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Biol Pharm Bull. 2017;40(9):1454-1462. doi: 10.1248/bpb.b17-00283.

Development of a High-Throughput Assay for Inhibitors of the Polo-Box Domain of Polo-Like Kinase 1 Based on Time-Resolved Fluorescence Energy Transfer.

Kim TG1,2, Lee JH1, Lee MY1, Kim KU1,3, Lee JH1,3, Park CH1,3, Lee BH1,2, Oh KS1,3.

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Bio-Organic Science Division, Korea Research Institute of Chemical Technology.
Graduate School of New Drug Discovery and Development, Chungnam National University.
Department of Medicinal and Pharmaceutical Chemistry, University of Science and Technology.


Although enzyme-linked immunosorbent assay (ELISA) technology has been widely accepted for binding assays against the polo-box domain (PBD) of polo-like kinase-1 (Plk1), these assays have a limitation-related heterogeneous procedure, such as multiple incubations and washing steps to apply high-throughput screenings (HTSs). In the present study, a Plk1-PBD binding assay based on time-resolved fluorescence energy transfer (TR-FRET) was developed for HTS of PBD-binding inhibitors. The TR-FRET-based Plk1-PBD binding assay is sensitive and robust and can be miniaturized into the 384-well plate-based format. Compared with the ELISA-based Plk1-PBD binding assay (Z' factor, 0.53; signal-to-background ratio, 4.19), the TR-FRET-based Plk1-PBD binding assay improved the Z' factor (0.72) and signal-to-background ratio (8.16). Using TR-FRET based Plk1-PBD binding assay, pilot library screening of 1019 natural compounds was conducted and five hit compounds such as haematoxylin, verbascoside, menadione, lithospermic acid and (1,3-dioxolo[4,5-g]isoquinolinium 5,6,7,8-tetrahydro-4-methoxy-6,6-dimethyl-5-[2-oxo-2-(2-pyridinyl)ethyl]-iodide) (DITMD) were identified as Plk1-PBD inhibitor. In a functional assay to validate the hit compounds, five hit compounds exhibited suppression of HeLa cells proliferation. These results suggest that TR-FRET-based Plk1-PBD binding assay can be applied for an efficient and less time-consuming HTS of compound libraries.


high-throughput screening; polo-box domain; polo-like kinase-1; protein–protein interaction

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