Development of a TR-FRET Ultra HTS Assay for Targeting the NSD3 and MYC Interaction

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Screening of NSD3-MYC inhibitors in (ultra) High-Throughput mode

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In all living cells, DNA can be summarized as being the complete set of instructions harbored by genes. The complex machinery within the nucleus allows coordinated reading of the different genes and triggers specific protein production. In that sense, cells are essentially very sophisticated biological factories.
Progress in biology today has introduced another level of complexity by regulating gene expression through specific assemblies of specialized proteins with DNA, in the transcription of complex units. This provides a means to fine-tune gene expression, switching protein production on and off to match cells’ needs. Studying these processes is the basis of a new specialty in biology called epigenetics.
However, grains of sand in cellular gear due to the misassembly of proteins (or their mutated forms) with DNA, can lead to impaired protein production and major cellular dysfunction, ultimately causing pathologies. Being able to control the protein machinery with specific inhibitors is one way to cure diseases through a targeted approach. This represents another angle for tackling cancer.
The NSD3-MYC protein complex has been studied extensively and is considered as key in controlling cell proliferation and tumorigenesis. In this article, Jinglin Xiong described the strategy she used to set up a screening assay to identify NSD3-MYC interaction inhibitors. She took a very smart approach using cell-based assays instead of the biochemical in vitro assays used more classically in epigenetic studies. She engineered a library of NSD3 mutants, performed co-transfection with MYC constructs in HEK283T cells, and optimized assay conditions in 384- and 1536-well formats.
A step-by-step procedure to set-up your HTS, or even uHTS, cell-based screening assays on your compelling epigenetic targets.

Abstract

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 fluorophore, 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.

Details

Assay and Drug Development Technology. 2018 Feb/Mar;16(2):96-106.

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