High Throughput Assay Development for ARFGAP Modulators

ARFGAP 调制器的高通量检测开发

• 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
• 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' 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

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（由申请方提供）：ADP-核糖基化因子的GTP酶激活蛋白（ARFGAP）形成一个蛋白质家族，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与各种疾病有关，包括癌症、阿尔茨海默病和自闭症。例如，与正常组织相比，ASAP 1（一种ARFGAP）表达在多种肿瘤中强烈上调，并且这种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在很大程度上是未知的。小分子ARFGAP调节剂将是分析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，这可能是由于目前可用的ARFGAP测定不适合高通量筛选。 在我们努力剖析ARFGAP调节的细胞信号转导中，已经开发了一种新的基于荧光偏振的ARFGAP测定法。在384孔格式中，测定的Z'因子为0.75。当应用于Prestwick库的约1，000种化合物的中试筛选时，该测定显示出高重现性，合理的命中率和自动化的适用性。 该提案旨在通过两个特定目标进一步开发这种新型测定以筛选ARFGAP抑制剂的小分子文库：1）优化适合于384孔格式的高通量筛选的测定条件;和2）配置在目标1中开发的测定用于小分子文库的高通量筛选。拟定的ARFGAP抑制剂测定法将代表第一个不基于放射性的此类测定法，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发挥的重要作用，从拟议的筛选中产生的任何ARFGAP抑制剂都可能在科学界获得广泛应用。此外，小分子ARFGAP抑制剂具有作为用于包括癌症和阿尔茨海默病在内的各种疾病的药物开发的先导化合物的潜力。 公共卫生关系：这些研究是ADP核糖基化因子GTP酶激活蛋白（ARFGAP）的一个重要和未充分研究的领域。 这项研究与公共卫生有关，因为ARFGAP与各种疾病有关，开发的小分子可用作探针，以了解ARFGAP相互作用网络和药物发现的潜在先导化合物。