More than an attachment module: covalent inhibitor warheads influence BTK dynamics and function.

Covalent inhibitors are rapidly becoming the standard of care for treatment of a range of disease states. Covalent inhibitors bind irreversibly to their target using a reactive electrophile (or warhead). Acrylamide and 2-butynamide are the most commonly used cysteine targeting electrophiles. These warheads are chosen for their efficient and selective modification of the protein and are presumed to be otherwise functionally inert. Using a panel of BTK covalent inhibitors (Tirabrutinib, Acalabrutinib, Ibrutinib and Zanubrutinib), we show that the 2-butynamide warhead on Tirabrutinib and Acalabrutinib, unlike the acrylamide warhead on Ibrutinib and Zanubrutinib, induces conformational heterogen