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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.

Author information

1
Bio-Organic Science Division, Korea Research Institute of Chemical Technology.
2
Graduate School of New Drug Discovery and Development, Chungnam National University.
3
Department of Medicinal and Pharmaceutical Chemistry, University of Science and Technology.

Abstract

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.

KEYWORDS:

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

PMID:
28867728
DOI:
10.1248/bpb.b17-00283
[Indexed for MEDLINE]
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