3.3.3. In Vitro EGFR Inhibitory Assay

The most potent analogs 3g, 4f, 4g, and 4h against MCF-7, HepG2 and HCT-116 cell lines were further tested for EGFR inhibitory activity using Enzy Chrom TM Kinase Assay Kit (EKIN-400) as previously described [60]. In addition, 20 µL reaction mixture containing the EGFR kinase, ATP, and substrate in the provided assay buffer. Set up a blank control that contains ATP and substrate but no enzyme. Incubate at desired temperature for 30 min. Prepare 900 µL 10µM (adenosine diphosphate) ADP premix by mixing 3 µL of 3 mM standard and 897 µL distilled water. Transfer 20 µL standards into separate wells of the plate. Prepare enough working reagent for each well. Add 40 µL working reagent to each assay well. Tap plate to mix. Incubate at room temperature for 10 min. Read fluorescence intensity at λ_exc = 530nm and λ_em = 590 nm, and then calculate kinase activity.