EGFR-TK inhibitory activity

Kinase activity was determined using Kinase-Glo Plus luminescence kinase assay kit (Promega Corporation, Madison, WI, USA) by calculating the amount of adenosine triphosphate (ATP) remaining in the kinase reactionsolution. The luminescent signal is correlated with the residual amount present and it was inversely associated with kinase activity. The tested compounds were diluted to 100 mM in 10% DMSO, then 5 ml the dilution was added to a 50 ml reaction. All of the enzymatic reactions were performed at 30°C for 40 min using a 50 ml reaction volume containing 10 mM MgCl₂, 40 mM Tris, (pH 7.4), 0.1 mg/ml bovine serum albumin, 0.2 mg/ml poly (Glu, Tyr) substrate, 10 mM ATP and EGFR. The plate was incubated for 5 min at room temperature then 50 ml Kinase-GloPlus Luminescence kinase assay regent was added to each reaction. ADP-Glo assay kit (Promega Corporation) is a protein kinase assays used to determine IC50 values in which adenosine diphosphate (ADP) generation was measured which leads to an increase in the luminescence signal. The reaction mixture was incubated in a 96-well plate at 30°C for 30 min, following the incubation period 25 ml ADP-Glo reagent was added to terminate the assay. The 96-well plate agitated for 30 min at ambient temperature and incubated, then 50 ml kinase detection reagentwas added. The 96-well plate was read using the ADP-Glo Luminescence reader. All the assay components were added to the blank control except the substrate. The blank control value was used to correct the activity for each protein kinase target is determined.