Using Chemical Biology to Study the Small GTPase Ra(RMI)

利用化学生物学研究小 GTP 酶 Ra(RMI)

• 批准号: 7020465
• 负责人: KARSTEN WEIS
• 金额: $ 19万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7020465
• 关键词: Xenopus; Xenopus oocyte; antineoplastics; bioassay; biotechnology; cell cycle; cell growth regulation; chemical registry /resource; drug discovery /isolation; enzyme inhibitors; fluorescence resonance energy transfer; growth inhibitors; guanosinetriphosphatases; high throughput technology; hydrolysis; nucleotides; protein protein interaction; protein structure function; receptor expression; small molecule

DESCRIPTION (provided by applicant): The goal of this project is to develop assays that will be applied to high throughput small molecule screens designed to target the small GTPase Ran and its associated proteins. Ran regulates nucleocytoplasmic transport during interphase and recently it has been demonstrated that Ran also regulates key event in mitosis such as spindle assembly, nuclear envelope assembly and nuclear pore complex assembly. In interphase and in mitosis RanGTP is enriched near the chromosomes. In the final stage of import through a nuclear pore, RanGTP binds the cargo receptor importin-beta, causing the release of cargo molecules into the nucleoplasm. It is likely that RanGTP functions in a similar manner during mitosis by causing importin-beta to release cargoes with important mitotic activities in the vicinity of chromatin. There is a growing body of evidence to support this hypothesis from experiments performed using extracts from the eggs of Xenopus laevis, however the details of how this process works in living cells are poorly understood. In addition, it is likely that Ran regulates events in mitosis that have yet to be identified. Our goal is to use chemical biology to identify small molecules that target the Ran pathway. We are proposing the following specific aims: Aim 1) To optimize a screening procedure to identify small molecules that disrupt the interaction between Ran and the transport receptor importin-beta. Aim 2) To develop an assay for use in a high throughput screen to identify compounds that inhibit RanGAP, a protein that regulates the nucleotide state of Ran. Aim 3) To adapt the Xenopus laevis egg extract system to a 384-well format for use in a high throughput screen to identify novel targets in the Ran pathway. Each of our assays takes advantage of fluorescence resonance energy transfer (FRET). In high throughput screens involving these assays we will select inhibitors based on their ability to affect the FRET signal. Our assays rely on both pure protein solutions and complex extracts, each of which has the potential to uncover different types of inhibitors. Inhibitors identified in these screens would serve as invaluable tools for dissecting the role of the Ran pathway during mitosis in live cells. Small molecules identified using high throughput approaches can be developed for the treatment of human disease. Because the Ran pathway is thought to plav an important regulatory role in mitosis, inhibitors that disrupt this pathway would have the potential to be developed as therapeutics for the treatment of cancer.

描述（由申请人提供）：该项目的目标是开发适用于高通量小分子筛选的检测方法，这些小分子筛选旨在靶向小 GTPase Ran 及其相关蛋白。 Ran 调节间期期间的核细胞质运输，最近已证明 Ran 还调节有丝分裂中的关键事件，例如纺锤体组装、核膜组装和核孔复合体组装。在间期和有丝分裂中，RanGTP 在染色体附近富集。在通过核孔输入的最后阶段，RanGTP 结合货物受体 importin-beta，导致货物分子释放到核质中。 RanGTP 很可能在有丝分裂期间以类似的方式发挥作用，通过导致 importin-beta 在染色质附近释放具有重要有丝分裂活性的货物。使用非洲爪蟾卵提取物进行的实验有越来越多的证据支持这一假设，但人们对这一过程如何在活细胞中发挥作用的细节知之甚少。此外，Ran 很可能调节有丝分裂中尚未确定的事件。我们的目标是利用化学生物学来识别针对 Ran 途径的小分子。我们提出以下具体目标： 目标 1) 优化筛选程序，以识别破坏 Ran 和转运受体 importin-beta 之间相互作用的小分子。目标 2) 开发一种用于高通量筛选的检测方法，以鉴定抑制 RanGAP（一种调节 Ran 核苷酸状态的蛋白质）的化合物。目标 3) 使非洲爪蟾卵提取物系统适应 384 孔格式，用于高通量筛选，以识别 Ran 途径中的新靶标。我们的每种检测均利用荧光共振能量转移 (FRET)。在涉及这些测定的高通量筛选中，我们将根据抑制剂影响 FRET 信号的能力来选择抑制剂。我们的检测依赖于纯蛋白质溶液和复杂提取物，每种溶液都有可能发现不同类型的抑制剂。在这些筛选中鉴定出的抑制剂将作为剖析 Ran 通路在活细胞有丝分裂过程中的作用的宝贵工具。使用高通量方法鉴定的小分子可用于治疗人类疾病。由于 Ran 途径被认为在有丝分裂中发挥着重要的调节作用，因此破坏该途径的抑制剂将有可能被开发为治疗癌症的疗法。