HIGH-THROUGHPUT ASSAY FOR INHIBITORS OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR-ASSOCIATED TYROSINE KINASE

We have developed a colorimetric assay for the examination of inhibito rs of epidermal growth factor (EGF) receptor-associated tyrosine kinas e in intact cells. EGF receptor from cells treated with inhibitors is captured by an anti-EGF receptor antibody and the phosphotyrosine cont ent is measured by an anti-phosphotyrosine antibody. The quantitative assay does not use radioactive substances and is configured for a high -throughput format. Since it is performed in intact cells, substances lower the phosphotyrosine content on the receptor by different mechani sms will be identified. One distinct feature of the assay is that it u ses the natural substrate inside the cell as compared to others using artificial substrates in an unphysiological environment. This assay is easy to perform, is reproducible, and is compatible with many organic solvents and tissue culture media. Thus, it is useful for the discove ry of EGF receptor kinase inhibitors from natural products or syntheti c compounds and is particularly suitable for large-scale screening.