‘Direct-to-biology’ drives optimisation of a cell-active covalent inhibitor of WRN helicase

We report a ‘direct-to-biology’ (D2B) approach for optimising covalent acrylamide binders of protein targets and apply this to the identification of a selective and cell-active inhibitor of Werner (WRN) helicase. Inhibition of WRN helicase activity exhibits a synthetic lethal relationship with cancers displaying high microsatellite instability (MSI-H) and is being pursued as a therapeutic strategy in the clinic. Using intact-protein liquid chromatography-mass spectrometry (LC-MS) screening, we identified acrylamide fragment binders of the WRN helicase domain and then used covalent D2B chemistry to optimise these initial hits. Our efforts ultimately afforded a potent covalent inhibitor of WRN-mediated DNA unwinding, which displays selective, concentration-dependent cellular engagement of WRN, and demonstrates synthetic lethality in an MSI-H setting. Furthermore, our inhibitor targets a distinct conformation of WRN helicase compared to the current clinical covalent inhibitor, presenting a complementary approach for covalent inhibition of WRN helicase. This work demonstrates how D2B chemistry platforms can be used to explore structure-activity relationships in a modular fashion, while reducing investment of human and material resources.