| Assay Method Information | |
| | Biochemical EGFR Inhibition Assays |
| Description: | Inhibitory effects of the compounds of the disclosure were measured in biochemical assays that measure the phosphorylation activity of EGFR enzyme phosphorylates 2.5 micromolar 5-FAM-EEPLYWSFPAKKK-CONH2 peptide substrate (ProfilerPro Kinase Peptide Substrate 22, PerkinElmer, Part #760366) in the presence of adenosine-5′-triphosphate (ATP) and varying concentrations of the test compound in 100 mM 2-[4-(2-hydroxyethyl) piperazin-1-yl]ethanesulfonic acid (HEPES), pH 7.5, 10 mM MgCl2, 0.015% Brij-35, 1 mM dithiothreitol (DTT), 1.0% dimehylsulfoxide (DMSO). Assays were performed at 1.0 mM ATP or at ATP Km of the EGFR enzymes. Reactions proceeded until between 10% to 20% total peptides were phosphorylated at room temperature (25° C.) and were terminated with 35 mM 2,2′,2″,2″-(ethane-1,2-diyldinitrilo)tetraacetic acid (EDTA). Product was detected using the Caliper mobility shift detection method where the phosphorylated peptide (product) and substrate were electrophoretically separated and measured. Percent activity was plotted against log concentration of compound and points to generate an apparent IC50. The following enzyme forms of EGFR were examples that were used in these assays:EGFR WT (SignalChem, E10-112G)EGFR L858R (SignalChem, E10-122BG)EGFR (d746-750) (SignalChem, E10-122JG)EGFR L858R C797S (SignalChem, E10-122ZG)EGFR (d746-750) C797S (SignalChem, E10-122TG) |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |