellent agreement was found between the results obtained by CE and those reported in the literature when using conventional radiometric enzyme assays. Moreover, CE was successfully used to determine the inhibitory effect of several potential inhibitors that was not yet assessed by conventional methods and is crucial for structure activity relation studies. This novel CE method is simple, rapid, very economic (few tens of nanoliters per IC50) and eco-friendly since no radioactivity was required. It could be extended to high-throughput screening of kinase inhibitors, which is of great interest for biomedical and pharmaceutical research fields.Journal of Chromatography A 09/2013; · 4.61 Impact Factor