High throughput screen for small molecule inhibitors of Ran regulated functions

使用FRET传感器Rango，之前我们进行了两次高通量筛选(HTS)来鉴定导入素β-输入素α结合的小分子抑制剂(Assay A)和RanGTP诱导的Importinβ与Importinα解离的抑制剂(Assay B)。然而，由于实验室人员有限，我们不得不将大部分时间和资源集中在实验室的主要项目上，即研究RAN的有丝分裂和细胞周期功能。自2011年完成初级筛选以来，我们改编或开发了几种类型的次级筛选，以在初级筛选中识别特定和选择性的抑制剂。通过测试几种不同的方法，我们确定了两种适合于这一目的的检测方法。第一个是基于活细胞的检测，稳定的HEK 293T细胞系表达钙调控的GFP-NFAT报告，用于核进出口。我们使用Importazole(Importinβ-RanGTP相互作用的抑制剂；Soderholm等人，ACS Chem)验证了这一方法。比奥尔。2011；6：700-8))作为阳性对照。作为二次筛查的第二种方法，我们发展了“珠悬浮法”。在本实验中，将Importinβ固定在微球上，并在显微镜下观察其与荧光标记的Importinβ结合结构域(IBB)的相互作用。由于IBB-Importinβ的相互作用受RanGTP的调节，因此通过持续的微珠结合荧光可以观察到抑制剂对RanGTP与Importin结合的破坏。我们用Importazole验证了这一分析，使用了适合高通量筛选的384孔玻璃底板。

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.