Figure 4.

Inhibition of BTK by tirabrutinib. The TR-FRET assay was used to detect free and total BTK using rBTK, Ramos B cells, purified human PBMCs, and whole blood (WB) samples preincubated for 2 h with increasing concentrations of tirabrutinib. Values were normalized to vehicle-treated samples to obtain percent BTK occupancy. The top left panel shows the standard dose–response curve generated using recombinant BTK. In Ramos B cells, purified human PBMCs, and WB samples, BTK binding to the biotinylated tirabrutinib probe was competitively inhibited in a dose-dependent manner by tirabrutinib. The EC₅₀ of tirabrutinib as measured by the TR-FRET assay is shown in the table (mean ± SD of n = 3 experiments, each in quadruplicate).