3.2. BTK Kinase Assay

To examine the enzyme activity, a kinase assay was performed using the ADP-Glo kinase assay kit (Promega, Madison, WI, USA) and the BTK enzyme system (Promega, Madison, WI, USA). Kinase reactions were performed using 10 μM ATP. The amount of the enzyme was determined as the concentration giving a signal-to-background ratio of 10 by titrating before using each enzyme batch. For 30 min, the inhibitor and enzyme in the kinase buffer were pre-incubated at room temperature. The ATP/substrate mixture was added and incubated for 30 min in a total kinase reaction volume of 10 μL at room temperature. The kinase reaction solution and ADP-Glo reagent were mixed in a 1:1 ratio (total 20 μL). After incubation for 30 min at room temperature, a kinase detection reagent (20 μL) was added for another 30 min. The luminescence intensity was read on a GloMax® Discover (Promega, Madison, WI, USA). For the single-dose screening enzyme inhibition assays, compounds were tested at 20 μM. Since the staurosporine completely inhibits BTK at 20 μM, it was used as the positive control and the relative percentage inhibition value was calculated compared to staurosporine as 100%. The IC₅₀ values were measured for a 7-dose, 10-fold serial dilution starting at 100 μM, and then calculated using four parameter logistic standard curve fitting using Sigmaplot software ver. 12.0.

In vitro profiling of BTK, BMX, EGFRK, ERBB2, ERBB4, ITK, JAK3, TEC, and TXK kinases was performed at Reaction Biology Corporation (Malvern, PA, USA) [18]. The kinase and substrate pairs were prepared in reaction buffer: 20 mM HEPES at pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.02% Brij 35, 0.02 mg/mL BSA, 0.1 mM Na₃VO₄, 2 mM DTT, and 1% DMSO. Compounds were added into the kinase reaction mixture and incubated for 20 min. Then, ³³P-ATP was delivered to the reaction mixture to initiate the reaction which was carried out at 10 μM total ATP. After 2 h at room temperature, the reaction mixture was spotted onto P81 ion exchange paper, and then kinase activity was detected by the filter-binding method. Compounds were tested for single-dose enzyme inhibition at a concentration of 20 μM. For the control compound, staurosporine was tested in a 10-dose IC₅₀ mode. To determine their IC₅₀ values, the compounds were tested at 10-concentrations with 3-fold serial dilution starting at 100 μM.