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High throughput screen for small molecule inhibitors of Ran regulated functions

Ran 调节功能的小分子抑制剂的高通量筛选

基本信息

批准号：
8937878
负责人：
Petr Kalab
金额：
$ 0.59万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8937878
关键词：
Antimitotic Agents Binding Bioinformatics Biological Assay Calcium Cell Cycle Cell Line Cell Nucleus Cell division Cells Chemical Structure Chemicals Chromatin Clinical Trials Complex Cytoplasm Development Dissociation Fluorescence Fluorescence Resonance Energy Transfer Funding Genomics Glass Goals Growth Human Human Resources Importins Interphase Lead Life Malignant Neoplasms Microscope Microspheres Mitosis Mitotic Mitotic Cell Cycle Monitor Monomeric GTP-Binding Proteins Normal Cell Nuclear Export Nuclear Import Oncogenic Phase Physiological Property Publishing Regulation Reporter Research Resources Role Running Somatic Cell Suspension substance Suspensions TACC3 gene Testing Therapeutic Time United States National Institutes of Health alpha Karyopherins aurora-A kinase base beta Karyopherins cancer cell chemotherapy design high throughput screening in vitro Assay inhibitor/antagonist karyopherin alpha 2 kinase inhibitor loss of function macromolecule research study screening sensor small molecule

项目摘要

Using FRET sensor Rango, previously we performed two primary high throughput screens (HTS) to identify small molecule inhibitors of importin beta-importin alpha binding (Assay A) and inhibitors of RanGTP-induced importin beta dissociation from importin alpha (Assay B). However, owing to the limited personnel in the lab, most of our time and resources had to be focused on our main project in the lab, the studies of the mitotic and cell cycle functions of Ran. Since the completion of the primary screens in 2011, we adapted or developed several types of secondary screens to identify specific and selective inhibitors among the primary screen hits. Through testing several different approaches, we identified two types of assays as suitable for this purpose. The first is a live cell-based assay with a stable HEK 293T cell line expressing calcium-regulated GFP-NFAT reporter for nuclear import and export. We validated this assay using Importazole (inhibitor of importin beta-RanGTP interaction; Soderholm et al., ACS Chem. Biol. 2011; 6:700-8)) as a positive control. As the second assay for the secondary screens, we developed the "bead suspension assay". In this assay, importin beta is immobilized on microbeads and its interaction with fluorescently tagged importin beta binding domain (IBB) is monitored under microscope. Since the IBB-importin beta interaction is regulated by RanGTP, the disruption of RanGTP binding to importin by an inhibitor is visualized via persistent bead-bound fluorescence. We validated this assay with importazole, using 384-well glass bottom plates that would be suitable for high throughput screening.

使用 FRET 传感器 Rango，我们之前进行了两次主要的高通量筛选 (HTS)，以鉴定输入蛋白 β-输入蛋白 α 结合的小分子抑制剂（检测 A）和 RanGTP 诱导的输入蛋白 β 与输入蛋白 α 解离的抑制剂（检测 B）。但由于实验室人员有限，我们的大部分时间和资源都集中在实验室的主要项目——Ran的有丝分裂和细胞周期功能的研究上。自 2011 年完成初级筛选以来，我们改编或开发了几种类型的二级筛选，以识别初级筛选命中中的特异性和选择性抑制剂。通过测试几种不同的方法，我们确定了两种适合此目的的检测方法。第一个是基于活细胞的测定，使用稳定的 HEK 293T 细胞系，表达钙调节的 GFP-NFAT 报告基因，用于核导入和导出。我们使用导入唑（导入蛋白 β-RanGTP 相互作用的抑制剂；Soderholm 等人，ACS Chem. Biol. 2011；6:700-8)）作为阳性对照验证了该测定。作为二次筛选的第二种测定，我们开发了“珠悬浮测定”。在此测定中，输入蛋白 β 固定在微珠上，并在显微镜下监测其与荧光标记的输入蛋白 β 结合域 (IBB) 的相互作用。由于 IBB-importin beta 相互作用受 RanGTP 调节，因此抑制剂对 RanGTP 与 importin 结合的破坏可通过持久的珠子结合荧光可视化。我们使用适合高通量筛选的 384 孔玻璃底板，用 importzo 验证了该测定。