Pif1 helicase plays multiple roles in maintaining genome stability, which is an attractive therapeutic target for helicase-related diseases, while small molecules targeting Pif1 are not yet available. In this study, we performed a fluorescence polarization-based high-throughput screening and identified that an FDA-approved drug, Tideglusib (TD), could inhibit the DNA-binding activity (IC50 = 6.2 ± 0.4 μM) and ATPase and helicase activity (IC50 = 2-4 μM) of Bacteroides sp. Pif1 (BaPif1), which was also confirmed with human Pif1. In addition, the TD analogue TDZD-8 displayed similar inhibitory effects on Pif1 activities. Notably, TD irreversibly inhibited BaPif1 and severely induced BaPif1 aggregation. Furthermore, inhibition of BaPif1 by TD was significantly attenuated in the presence of dithiothreitol, indicating that TD could be a thiol-reactive compound. We also identified that Cys-380 of BaPif1 is critical for the inhibition by TD, suggesting that TD inhibits BaPif1 via an irreversible and Cys-380-dependent mechanism.
© 2022 The Authors. Published by American Chemical Society.