We performed an in vitro kinase activity assay with both wild type BTK and the C481S mutant, (Fig. ). The assay was performed with a preincubation period of two hours, equivalent to the timescale of degradation induced by the PROTACs in cells (Supp. Fig. ). As expected, Ibrutinib was both highly potent against the WT and sensitive to the mutation, with a 74-fold reduction in potency, indicating efficient covalent engagement. The non-covalent NC-1 showed very mild sensitivity to the mutation, with < 2-fold reduction in affinity. The acrylamide IR-2 did not inhibit BTK more potently than NC-1 and also showed only slight sensitivity to the mutation, indicating inefficient covalent bond formation, possibly due to the lowered reactivity of the b-substituted acrylamide. On the other hand, the cyanoacrylamides RC-2 and RC-3 were an order of magnitude more potent than NC-1 and also highly sensitive to the mutation with 68-fold and >1000-fold reduction in potency, respectively, indicating rapid covalent binding to BTK on the timescale of degradation. All the PROTACs tested except RC-3 exhibited sub-100 nM binding to BTK even after mutation of C481. This may explain why only degradation by RC-3 was significantly impaired by the mutation.