Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors |
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Authors: | Yamamoto Noriyuki Hasegawa Haruki Seki Hitomi Ziegelbauer Karl Yasuda Takahiro |
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Institution: | Research Center Kyoto, Bayer Yakuhin, Ltd, 6-5-1-3, Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan. noriyuki.yamamoto.ny@bayer.co.jp |
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Abstract: | Syk is a tyrosine kinase which is indispensable in immunoglobulin Fc receptor- and B cell receptor-mediated signal transduction in various immune cells. This pathway is important in the pathophysiology of allergy. In this study we established a quantitative nonradioactive kinase assay to identify inhibitors of Syk. We used recombinant GST-tagged Syk purified from baculovirus-infected insect cells. As a substrate, biotinylated peptide corresponding to the activation loop domain of Syk, whose tyrosine residues are autophosphorylated upon activation, was employed to screen both ATP- and substrate-competitive inhibitors. After the kinase reaction in solution phase, substrate was trapped on a streptavidin-coated plate, followed by detection of the phosphorylated tyrosine with europium-labeled anti-phosphotyrosine antibody. The kinase reaction in solution phase greatly enhanced phosphorylation of substrate compared to that of plate-coated substrate. High signal-to-background ratio and low data scattering were obtained in the optimized high-throughput screening (HTS) format. Further, several kinase inhibitors showed concentration-dependent inhibition of recombinant Syk kinase activity with almost the same efficacy for immunoprecipitated Syk from a human cell line. These data suggest that this assay is useful to screen Syk kinase inhibitors in HTS. |
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Keywords: | Syk Europium-based fluoroimmunoassay High-throughput screening Asthma |
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