Development of a Time-Resolved Fluorescence Resonance Energy Transfer Ultrahigh-Throughput Screening Assay for Targeting the NSD3 and MYC Interaction
Development of a Time-Resolved Fluorescence Resonance Energy Transfer Ultrahigh-Throughput Screening Assay for Targeting the NSD3 and MYC Interaction
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DOI：
10.1089/adt.2017.835
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发表时间：
2018-03-01
影响因子：
1.8
通讯作者：
Du, Yuhong
中科院分区：
文献类型：
--
作者：
Xiong, Jinglin;Pecchi, Valentina Gonzalez;Du, Yuhong
表观遗传调节剂在细胞功能重编程中起着关键作用，成为一类新的有前途的治疗靶点。核受体结合SET结构域蛋白3（NSD 3）是赖氨酸甲基转移酶家族的成员。有趣的是，没有甲基转移酶片段的NSD 3的短同种型NSD 3S在广泛的癌症中表现出致癌活性。我们最近发现NSD 3S与MYC相互作用，MYC是肿瘤发生的中心调节因子，这表明NSD 3S通过参与MYC调节细胞增殖的机制。因此，NSD 3S/MYC相互作用的小分子抑制剂将是理解NSD 3在肿瘤发生中的功能的有价值的工具，用于潜在的癌症治疗发现。在这里，我们报告了一种基于细胞裂解物的时间分辨荧光共振能量转移（TR-FRET）测定的发展，以超高通量筛选（uHTS）的形式来监测NSD 3S与MYC的相互作用。在我们的TR-FRET测定中，使用抗Flag-铽和抗谷胱甘肽S-转移酶（GST）-d2（成对的荧光团）间接标记HEK 293 T细胞裂解物中的Flag标记的NSD 3和GST-MYC。该TR-FRET测定在1，536孔uHTS形式中是稳健的，信号背景比>8且Z'因子>0.7。对含有2,000种化合物的光谱库进行了初步筛选，确定了几种阳性结果。在正交蛋白质-蛋白质相互作用测定中，证实一种阳性化合物破坏NSD 3/MYC相互作用。因此，我们优化的uHTS测定可应用于筛选活动的未来扩大，以鉴定靶向NSD 3/MYC相互作用的小分子抑制剂。
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.