First, we tested the degradation activity of the PROTACs against overexpressed wild type BTK and the C481S mutant, which cannot form covalent complexes (Fig. ). As expected, the degradation by non-covalent NC-1 was only mildly affected by the mutation. However, the covalent PROTACs IR-1, IR-2 and RC-2 also showed low sensitivity to the mutation. In contrast, degradation by RC-3 was severely impaired by the mutation, indicating an important role for covalent engagement in the degradation process by RC-3. Second, we tested the ability of the compounds to covalently bind and inhibit BTK.