Development of a high-throughput fluorescence polarization DNA cleavage assay for the identification of FEN1 inhibitors

J Biomol Screen. 2013 Jun;18(5):567-75. doi: 10.1177/1087057113476551. Epub 2013 Feb 20.

Abstract

Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5' overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z' factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.

Keywords: DNA damage repair; FEN1; HTS; artifact assay; fluorescence polarization; screening.

MeSH terms

  • DNA Cleavage* / drug effects
  • Dose-Response Relationship, Drug
  • Drug Discovery / methods*
  • Enzyme Inhibitors / isolation & purification*
  • Flap Endonucleases / antagonists & inhibitors*
  • Flap Endonucleases / metabolism
  • Fluorescence Polarization / methods
  • High-Throughput Screening Assays / methods*
  • Humans
  • Models, Biological
  • Oligonucleotides / chemistry
  • Oligonucleotides / metabolism
  • Osmolar Concentration
  • Small Molecule Libraries / analysis
  • Substrate Specificity

Substances

  • Enzyme Inhibitors
  • Oligonucleotides
  • Small Molecule Libraries
  • Flap Endonucleases
  • FEN1 protein, human