Development of a high-throughput fluoroimmunoassay for Syk kinase and Syk kinase inhibitors.
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.
Yamamoto
Noriyuki
N
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
Hasegawa
Haruki
H
Seki
Hitomi
H
Ziegelbauer
Karl
K
Yasuda
Takahiro
T
D002021:Buffers; D004791:Enzyme Inhibitors / Q000032:analysis*; D004792:Enzyme Precursors / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015200:Fluoroimmunoassay / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000072377:Syk Kinase; D013816:Thermodynamics
D002021:Buffers; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase