Off-target suppression of cell proliferation by commercially available DNA2 inhibitors

The disease-associated DNA2 nuclease-helicase is critical for the proper processing of stalled DNA replication forks (RFs). DNA2 inhibition therefore has the potential to cause synthetic lethality in cancer cells in which DNA replication stress and RF stalling are prevalent. Several small molecule biochemical inhibitors of DNA2 have been identified. 4-hydroxy-8-nitroquinoline-3-carboxylic acid, known as DNA2 inhibitor C5, has been widely adopted as a tool compound to test the anti-cancer potential of targeting DNA2 in cellular models, and to study the dependence of molecular processes on DNA2 in cells. Here, we show that C5 treatment does not phenocopy DNA2 loss or inactivation in cells. Independently of DNA2, C5, and its derivative d16, strongly inhibit DNA synthesis in S phase of the cell cycle, revealing off-target anti-proliferative activity. Finally, C5 and d16 are unable to inhibit helicase-dependent single-stranded DNA (ssDNA) accumulations generated by a nuclease-dead version of DNA2. These data show that commonly used compounds fail to effectively inhibit DNA2 in cells and highlight potential caveats in the interpretation of published cellular data obtained with C5 and d16. Moreover, our experimental workflow provides an in cellulo analysis pipeline suitable for identifying biologically active DNA2 inhibitors as a tool for fundamental research and validation of DNA2 as an emerging anti-cancer therapeutic target.