High Throughput Assay Development for ARFGAP Modulators

ARFGAP 调制器的高通量检测开发

• 批准号: 8049810
• 负责人: Qisheng Zhang
• 金额: $ 7.34万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-27 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8049810
• 关键词: ADP-Ribosylation Factors; Alzheimer' s Disease; Area; Autistic Disorder; Automation; Biological Assay; Breast Cancer Cell; Cancer Patient; Cell Adhesion; Cells; Cellular biology; Collection; Colorectal Cancer; Communities; Dimethyl Sulfoxide; Disease; Drug Delivery Systems; Enzymes; Fluorescence Polarization; GTPase-Activating Proteins; Goals; Human; Lead; Libraries; Life; Literature; Malignant Neoplasms; Measures; Migration Assay; Neoplasm Metastasis; Normal tissue morphology; Play; Protein Family; Public Health; Radioactivity; Radiolabeled; Reaction; Reproducibility; Research; Role; Signal Transduction; Temperature; Testing; Time; abstracting; assay development; base; drug development; drug discovery; high throughput screening; inhibitor/antagonist; migration; neuron development; novel; outcome forecast; overexpression; radiotracer; small molecule; small molecule libraries; tool; tumor; tumor progression

DESCRIPTION (provided by applicant): GTPase-activating proteins of ADP-ribosylation factors (ARFGAPs) form a family of proteins that play key roles in cell adhesion and migration, tumor progression, as well as neuronal development. A body of evidence also indicates that ARFGAPs are involved in various diseases, including cancer, Alzheimer's disease, and autism. For example, ASAP1 (one ARFGAP) expression is strongly up-regulated in a variety of tumors in comparison with normal tissue and that this expression correlates with poor metastasis-free survival and prognosis in colorectal cancer patients. However, the detailed mechanism by which ARFGAPs regulate different diseases is largely unknown. Small molecule ARFGAP modulators will be invaluable tools to dissect ARFGAP-regulated cell signaling and further validate ARFGAPs as drug targets. However, there are no small molecule ARFGAP inhibitors in the literature possibly due to the fact that the currently available ARFGAP assays are not amenable to high throughput screening. In our efforts to dissect ARFGAP-regulated cell signaling, a novel fluorescence polarization-based ARFGAP assay has been developed. The Z' factor of the assay is 0.75 in 384-well format. When applied to a pilot screen of the Prestwick library of around 1,000 compounds, the assay demonstrated high reproducibility, reasonable hit rates, and suitability for automation. This proposal seeks to further develop this novel assay to screen libraries of small molecules for ARFGAP inhibitors through two specific aims: 1) Optimize assay conditions that are suitable for high throughput screen in 384-well format; and 2) Configure the assay that is developed in aim 1 for high throughput screening of small molecule library. The proposed assay for ARFGAP inhibitors will represent the first such assay that is not based on radioactivity and will likely generate ARFGAP-selective small molecule inhibitors. Given the important roles ARFGAPs played in basic cell biology, any ARFGAP inhibitor that is generated from the proposed screen will likely find broad applications in the scientific community. Furthermore, the small molecule ARFGAP inhibitors have the potential to function as lead compounds for drug development in various diseases including cancer and Alzheimer's disease. PUBLIC HEALTH RELEVANCE: The proposed studies are of an important and under-investigated area of GTPase-activating proteins of ADP-ribosylation factors (ARFGAPs). The proposed research has relevance to public health, because ARFGAPs are involved in various diseases, and the small molecules developed could be used as probes to understand the ARFGAP interaction network and potential lead compounds for drug discovery.

描述（由申请人提供）：adp核糖基化因子的gtpase激活蛋白（ARFGAPs）形成了一个蛋白家族，在细胞粘附和迁移、肿瘤进展以及神经元发育中发挥关键作用。大量证据还表明，arfgap与多种疾病有关，包括癌症、阿尔茨海默病和自闭症。例如，与正常组织相比，ASAP1（一种ARFGAP）在多种肿瘤中的表达强烈上调，并且该表达与结直肠癌患者较差的无转移生存和预后相关。然而，ARFGAPs调控不同疾病的详细机制在很大程度上是未知的。小分子ARFGAP调节剂将成为解剖ARFGAP调控的细胞信号传导和进一步验证ARFGAP作为药物靶点的宝贵工具。然而，文献中没有小分子ARFGAP抑制剂，这可能是由于目前可用的ARFGAP检测方法不适合高通量筛选。为了剖析ARFGAP调控的细胞信号，我们开发了一种新的基于荧光偏振的ARFGAP检测方法。在384孔格式下，该分析的Z′因子为0.75。当应用于Prestwick库中约1000种化合物的先导筛选时，该分析显示出高重复性，合理的命中率和自动化适用性。本提案旨在通过两个特定目标进一步开发这种新的检测方法来筛选ARFGAP抑制剂的小分子文库：1)优化适合384孔格式高通量筛选的检测条件；2)配置目标1中开发的检测方法，用于小分子文库的高通量筛选。拟议的ARFGAP抑制剂检测将是第一个不基于放射性的检测，并可能产生ARFGAP选择性小分子抑制剂。鉴于ARFGAP在基础细胞生物学中的重要作用，任何由该筛选产生的ARFGAP抑制剂都可能在科学界找到广泛的应用。此外，小分子ARFGAP抑制剂有潜力作为包括癌症和阿尔茨海默病在内的各种疾病药物开发的先导化合物。