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Development of a High-Throughput Fluoroimmunoassay for Syk Kinase and Syk Kinase Inhibitors

Development of a High-Throughput Fluoroimmunoassay for Syk Kinase and Syk Kinase Inhibitors

Noriyuki Yamamoto, Haruki Hasegawa, Hitomi Seki, Karl Ziegelbauer, Takahiro Yasuda

Anal Biochem. 2003 Apr 15;315(2):256-61.

PMID: 12689835

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
IAR42411935	SYK, active, GST tagged human			Price

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