Development of a Time-Resolved Fluorescence Resonance Energy Transfer Ultrahigh-Throughput Screening Assay for Targeting the NSD3 and MYC Interaction

• Published in: Assay and drug development technologies Vol. 16; no. 2; p. 96
• Main Authors: Xiong, Jinglin, Pecchi, Valentina Gonzalez, Qui, Min, Ivanov, Andrey A, Mo, Xiulei, Niu, Qiankun, Chen, Xiang, Fu, Haian, Du, Yuhong
• Format: Journal Article
• Language: English
• Published: United States 01.03.2018
• Subjects: Fluorescence Resonance Energy Transfer; HEK293 Cells; High-Throughput Screening Assays; Histone-Lysine N-Methyltransferase - chemistry; Humans; Nuclear Proteins - chemistry; Protein Binding; Proto-Oncogene Proteins c-myc - chemistry; Time Factors; NSD3; protein–protein interaction; screening; MYC; TR-FRET; epigenetics
• ISSN: 1557-8127
• DOI: 10.1089/adt.2017.835

Epigenetic modulators play critical roles in reprogramming of cellular functions, emerging as a new class of promising therapeutic targets. Nuclear receptor binding SET domain protein 3 (NSD3) is a member of the lysine methyltransferase family. Interestingly, the short isoform of NSD3 without the methyltransferase fragment, NSD3S, exhibits oncogenic activity in a wide range of cancers. We recently showed that NSD3S interacts with MYC, a central regulator of tumorigenesis, suggesting a mechanism by which NSD3S regulates cell proliferation through engaging MYC. Thus, small molecule inhibitors of the NSD3S/MYC interaction will be valuable tools for understanding the function of NSD3 in tumorigenesis for potential cancer therapeutic discovery. Here we report the development of a cell lysate-based time-resolved fluorescence resonance energy transfer (TR-FRET) assay in an ultrahigh-throughput screening (uHTS) format to monitor the interaction of NSD3S with MYC. In our TR-FRET assay, anti-Flag-terbium and anti-glutathione S-transferase (GST)-d2, a paired fluorophores, were used to indirectly label Flag-tagged NSD3 and GST-MYC in HEK293T cell lysates. This TR-FRET assay is robust in a 1,536-well uHTS format, with signal-to-background >8 and a Z' factor >0.7. A pilot screening with the Spectrum library of 2,000 compounds identified several positive hits. One positive compound was confirmed to disrupt the NSD3/MYC interaction in an orthogonal protein-protein interaction assay. Thus, our optimized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the NSD3/MYC interaction.