Inhibitors of protein tyrosine phosphorylation: preliminary assessment of activity by time-resolved fluorescence

Epidermal growth factor (EGF) receptor (ErbB1)-associated tyrosine kinase i nhibitors may act as potential chemotherapeutic agents. In order to assess the inhibitory activity of these compounds, we developed a simple and sensi tive assay based on time-resolved fluorescence. In this technique, crude ce ll lysates bearing the ErbB1 receptor were captured in microtitre plates im mobilized with monoclonal anti-ErbB1 antibody SG 565. Subsequently, the pho sphotyrosine content of the cell lysates was quantified by a europium-label led anti-phosphotyrosine antibody. Thus, genistein, a tyrosine kinase inhib itor, was capable of reducing by half the tyrosine phosphorylation caused b y the binding of EGF to A431 cells, whereas 6-carboxymethyl genistein did n ot inhibit protein tyrosine phosphorylation. This assay is simple to perfor m, does not use radioactive substrates, and can be useful for screening EGF receptor tyrosine kinase inhibitors from natural products or synthetic com pounds. Moreover, the assay has a high signal:noise ratio and is suitable f or large-scale screening. Copyright (C) 2000 John Wiley & Sons, Ltd.