EGFR Drug Discovery

Biochemical screening of EGFR wildtype and more than 50 mutants is performed with our gold-standard radiometric assay formats. The HotSpot assay, performed at our US site, and the ³³PanQinase assay, performed at our German site, both utilize ³³P-labeled ATP as a phosphate donor to measure the enzymatic activity of kinases phosphorylating peptide or protein substrates.

Biophysical assays such as SPR (Surface Plasmon Resonance) enable real-time kinetic studies of inhibitor-target interaction to provide information on on-and off-rate, residence time, and binding affinity. Thermal shift and Microscale Thermophoresis are preferred orthogonal methods for testing the binding affinity of compounds.

Reaction Biology offers three cell-based options for compound testing against EGFR activity.

1. Our Cellular Phosphorylation Assay format comprises a number of EGFR assays that allow testing of EGFR activity in the physiologic environment of intact cells. We provide an assay performed in A431 tumor cells enabling the testing of inhibitors on endogenous, constitutively overexpressed, EGFR protein.
   Our EGFR mutant assays, on the other hand, are performed in Rat1 fibroblasts that express the cellular domain of EGFR mutants fused to a transmembrane domain which causes constitutive EGFR autophosphorylation.
    
2. Our Kinase-dependent Transformation Assay is performed with interleukin 3-dependent pro-B-cells that express EGFR as the driver for their proliferation. This assay allows investigating the inhibition of EGFR signaling in a high-throughput format in the physiologic environment of intact cells.
    
3. Our Cell Proliferation Assay allows investigation of the potency of EGFR inhibitors on malignant cells that carry EGFR mutations such as HCC827 or PC9 for differential efficacy effects depending on the mutation status of EGFR.

Reaction Biology provides support for the entire EGFR Drug Discovery Process from Hit identification, Hit-to-Lead, Lead Optimization, and In Vivo Proof-of-Concept studies with the support of our medicinal chemistry, cardiac safety assessment, and in vitro DMPK partners.

In the poster below, we show a comparative analysis of approved EGFR inhibitors of four generations with respect to their biochemical and cellular potency against different EGFR mutants as well as their selectivity against the human kinome.

Download here.