4.5. In Vitro Assay for PAK1 Inhibition

PAK1 kinase activity was measured using the ADP-Glo™ kinase assay kit (Promega, Madison, WI, USA) according to manufacturer’s instructions. Briefly, human PAK1 (25 ng/reaction) was incubated with test compounds (5 μL) for 10 min. The kinase reaction was started by adding 2.5× adenosine triphosphate (ATP)/substrate mix (10 μL). The reaction was terminated by adding 25 μL ADP-Glo™ reagent, followed by an incubation time of 40 min. The kinase detection reagent (50 μL) was added to the reaction mixture, and after 30 min of incubation, the luminescence was recorded by MTP-880Lab microplate reader (Corona, Ibaraki, Japan) with an integration time of 0.5 s per well. The percentage inhibition was calculated relative to the control kinase activity without any inhibitor.