High Throughput Assay Development for ARFGAP Modulators

ARFGAP 调制器的高通量检测开发

基本信息

批准号：
8321745
负责人：
Qisheng Zhang
金额：
$ 3.7万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
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/8321745
关键词：
ADP-Ribosylation Factors Alzheimer&apos s Disease Area Autistic Disorder Automation Biological Assay Breast Cancer Cell Cancer Patient Cell-Cell Adhesion Cells Cellular biology Collection Colorectal Cancer Communities Dimethyl Sulfoxide Disease Drug Delivery Systems Enzymes Fluorescence Polarization GTPase-Activating Proteins Goals Guanosine Triphosphate 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 assay development base cell motility drug development drug discovery high throughput screening inhibitor/antagonist neuron development novel outcome forecast overexpression public health relevance radiotracer small molecule small molecule libraries tool tumor tumor progression

项目摘要

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 GTP 酶激活蛋白 (ARFGAP) 形成一个蛋白家族，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）表达在多种肿瘤中强烈上调，并且该表达与结直肠癌患者的不良无转移生存和预后相关。然而，ARFGAP 调节不同疾病的详细机制尚不清楚。小分子 ARFGAP 调节剂将成为剖析 ARFGAP 调节的细胞信号传导并进一步验证 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，可能是因为目前可用的 ARFGAP 测定不适合高通量筛选。在我们努力剖析 ARFGAP 调节的细胞信号传导过程中，开发了一种新型的基于荧光偏振的 ARFGAP 检测方法。在 384 孔格式中，测定的 Z' 因子为 0.75。当应用于 Prestwick 文库约 1,000 种化合物的初步筛选时，该测定法表现出高重现性、合理的命中率以及自动化的适用性。该提案旨在通过两个具体目标进一步开发这种新的检测方法，以筛选小分子文库中的 ARFGAP 抑制剂： 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 抑制剂测定法将代表第一个不基于放射性的测定法，并且可能会产生 ARFGAP 选择性小分子抑制剂。鉴于 ARFGAP 在基础细胞生物学中发挥的重要作用，从所提出的筛选中产生的任何 ARFGAP 抑制剂都可能在科学界找到广泛的应用。此外，小分子 ARFGAP 抑制剂有潜力作为先导化合物，用于各种疾病（包括癌症和阿尔茨海默氏病）的药物开发。公共健康相关性：拟议的研究涉及 ADP 核糖基化因子 (ARFGAP) 的 GTP 酶激活蛋白 (ARFGAP) 的一个重要且研究不足的领域。拟议的研究与公共卫生相关，因为 ARFGAP 涉及多种疾病，开发的小分子可用作探针来了解 ARFGAP 相互作用网络和药物发现的潜在先导化合物。