In Vitro Pim Kinase Activity Assay

The inhibition ratios of test compounds for Pim-1, 2 (Life Technologies; Thermo Fisher Scientific Inc., MA, United States), 3 (Merck KgaA), and FLT3 (Life Technologies; Thermo Fisher Scientific Inc., MA, United States) were determined using a modified FRET-based Z′-LYTE kinase assay kit (Invitrogen; Thermo Fisher Scientific Inc.). The Ser/Thr 7 peptide and Tyr-2 peptide were used as substrates for Pim kinases and FLT3, respectively. The reactions were carried out in a 384-well plate with a 10 µL reaction volume per well containing 1 µM substrates in 50 mM HEPES buffer with pH 7.5, 0.01% BRIJ-35, 10 mM MgCl₂, 1 mM EGTA, and an appropriate amount of kinases (Pim-1: 0.03 μg/ml, Pim-2: 0.2 μg/ml, Pim-3: 0.2 μg/ml, FLT3: 0.1 μg/ml) with test compounds. The final reaction concentrations of ATP were 400, 5, 100, and 500 µM for Pim-1, 2, 3, and FLT3 respectively. After 1 h incubation, reactions were developed and terminated, and fluorescence ratios were calculated following the manufacturer’s protocol.