In vitro kinase assay

BTK kinase activity inhibition IC₅₀ was measured by PhosphoSens® Kinase Assay Kit (Assay Quant Technologies Inc., Cat.# CSKS-AQT0101K). This assay was performed in 384-well, white flat bottom polystyrene NBS microplates (Corning #3824) at room temperature. Active recombinant BTK was purchased from SignalChem (Cat.# B10-10H-10). All the drugs (3 warhead control and 4 PROTACs) were dissolved in DMSO (10 mM). Duplicate drug titrations (1000 nM, 200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM 0.64 nM, 0.128 nM, and 0.0256 nM) were used to generate each IC₅₀. Typical final concentrations of each reaction component are as follows: 2.5 nM BTK, and 10 μM PhosphoSens® Substrate, 54 mM HEPES, pH 7.5, 1 mM ATP, 1.2 mM DTT, 0.012% Brij-35, 10 mM MgCl₂, 1% glycerol, and 0.2 mg mL⁻¹ BSA. Incubate the mixture at room temperature for 15 min and collect the fluorescence intensity readings (Ex 360 nm/Em 492 nm) for 60 min with 3 min interval in a BioTek Synergy H1 fluorescence microplate reader. To calculate the IC₅₀, subtract the background fluorescence determined with the “no kinase” control for each time point from the total signal to obtain corrected Relative Fluorescence Units (RFU) values. Plot the corrected RFU vs. Time for each inhibitor concentration and determine the initial reaction rates (slope of the linear portion) for each progress curve for each inhibitor concentration. Then plot velocity (RFU corrected/minute) vs [inhibitor] and determine the IC₅₀ using a 4-parameter logistic fit.