High throughput screen for small molecule inhibitors of Ran regulated functions

使用FRET传感器Rango，之前我们进行了两个主要的高通量筛选（HTS）来鉴定进口蛋白β -进口蛋白α结合的小分子抑制剂（实验A）和rangtp诱导的进口蛋白β与进口蛋白α分离的抑制剂（实验B）。随后，我们还开发了化学发光二次筛选，以确定特异性作用的抑制剂。因为在2012- 2013年我们几乎完全专注于检测Ran的细胞功能的项目，所以Ran的小分子抑制剂的开发被搁置了。然而，我们在开发两种二级筛选方面取得了一些进展，这些筛选可在原发性HTS的数百个hit中识别特异性和选择性抑制剂。1)基于活细胞的RanGTP和进口蛋白β调控核进口小分子抑制剂检测本实验是基于表达钙调控GFP-NFAT报告基因的稳定HEK 293T细胞系应用于核进出口。我们使用Importazole（进口蛋白β - rangtp相互作用抑制剂）、Soderholm等人、ACS Chem验证了该方法。医学杂志。2011;（6:700-8）)作为积极对照。2)基于体外筛选RanGTP和进口蛋白复合物小分子抑制剂。该试验旨在鉴定作为蛋白质-蛋白质解离抑制剂而不是蛋白质-蛋白质结合抑制剂的化合物。例如，虽然Importazole在细胞中特异性地破坏了RanGTP调控的进口蛋白β功能，但在经典的生化下拉试验中，它无法检测到破坏RanGTP与进口蛋白β的结合，而且我们的化学发光试验也无法检测到它的活性。因此，我们设计了一种检测方法，在显微镜下直接观察rangtp诱导的荧光产物与微珠结合的进口蛋白β的解离，消除了破坏弱化合物-蛋白质相互作用的洗涤步骤。作为阳性对照，我们在本实验中使用Importazole，发现它确实破坏了rangtp诱导的进口β货物的释放，正如预期的那样。该试验在384孔玻璃底板中进行测试，可用于自动化半高通量显微镜屏幕。2013年6月，我的实验室加入了Pavol Cekan博士，他拥有化学博士学位，并计划继续这个项目。

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). Subsequently we also developed chemiluminescence secondary screen to identify specifically acting inhibitors. Because in 2012-13 we focused almost exclusively on the project examining the cellular functions of Ran, the development of small molecule inhibitors of Ran was on hold. However, we made some progress in developing two secondary screens to identify specific and selective inhibitors among the several hundred hits from the primary HTS. 1) Live cell-based assay for the small molecule inhibitors of RanGTP and importin beta-regulated nuclear import This assay is based on the application of a stable HEK 293T cell line expressing calcium-regulated GFP-NFAT reporter for nuclear import and export. We validated the assay using Importazole (inhibitor of importin beta-RanGTP interaction; Soderholm et al., ACS Chem. Biol. 2011; 6:700-8)) as a positive control. 2) In vitro based screen for small molecule inhibitors of RanGTP and importin beta complex. This assay was designed to identify compounds acting as inhibitors of protein-protein dissociation rather than protein-protein binding. For example, while Importazole specifically disrupts RanGTP-regulated importin beta function in cells, it does not detectably disrupt RanGTP binding to importin beta in classical biochemical pull-downs assays and its activity was also not detectable by our chemiluminescence assay. We therefore designed an assay in which the RanGTP-induced dissociation of fluorescent cargos from microbead-bound importin beta is directly observed under microscope, eliminating washing steps that disrupt weak compound-protein interactions. As a positive control, we used Importazole in this assay and found that it indeed disrupted RanGTP-induced release of importin beta cargos, as expected. This assay was tested in 384-well glass bottom plates that are amenable for automated semi-high throughput microscopy screens. In June 2013, my lab was joined by Dr. Pavol Cekan, who has a Ph.D. in chemistry and is planning to continue in this project.