A Continuous Fluorescence Microplate Assay for Epidermal Growth Factor Receptor (EGFR) Wild-Type and Mutant Tyrosine Kinase Activity

As a member of the ErbB/HER protein family, the human epidermal growth factor receptor (EGFR) includes four structurally related receptor tyrosine kinase (RTK) enzymes that are activated by different ligands. The binding of EGF to the extracellular domain of the EGFR RTK results in autophosphorylation of the dimer, which activates a series of downstream signaling cascades that regulate multiple cellular functions. When deregulated, it is one of the most frequent causes of solid tumors². This family of ligand-binding, activatable, receptor-based enzymes are active targets for research and drug discovery³, particularly in combination with other small molecule drugs or antibody-based therapies⁴.

The efficacy of a particular kinase inhibitor, when combined with a particular monoclonal antibody or another small molecule drug, is highly dependent on the mechanism of the inhibitor. Therefore, tyrosine kinase activity assays enable the identification of diverse inhibitor properties, allowing profiling and ranking of lead candidates early in the discovery process. They are likely to be more fruitful for developing combined therapeutic approaches due to the careful characterization of structural variants during candidate optimization.

Below we describe our development of a continuous, real-time, fluorescence-based assay for EGFR tyrosine kinase inhibitors using PhosphoSens^® Technology to provide critical kinetic data necessary for the detailed characterization and ranking of lead candidates. Designed for use with a simple add-and-read workflow and standard fluorescence microplate reader, PhosphoSens Technology is both novel and non-disruptive, delivering a progress curve in every well. Our continuous assay format can readily distinguish covalent from reversible inhibitors based on residence time, following our Time-Dependent Inhibition (TDI) workflow. While being highly reproducible and easy to use, our methodology works with wild-type (natural) EGFR and a panel of clinically-relevant mutants. In addition to our continuous, kinetic format, we developed a partner end-point assay, PhosphoSens-Red, to allow for the structural optimization of lead candidates in high-throughput mode, even for highly auto-fluorescent compounds.

Key Takeaways

We developed a robust Sox-based fluorogenic substrate assay for the EGFR tyrosine kinase and relevant clinical mutants, that provides a continuous, real-time, one-step, format suitable for standard fluorescence microplate readers, in 96-well, 384-well or 1536-well mode.  This EGFR PhosphoSens®assay provides detailed kinetic information about inhibitor candidates, allowing rapid, in-depth analysis to rank and profile lead compounds.  In addition, the compatibility of the method with automation equipment makes it applicable for primary or secondary high-throughput screens, where both the continuous and Endpoint/Red (TRF) formats eliminate compound autofluorescence.  Using kinetic parameters to elucidate mechanism of action for new inhibitor candidates is enabling more rapid development of improved drugs that also  creates the opportunity for multi-drug and antibody-drug combinations to address the challenges seen in the clinic.