Regulation of Cullin-RING Ligases by Nedd8

Nedd8 对 Cullin-RING 连接酶的调节

• 批准号: 7812495
• 负责人: RAYMOND J DESHAIES
• 金额: $ 20.14万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7812495
• 关键词: Antineoplastic Agents; Biological Assay; Cells; Characteristics; Chemicals; Clinical; Complex; Cullin Proteins; Development; Enzymes; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Funding; Gene Expression; Goals; Human; In Vitro; Inhibitory Concentration 50; Label; Lead; Libraries; Ligase; Link; Malignant Neoplasms; Mediating; Methods; Modification; Monitor; Mutate; Optics; Parents; Peer Review; Pharmaceutical Preparations; Physiological; Recovery; Regulation; Research; Research Design; Role; Screening procedure; Secondary to; Substrate Specificity; Ubiquitin Like Proteins; United States National Institutes of Health; Ursidae Family; design; high throughput screening; human disease; in vitro Assay; in vivo; inhibitor/antagonist; isopeptidase; malignant breast neoplasm; novel therapeutics; outcome forecast; overexpression; parent grant; programs; public health relevance; research study; scaffold; small molecule libraries; tool; ubiquitin ligase

DESCRIPTION (provided by applicant): This application is responsive to NOT-OD-09-058 (NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications). We seek support from the Recovery Act Funds to expand the scope of the specific aims, research design, and methods of GM065997, which bears the title: Regulation of cullin-RING ligases by Nedd8. Nedd8 is a ubiquitin-like protein that is reversibly conjugated to the cullin subunit of cullin-RING ubiquitin ligases (CRLs). Both the conjugation and deconjugation of Nedd8 are essential to maintain the activity of CRLs in human cells. In the second Aim of the parent application, we proposed to investigate how Nedd8 deconjugation of cullins by the COP9 Signalosome (CSN) is regulated by the recruitment of substrates and other subunits of the ligase complex to the cullin scaffold. In the present application, we propose to expand upon this goal by developing a potent and specific inhibitor of the Nedd8 isopeptidase activity of CSN. We will devise homogeneous fluorescence polarization and fluorescence resonance energy transfer assays to monitor cleavage of Nedd8-Cul1 both in vitro and in vivo, and will employ the in vitro assay to conduct high- throughput screens of chemical libraries. Hits will be subjected to secondary screens designed to evaluate their in vivo efficacy, potency, and selectivity. Our goal is to identify a lead compound with an IC50 of at least 1 ?M in both in vitro and in vivo assays. A lead with these characteristics would be a very useful tool to study the physiological role of CSN-dependent Nedd8 deconjugation in vivo, and to evaluate how this function relates to the substrate specificity of CSN, which is the subject of Aim 2 of the parent application. The experiments proposed here substantially expand the scope of the original peer reviewed Aims of the parent grant by introducing new new methods and approaches that will yield new tools to study the function of CSN. Pursuit of the proposed experiments will accelerate the tempo of research in my lab on the cellular consequences of CRL regulation by Nedd8 deconjugation, and will enable the recruitment of new staff. PUBLIC HEALTH RELEVANCE: Subunits of cullin-RING ubiquitin ligases (CRLs) are mutated or overexpressed in a variety of human diseases including cancer and overexpression of Csn5 drives a gene expression program that is tightly linked to poor prognosis in breast cancer. In addition, the Nedd8 activating enzyme that together with Csn5 mediates the cycle of cullin modification by Nedd8 is the target of a promising anti-cancer drug that is in clinical development. Identification of a chemical that blocks cleavage of Nedd8 from cullins by Csn5 will lead to a deeper understanding of the physiological role of Nedd8 deconjugation and will lead to a better understanding of how overactive Csn5 underlies human disease. Besides being an important research tool, a Csn5 inhibitor has great potential to serve as a starting point for development of new therapeutic drugs to modulate CRL activity for clinical benefit.

描述(由申请人提供)：本申请响应NOT-OD-09-058(NIH宣布恢复法资金可用于竞争性修订申请)。我们寻求复苏法案基金的支持，以扩大GM065997的具体目标、研究设计和方法的范围，其标题为：Nedd8对剔除环连合酶的调节。NEDD8是一种泛素样蛋白，它可逆地与cullin环泛素连接酶(CRL)的cullin亚基结合。Nedd8的结合和去结合都是维持CRLS在人类细胞中的活性所必需的。在亲本应用的第二个目标中，我们建议研究COP9信号体(CSN)对Cullins的Nedd8去结合是如何通过将底物和连接酶复合体的其他亚单位招募到cullin支架上来调节的。在目前的应用中，我们建议通过开发一种有效和特异的CSN Nedd8异肽酶活性的抑制剂来扩大这一目标。我们将设计均相荧光偏振和荧光共振能量转移方法来监测Nedd8-Cul1在体外和体内的切割情况，并将利用体外实验进行高通量的化学文库筛选。HITS将接受二次筛选，以评估它们在体内的疗效、效力和选择性。我们的目标是在体外和体内试验中鉴定一种IC50至少为1？M的先导化合物。具有这些特征的铅将是一个非常有用的工具，可以在体内研究依赖CSN的Nedd8去共轭的生理作用，并评估这一功能如何与CSN的底物专一性有关，这是父本申请的目标2的主题。这里提出的实验通过引入新的方法和途径，极大地扩大了父母赠款最初同行评审目标的范围，这些新方法和途径将产生新的工具来研究CSN的功能。对拟议的实验的追求将加快我实验室研究Nedd8去结合调节CRL的细胞后果的速度，并将使招聘新员工成为可能。 公共卫生相关性：在包括癌症在内的各种人类疾病中，剔除环泛素连接酶(CRL)的亚单位发生突变或过度表达，Csn5的过度表达驱动了一种与乳腺癌预后不良密切相关的基因表达程序。此外，Nedd8激活酶与Csn5一起介导Nedd8修饰cullin的循环，是一种有希望的抗癌药物的靶点，该药物正在临床开发中。鉴定出一种化学物质可以阻止Nedd8从Cullins裂解，这将有助于更深入地理解Nedd8去结合的生理作用，并将更好地理解过度活跃的Csn5是如何导致人类疾病的。除了作为一种重要的研究工具外，CsN5抑制剂还具有巨大的潜力，可以作为开发新的治疗药物的起点，以调节CRL的活性，从而使临床受益。