Figure 1.

Vecabrutinib kinome profile and effect on B-cell receptor pathway protein phosphorylation. (A) Vecabrutinib and ibrutinib kinase inhibition profiles. Vecabrutinib kinase selectivity and kinase inhibitory activities were evaluated in the KinaseProfiler™ panel of 234 individual kinases (Eurofins Pharma Discovery Services, Dundee, UK). For TEC and LCK, vecabrutinib activity was assessed against the activated kinase. (B) Phospho-BTK inhibition in human whole blood ex vivo. Peripheral blood mononuclear cells were isolated from peripheral blood of healthy donors and were incubated with 0.3 to 10,000 nM vecabrutinib for 0.5 h. Experiments were conducted on samples from 145 individuals to calculate IC₅₀ values. Mean and standard deviation (SD) values are shown with horizontal lines and error bars. (C) Phospho-PLCg2 inhibition in the Ramos cell line. Cells were incubated with 0.3 to 10,000 nM vecabrutinib. Experiments were conducted on four biological replicates in the Ramos cell line to calculate IC₅₀ values. Mean and SD values are shown with horizontal lines and error bars. (D-F). Effects of vecabrutinib on BCR pathway proteins in MEC-1 cells that overexpress wild-type or mutant BTK. Green fluorescence protein (GFP)-labeled MEC-1 cell lines that stably overexpress wild-type BTK (BTK^WT) and mutant BTK (BTK^C481S or BTK^C481R) were generated by using standard lentiviral transfection methods. Cells were sorted by a BD FACSAria (BD Biosciences) for the enrichment of transduced GFP⁺ cell populations in each cell line. For all experiments, >75% GFPpositive cell populations were used. Cells were treated with indicated concentrations of vecabrutinib or ibrutinib and incubated for 24 h. Protein extracts were subjected to immunoblot assays to determine levels of phospho-BTK (Y223), BTK, phospho-ERK (T202/Y204), ERK in MEC-1 cells overexpressing (D) BTK^WT, (E) BTK^C481S, and (F) BTK^C481R cells. Vinculin was used as the loading control.