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.