Development of a Real-Time, Continuous, Fluorescence Microplate Assay for Epidermal Growth Factor Receptor Tyrosine Kinase Activity

Development of a Real-Time, Continuous, Fluorescence Microplate Assay for Epidermal Growth Factor Receptor Tyrosine Kinase Activity

Erik Schaefer, Ph.D. and Bill Lu, Ph.D.

 AssayQuant Technologies, Inc., 260 Cedar Hill Street, Marlboro, MA 01752

Epidermal Growth Factor Receptor (EGFR) is a member of the HER (Human Epidermal growth factor Receptor) network of receptor tyrosine kinases (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5215954/).  It is a self-phosphorylating dimer that is one of the most commonly found modified oncogenes in solid tumors (https://pubmed.ncbi.nlm.nih.gov/22785351/).  This family of ligand-binding, activatable, receptor-based enzymes are active targets for drug discovery (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3359794/), particularly in combination with antibody therapies (https://pubmed.ncbi.nlm.nih.gov/15289342/). Yet, antibody-dependent cell-mediated cytotoxicity is a complex phenomenon and the efficacy of a particular kinase inhibitor, when combined with a particular monoclonal antibody, appears to be highly dependent on mechanism of action of the inhibitor (https://clincancerres.aacrjournals.org/content/27/3/807).  Therefore, tyrosine kinase activity assays that enable analysis of this mechanism of action, allowing profiling and ranking of lead candidates early in the discovery process, are likely to be more fruitful for developing such combined therapeutic  approaches than end-point inhibitor screens.  We describe here the development of a continuous, real-time, fluorescence-based assay for EGFR tyrosine kinase inhibitors, designed for use in combination with standard lab-automation equipment, which provides the key kinetic data necessary for ranking and dissecting the mechanism of action of lead candidates.  We found that we could readily distinguish covalent from reversible inhibitors based on residence time and that the method worked with wild type (natural) EGFR and five different mutants.  The method is highly reproducible and easy to use, identifying known inhibitors readily, along with elucidating their modes of action.