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| | Potency Assessment EGFR (WT) and EGFR Mutants |
| Description: | The inhibition activity and the selectivity of the compounds against mutant EGFR and WT EGFR were assessed by using WT EGFR cell line A-431 (American Type Culture Collection (ATCC), CRL-1555) and mutant cell lines as described in Example 99.One day before the test, WT or mutant cells were seeded in 96 well plates at appropriate concentrations with corresponding growth media supplemented with 1% FBS, and incubated overnight. The next day, tested compounds at a series of concentrations were added into each individual well of the plates, and the plates were incubated for 4 h at 37° C. with 5% CO2. For WT EGFR cell line, an additional stimulation with 100 ng/ml recombinant hEGF (RD, Cat #236-EG) for 10 min should be performed after the incubation with the tested compound and before the analysis by MSD kits.The EGFR (Y1068) phosphorylation level (activity) of cells in each well were then measured with MSD kit named MULTI-SPOT 96 4-Spot HB Prototype EGFR Triplex ANALYTES: pEGFR(Tyr1068),pEGFR(Tyr1173), Total EGFR according to the manufacturer's instruction (MESO SCALE DISCOVERY, Cat #N45ZB-1). The assay is an electrochemiluminescent method for determining both phosphorylated and total EGFR of cells with an MSD SECTOR Imager and the ratio of p-EGFR/total EGFR can be generated by the machine. The Her2 (Y1248) phosphorylation level (activity) of cells in each well were measured with MSD Kit named Phospho-ErbB2 (Tyr1248) Assay Whole Cell Lysate Kit according to the manufacturer's instruction (MESO SCALE DISCOVERY, Cat #K151CLD-3). The assay is an electrochemiluminescent method (MESO SCALE DISCOVERY) for determining both phosphorylated and total Her2 of cells with an MSD SECTOR Imager and then the ratio of p-Her2/total Her2 can be generated by the machine. The percentage of inhibition was calculated based on the formula: % inhibition=100×[1−(ratio of sample well−ratio of Min ctrl well)/(ratio of Max−Ratio of Min ctrl well)]. The IC50 values were calculated as the compounds' concentration required for 50% inhibition in best-fit curves using Prism GraphPad 7.0 or Microsoft Xlfit software. |
| Affinity data for this assay | |
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