Development of a FOXM1-DBD Binding Assay for High-Throughput Screening Using TR-FRET Assay

• Published in: Biological & pharmaceutical bulletin 2021/10/01, Vol.44(10), pp.1484-1491
• Main Authors: Lee, Mi Young, Haam, Chae Eun, Mun, Jihye, Lim, Gyutae, Lee, Byung Ho, Oh, Kwang-Seok
• Format: Article
• Language: English
• Subjects: Cell lines; Cell proliferation; Cell viability; Deoxyribonucleic acid; DNA; DNA - metabolism; Drug Discovery - methods; Electrophoretic mobility; Fluorescence Resonance Energy Transfer; Forkhead box protein M1; Forkhead Box Protein M1 - antagonists & inhibitors; Forkhead Box Protein M1 - metabolism; Forkhead protein; High-throughput screening; High-Throughput Screening Assays - methods; Humans; MCF-7 Cells; Protein Binding - drug effects; Protein Interaction Domains and Motifs; protein–DNA interaction; Radioisotopes; Transcription factors
• ISSN: 0918-6158; 1347-5215
• DOI: 10.1248/bpb.b21-00322

Electrophoretic mobility shift assay (EMSA) technology has been widely employed for the analysis of transcription factors such as Forkhead box protein M1 (FOXM1). However, the application of high-throughput screening (HTS) in performing, such analyses are limited as it uses time consuming electrophoresis procedure and radioisotopes. In this study, we developed a FOXM1-DNA binding domain (DBD) binding assay based on time-resolved fluorescence energy transfer (TR-FRET) that enables HTS for the inhibitors of FOXM1–DNA interaction. This assay was robust, highly reproducible and could be easily miniaturized into 384-well plate format. The signal-to-background (S/B) ratio and Z′ factor were calculated as 7.46 and 0.74, respectively, via a series of optimization of the assay conditions. A pilot library screening of 1019 natural compounds was performed using the FOXM1-DBD binding assay. Five hit compounds, namely, AC1LXM, BRN5, gangaleoidin, leoidin, and roemerine were identified as the inhibitors of FOXM1. In a cell viability assay, it was demonstrated that cell proliferation of FOXM1 overexpressed cell lines was suppressed in cell lines such as MDA-MB-231 and MCF-7 by five hit compounds. These results indicate that developed FOXM1-DBD binding assay can be applied to highly efficiency HTS of compound libraries.