4.3.5. EGFR Kinase Inhibition

To determine the EGFR tyrosine kinase-inhibitory potency of 3BrQuin-SAHA and 3ClQuin-SAHA, a cell-free EGFR kinase assay from Promega was employed (#3831) [38]. The clinically approved EGFR tyrosine kinase-inhibitor gefitinib (1 µM) was used as a positive control. Kinase reactions were carried out with EGFR (5 ng/μL), ATP (5 μΜ), and substrates (0.2 μg/μL) in kinase reaction buffer using kinase enzyme systems (Promega, Madison, WI, USA). Before the kinase reaction was started, enzyme and inhibitors were incubated at room temperature (22–25 °C) for 0.5 h. All kinase reactions were performed in 384-well plates with a volume of 5 μL and an incubation time of 1 h. To deplete the remaining ATP, 5 μL of ADP-Glo reagent (ADP-Glo kinase assay kit; Promega) was added to each well at RT for 40 min. Finally, 10 μL of kinase detection solution was added into each well of the 384-well plate. Luminescence was measured with a VarioSkan Flash 40053 microplate luminometer (Thermo Fisher Scientific, Waltham, Mass., USA) for 1 s. Measurements were performed in triplicate. Data of n = 3 independent experiments are given as means ± SEM of the percentage decrease in EGFR-TK activity, as compared to untreated controls, whose activity was set to 100%.