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Inhibitors of protein tyrosine phosphorylation: preliminary assessment of activity by time‐resolved fluorescence
Author(s) -
AmirZaltsman Yehudit,
Mazor Ohad,
Gayer Batya,
Scherz Avigdor,
Salomon Yoram,
Kohen Fortune
Publication year - 2000
Publication title -
luminescence
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.428
H-Index - 45
eISSN - 1522-7243
pISSN - 1522-7235
DOI - 10.1002/1522-7243(200011/12)15:6<377::aid-bio619>3.0.co;2-6
Subject(s) - tyrosine kinase , genistein , receptor tyrosine kinase , tyrosine phosphorylation , tyrosine kinase inhibitor , phosphorylation , tyrosine , chemistry , microbiology and biotechnology , platelet derived growth factor receptor , proto oncogene tyrosine protein kinase src , biochemistry , monoclonal antibody , protein tyrosine phosphatase , epidermal growth factor , sh2 domain , biology , signal transduction , receptor , antibody , growth factor , cancer , endocrinology , immunology , genetics
Epidermal growth factor (EGF) receptor (ErbB1)‐associated tyrosine kinase inhibitors may act as potential chemotherapeutic agents. In order to assess the inhibitory activity of these compounds, we developed a simple and sensitive assay based on time‐resolved fluorescence. In this technique, crude cell lysates bearing the ErbB1 receptor were captured in microtitre plates immobilized with monoclonal anti‐ErbB1 antibody SG 565. Subsequently, the phosphotyrosine content of the cell lysates was quantified by a europium‐labelled anti‐phosphotyrosine antibody. Thus, genistein, a tyrosine kinase inhibitor, was capable of reducing by half the tyrosine phosphorylation caused by the binding of EGF to A431 cells, whereas 6‐carboxymethyl genistein did not inhibit protein tyrosine phosphorylation. This assay is simple to perform, does not use radioactive substrates, and can be useful for screening EGF receptor tyrosine kinase inhibitors from natural products or synthetic compounds. Moreover, the assay has a high signal:noise ratio and is suitable for large‐scale screening. Copyright © 2000 John Wiley & Sons, Ltd.