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Published April 2016 | Published + Supplemental Material
Journal Article Open

A Selective Small Molecule DNA2 Inhibitor for Sensitization of Human Cancer Cells to Chemotherapy

Abstract

Cancer cells frequently up-regulate DNA replication and repair proteins such as the multifunctional DNA2 nuclease/helicase, counteracting DNA damage due to replication stress and promoting survival. Therefore, we hypothesized that blocking both DNA replication and repair by inhibiting the bifunctional DNA2 could be a potent strategy to sensitize cancer cells to stresses from radiation or chemotherapeutic agents. We show that homozygous deletion of DNA2 sensitizes cells to ionizing radiation and camptothecin (CPT). Using a virtual high throughput screen, we identify 4-hydroxy-8-nitroquinoline-3-carboxylic acid (C5) as an effective and selective inhibitor of DNA2. Mutagenesis and biochemical analysis define the C5 binding pocket at a DNA-binding motif that is shared by the nuclease and helicase activities, consistent with structural studies that suggest that DNA binding to the helicase domain is necessary for nuclease activity. C5 targets the known functions of DNA2 in vivo: C5 inhibits resection at stalled forks as well as reducing recombination. C5 is an even more potent inhibitor of restart of stalled DNA replication forks and over-resection of nascent DNA in cells defective in replication fork protection, including BRCA2 and BOD1L. C5 sensitizes cells to CPT and synergizes with PARP inhibitors.

Additional Information

© 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Received 22 February 2016; Received in revised form 29 February 2016; Accepted 29 February 2016; Available online 10 March 2016. Funding Sources: The work was supported by an NIH grant RO1 CA085344, the Margaret E. Early Medical Research Trust grant ADF-1481M to B.H.S., Margaret Early Medical Research Trust grant and ARO W81XWH-09-1-0041 to J.L.C., a Caltech-City of Hope Biomedical Research Initiative to B.H.S. and J.L.C., and K22 grant 5K22CA175262–04 to K.S. Wenpeng Liu is supported by the China Scholarship Council (CSC) during his visit to California Institute of Technology and City of Hope, and K.S. is a CPRIT scholar in cancer biology. We thank the City of Hope Microscopy and Bioinformatics Core Facilities, National Cancer Institute (NCI) designated cancer center support grant P30 CA033572, and Nancy Linford, Ph.D. for her critical reading and editing of the manuscript and S. Howard and J. Stark for their technical support. Author Contributions: L.Z., J.L.C. and B.S. designed the study and supervised the entire project. L.Z., J.L.C., B.S. and K.S. wrote the manuscript. H.L. established the 3-D model and virtually screened the chemical inhibitors. W.L., P.P., J.L.C., Q.W., C.L, and K.K.J. purified the proteins and performed all of the biochemical experiments in the current study. K.S. and V.P. designed and executed the fork protection experiments. Z.L. performed the DNA fiber experiments. Correspondence concerning fork protection experiments may be addressed to K.S. and V.P. M.Z., Z.L., H.D., K.K.J., L.C., performed the cellular functional (SSA, HR, P-RPA resection, replication fork restart) and toxicological assays. S.S. supervised the enzyme kinetics experiments. We declare no financial or other relationships that may lead to a conflict of interest in this study.

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August 22, 2023
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