Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

作者：McWhirter Claire^*; Tonge Michael; Plant Helen; Hardern Ian; Nissink Willem; Durant Stephen T

来源：Journal of Biomolecular Screening, 2013, 18(5): 567-575.

DOI：10.1177/1087057113476551

• FEN1
• HTS
• screening
• fluorescence polarization
• DNA damage repair
• artifact assay

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.

• 出版日期2013-6