Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors.

Authors:
Noriyuki Yamamoto
Noriyuki Yamamoto
Research Center Kyoto
Japan
Haruki Hasegawa, PhD
Haruki Hasegawa, PhD
Amgen Inc.
Principal Scientist
Protein trafficking Protein biosynthesis
South San Francisco, CA | United States
Karl Ziegelbauer
Karl Ziegelbauer
and Institute of Cardiovascular Research
Germany
Takahiro Yasuda
Takahiro Yasuda
Center for Clinical Research

Anal Biochem 2003 Apr;315(2):256-61

Research Center Kyoto, Bayer Yakuhin, Ltd, 6-5-1-3, Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan.

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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April 2003
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