title: Development of a high-throughput fluorescence polarization DNA cleavage assay for the identification of FEN1 inhibitors.
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.
authors: Discovery Sciences, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire, UK. claire.mcwhirter@astrazeneca.com
authors: Claire
authors: C
authors: McWhirter
authors: Michael
authors: M
authors: Tonge
authors: Helen
authors: H
authors: Plant
authors: Ian
authors: I
authors: Hardern
authors: Willem
authors: W
authors: Nissink
authors: Stephen T
authors: ST
authors: Durant
mesh_terms: D053837:DNA Cleavage / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D045585:Flap Endonucleases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D005454:Fluorescence Polarization / Q000379:methods; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008954:Models, Biological; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D009994:Osmolar Concentration; D054852:Small Molecule Libraries / Q000032:analysis; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D009841:Oligonucleotides; D054852:Small Molecule Libraries; D045585:Flap Endonucleases; C468317:FEN1 protein, human
keywords: DNA damage repair; FEN1; HTS; artifact assay; fluorescence polarization; screening