Screening for inhibitors of NSD3 as a treatment for lung cancer

摘要 核受体结合定域蛋白3(NSD3)属于NSD组蛋白家族 甲基转移酶，负责组蛋白3上赖氨酸36的单甲基化和双甲基化(H3K36me1/2)。 越来越多的研究将NSD3与多种癌症联系起来，包括肺癌、乳腺癌、胰腺癌和膀胱癌 癌症。最近的研究表明，NSD3是侵袭性肺鳞状细胞癌的癌基因， 这代表了非小细胞肺癌的一种亚型，临床结果非常差。扩增的NSD3基因 和/或在LUSC中高表达，其作为癌基因在该亚型肺癌中的作用一直很好 通过体外和体内的遗传学研究进行了验证。此外，NSD3的催化活性也起到了 在LUSC肿瘤发生中关键作用，支持NSD3催化集结构域的小分子抑制物 可以代表肺癌患者的新疗法。然而，NSD3抑制剂尚未被报道为 Date，但作为治疗LUSC和其他NSD3癌症患者的新的潜在疗法是非常受欢迎的 放大。在这个项目中，我们建议通过进行高通量筛选来开发NSD3抑制剂 (HTS)，密歇根大学CCG。HTS将专注于识别小分子抑制物 NSD3催化集结构域。到目前为止，我们开发并验证了一种荧光偏振(FP)分析方法 将被用作HTS的初步筛选试验，以确定NSD3抑制剂。HTS鉴定的小分子 将验证与NSD3结合和抑制其催化活性使用一系列生化和 生物物理实验，包括核磁共振、ITC和组蛋白甲基转移酶分析。活性最强的 化合物将在基于细胞的研究中进行表征，以评估NSD3在哺乳动物细胞和 了解他们的作用机制。选定的NSD3抑制剂将在一组肺癌细胞系中进行测试 具有NSD3扩增和/或过度表达的活性、选择性和作用机制。在……里面 综上所述，我们希望找到有选择性地靶向催化集结构域的高价值化学探针 NSD3，这将适用于癌细胞的机制研究。这个项目将为 开发更有效的NSD3抑制剂，适用于肺癌模型的体内研究 以及未来的治疗意义。

Abstract The nuclear receptor binding SET domain protein 3 (NSD3) belongs to the family of NSD histone methyltransferases, which are responsible for mono- and di-methylation of lysine 36 on histone 3 (H3K36me1/2). Increasing number of studies link NSD3 to various cancers, including lung, breast, pancreatic and bladder cancers. Resent study implicates NSD3 as an oncogene in aggressive lung squamous cell carcinoma (LUSC), which represents a sub-type of non-small cell ling cancer with very poor clinical outcome. NSD3 in amplified and/or overexpressed in LUSC, and its role as an oncogene in this sub-type of lung cancer has been well validated using genetic studies, both in vitro and in vivo. Furthermore, the catalytic activity of NSD3 plays a critical role in LUSC oncogenesis, supporting that small molecule inhibitors of the catalytic SET domain of NSD3 could represent new therapeutics for lung cancer patients. However, NSD3 inhibitors have not been reported to date, but are highly desired as new potential therapeutics for patients with LUSC and other cancers with NSD3 amplifications. In this project we propose to develop NSD3 inhibitors by performing high throughput screening (HTS) at CCG, University of Michigan. The HTS will focus on identification of small molecule inhibitors of the catalytic SET domain of NSD3. To date, we developed and validated a fluorescence polarization (FP) assay that will be used as a primary screening assay for HTS to identify NSD3 inhibitors. Small molecules identified by HTS will be validated for binding to NSD3 and for inhibition of its catalytic activity using a series of biochemical and biophysical experiments, including NMR, ITC, and histone methyltransferase assays. Activity of the most potent compounds will be characterized in cell-based studies to assess inhibition of NSD3 in mammalian cells and understand their mechanism of action. Selected NSD3 inhibitors will be tested in a panel of lung cancer cell lines with NSD3 amplifications and/or overexpression for their activity, selectivity, and mechanism of action. In summary, we expect to identify highly valuable chemical probes selectively targeting catalytic SET domain of NSD3, which will be suitable for mechanistic studies in cancer cells. This project will pave the way towards development of more potent NSD3 inhibitors that will be appropriate for in vivo studies in lung cancer models and for future therapeutic implications.