2.6. Kinase Profiling Assay

The kinase inhibition profile of the compounds was determined by testing 96 protein kinases’ activity using a radiometric protein kinase assay (33 PanQinase^® Activity Assay, Proqinase, Germany). In brief, 10 µL of non-radioactive ATP solution (in H₂O), 25 µL of assay buffer/[γ-33 P]-ATP mixture, 5 µL of 0.1 μM the tested compound in 10% DMSO and 10 µL of enzyme/substrate were mixed in 96-well FlashPlatesTM (Perkin Elmer, Boston, MA, USA). The reaction was conducted at 30 °C for 60 min. To stop the reaction, 50 µL of 2% (v/v) H₃PO₄ was added into the protein kinase reaction cocktails. After which, with 200 µL 0.9% (w/v) NaCl twice, the incorporation of ³³ Pi (counting of “cpm”) was determined with a microplate scintillation counter (Wallac Microbeta, Boston, MA, USA). All protein kinase assays were performed using a BeckmanCoulter Biomek 2000/SL robotic system (Brea, CA, USA). The residual activity (in %) for each compound was normalized to 100% enzyme activity (untreated control) and background (negative control).