Vecabrutinib is a noncovalent BTK inhibitor that inhibits C481S mutant BTK. Crystal structure showing binding of vecabrutinib (A) and ibrutinib (B) to the active site of BTK. Inhibition of kinase activity of WT and C481S mutant BTK by vecabrutinib (C), ibrutinib (D), and acalabrutinib (E), as measured by a direct kinase assay. Data in (C) and (D) are averages of 3 biological replicates (supplemental Figure 3A-B). (F) Changes in BCR-mediated increases in cytosolic Ca²⁺ in BTK^−/− DT40 cells reconstituted with WT or C481S mutant BTK treated with vecabrutinib or ibrutinib. (G) Changes in inositol phosphate formation in COS-7 cells coexpressing PLCγ₂ and BTK E41K, its ibrutinib-resistant variant BTK E41K|C481S, or its kinase-dead variant BTK E41K|K430R, following treatment with vehicle, vecabrutinib, or acalabrutinib. Data in (F-G) are representative of 3 biological replicates. Analysis of changes in cell viability of WT BTK (n = 8) and C481S BTK mutant (n = 4) primary CLL cells, as measured by DiOC6/PI staining and flow cytometry, after 4 days of treatment with 0.5 µM (H) and 1 µM (I) ibrutinib (Ibru) or vecabrutinib (Veca). Dashed line in (H-I) indicates normalization to dimethyl sulfoxide. P values for WT BTK vs C481S BTK mutants were calculated using a Mann-Whitney U test, whereas P values for comparison of treatments between paired samples were calculated using a paired Student t test. *P ≤ .05, **P ≤ .01. Mut, mutant; ns, not significant (P > .05); WT, wild-type.