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

EBioMedicine. 2016 Apr;6:73-86. doi: 10.1016/j.ebiom.2016.02.043. Epub 2016 Mar 10.

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.

Keywords: Camptothecin; Cancer; Chemotherapy; DNA binding; DNA end resection; DNA replicatoin fork protection; DNA2 inhibitor; Helicase; Nuclease; PARP inhibitor; Sensitizer.

MeSH terms

  • A549 Cells
  • Binding Sites
  • Camptothecin / pharmacology*
  • Carboxylic Acids / chemistry
  • Carboxylic Acids / pharmacology
  • Cell Line, Tumor
  • Computer Simulation
  • DNA Helicases / antagonists & inhibitors
  • DNA Helicases / chemistry*
  • DNA Replication / drug effects
  • Drug Screening Assays, Antitumor
  • Drug Therapy
  • Enzyme Inhibitors / chemistry
  • Enzyme Inhibitors / pharmacology*
  • High-Throughput Screening Assays
  • Humans
  • MCF-7 Cells
  • Neoplasms / drug therapy
  • Neoplasms / enzymology*
  • Nitroquinolines / chemistry
  • Nitroquinolines / pharmacology*
  • Small Molecule Libraries / chemistry
  • Small Molecule Libraries / pharmacology*

Substances

  • 4-hydroxy-8-nitroquinoline-3-carboxylic acid
  • Carboxylic Acids
  • Enzyme Inhibitors
  • Nitroquinolines
  • Small Molecule Libraries
  • DNA Helicases
  • DNA2 protein, human
  • Camptothecin