Figure 6.

All PROTACs are potent BTK inhibitors invitro, and the cyanoacrylamides show slow dissociation kinetics and sensitivity to the C481S mutation. (A) In vitro kinase activity assay using wild-type BTK (0.6 nM BTK, 5 μM ATP). (B) Summary of IC₅₀ values for the PROTACs against wild-type BTK and C481S BTK. (C) Time course LC-MS binding assay (3 μM compound + 2 μM BTK at room temperature). (D) Ibrutinib competition assay validates reversible binding by cyanoacrylamides; 40 μM ibrutinib was added to the preformed complex and incubated at 37 °C, and the different species were quantified by LC-MS.