High throughput assay for inhibitors of the epidermal growth factor receptor-associated tyrosine kinase

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Epidermal Growth Factor (EGF) receptor tyrosine kinase plays a crucial role in cell signaling.
• Inhibitors of this kinase are important therapeutic targets for various diseases.
• Existing assays often use artificial substrates in non-physiological conditions.

Purpose of the Study:

• To develop a novel colorimetric assay for identifying inhibitors of EGF receptor-associated tyrosine kinase.
• To establish a high-throughput screening method using intact cells and natural substrates.
• To facilitate the discovery of novel kinase inhibitors from diverse compound libraries.

Main Methods:

• A colorimetric assay was developed utilizing intact cells.
• EGF receptor from treated cells was captured using an anti-EGF receptor antibody.
• Phosphotyrosine content was quantified using an anti-phosphotyrosine antibody.

Main Results:

• The assay is quantitative, does not require radioactive substances, and is suitable for high-throughput screening.
• It can identify inhibitors that lower phosphotyrosine content through various mechanisms within intact cells.
• The assay uses the natural substrate, offering a more physiologically relevant environment compared to artificial substrate assays.

Conclusions:

• This assay is a robust, reproducible, and easy-to-perform method for discovering EGF receptor kinase inhibitors.
• Its compatibility with organic solvents and tissue culture media makes it suitable for screening natural products and synthetic compounds.
• The assay is particularly valuable for large-scale screening initiatives in drug discovery.