A high throughput screening assay for the identification of SUMOylation inhibitor

描述（由申请人提供）：本提案的总体目标是执行高通量筛选试验（HTS）以确定SUMOylation抑制剂。SUMO （Small Ubiquitin-like MOdifier）蛋白家族作为一种重要的翻译后修饰蛋白，在基因转录、细胞周期进程、DNA修复、病毒和细菌感染以及神经退行性疾病的发生等许多功能中发挥着重要作用。SUMOylation的抑制剂将作为研究SUMOylation在不同细胞功能中的作用所需的试剂，这在很大程度上仍不清楚。这种抑制剂还具有治疗癌症和病毒感染等疾病的潜力。然而，这些抑制剂尚未被报道，也尚未提供给科学界。我们已经开发了几种测定方法来鉴定这种抑制剂。初级HTS检测采用ALPHA筛选技术。二次筛选试验使用荧光共振能量转移（FRET）来消除在ALPHA筛选中获得的假阳性命中。此外，泛素化试验将用于识别特异性抑制sumo酰化而不是其他泛素样修饰系统的命中。筛选中确定的抑制剂的直接应用是：1)探索SUMOylation在不同细胞功能中的作用，例如在DNA修复途径中；2)在SUMOylation已被证明发挥重要作用的疾病的细胞和动物模型中测试抑制剂，例如在癌症和病毒感染中。我们的长期目标是基于本研究中发现的hit的结构-活性关系研究开发有效的SUMOylation抑制剂。同源泛素-蛋白体途径的抑制剂在剖析泛素-蛋白体途径在许多细胞功能中的作用方面至关重要，并且作为抗癌治疗剂取得了惊人的成功。SUMOylation抑制剂在研究中具有广泛的影响，并可能导致在开发治疗破坏性疾病（如癌症和传染病）的治疗药物方面建立新的范例。公共卫生相关性：

DESCRIPTION (provided by applicant): The overall goal of this proposal is to perform high throughput screening assays (HTS) to identify inhibitors for SUMOylation. Protein modification by the SUMO (Small Ubiquitin-like MOdifier) family of proteins has recently been established as an important post- translational modification that plays an essential role in many functions including gene transcription, cell cycle progression, DNA repair, viral and bacterial infection, and the development of neurodegenerative diseases. Inhibitors of SUMOylation will serve as much needed reagents for investigating the role of SUMOylation in different cellular functions, which remain largely unclear. The inhibitors will also have therapeutic potential for diseases, such as cancer and viral infection. However, such inhibitors have not been reported and are not yet available to the scientific community. We have developed several assays to identify such inhibitors. The primary HTS assay uses the ALPHA screening technology. The secondary screening assay uses fluorescence resonance energy transfer (FRET) to eliminate false positive hits obtained in the ALPHA screen. Additionally, an ubiquitination assay will be used to identify the hits that are specific for inhibiting SUMOylation and not other ubiquitin-like modification systems. The immediate applications of the inhibitors identified in the screen are: 1) to probe the role of SUMOylation in different cellular functions, such as in the DNA repair pathways, 2) to test the inhibitors in cellular and animal models of diseases where SUMOylation has been shown to play important roles, such as in cancer and viral infections. Our long term goal is to develop potent SUMOylation inhibitors based on the structure-activity relationship studies of the hits identified from this study. Inhibitors of the homologous ubiquitin-proteosome pathway have been critical in dissecting the role of the ubiquitin-proteosome pathway in many cellular functions and are surprisingly successful as anti-cancer therapeutic agents. SUMOylation inhibitors hold similar promise in having a broad impact in research and could lead to the establishment of a new paradigm in developing therapeutic agents for devastating diseases, such as cancer and infectious diseases. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to perform high throughput screening assays (HTS) to identify inhibitors for SUMOylation.