High throughput screen for small molecule inhibitors of Ran regulated functions

使用FRET传感器Rango，先前我们进行了两次初级高通量筛选（HTS）以鉴定输入蛋白β-输入蛋白α结合的小分子抑制剂（测定A）和RanGTP诱导的输入蛋白β从输入蛋白α解离的抑制剂（测定B）。然而，由于实验室人员有限，我们的大部分时间和资源不得不集中在我们的主要项目在实验室，有丝分裂和细胞周期的功能的研究Ran。自2011年完成初步筛选以来，我们调整或开发了几种类型的二次筛选，以在初步筛选中鉴定特异性和选择性抑制剂。通过测试几种不同的方法，我们确定了两种类型的测定适合于此目的。第一种是基于活细胞的测定，其使用表达用于核输入和输出的钙调节的GFP-NFAT报告基因的稳定HEK 293 T细胞系。我们使用Importazole（输入蛋白β-RanGTP相互作用的抑制剂; Soderholm等人，2011; 6：700-8））作为阳性对照。作为二次筛选的第二次测定，我们开发了“珠悬浮测定”。在该测定中，输入蛋白β被固定在微珠上，并在显微镜下监测其与荧光标记的输入蛋白β结合结构域（伊布）的相互作用。由于IBB-输入蛋白β相互作用受RanGTP调节，抑制剂对RanGTP与输入蛋白结合的破坏通过持续的珠结合荧光显现。我们使用适合于高通量筛选的384孔玻璃底板，用Importazole验证了该测定。

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.