In vitro activity: Importazole is an effective inhibitor of the Ran/importin-β interaction in vitro. It inhibits proliferation and induces apoptosis of multiple myeloma cells by blocking the NF-κB signal pathway in vitro. Importazole specifically blocks importin-β-mediated nuclear import both in Xenopus egg extracts and cultured cells, without disrupting transportin-mediated nuclear import or CRM1-mediated nuclear export. When added during mitosis, importazole impairs the release of an importin-β cargo FRET probe and causes both predicted and novel defects in spindle assembly.

Kinase Assay: Importazole, a 2,4-diaminoquinazoline identified from a FRET-based, high-throughput small molecule screen for compounds that interfere with the interaction between RanGTP and importin-β, is a small molecule inhibitor of the transport receptor importin-β that specifically blocks importin-β-mediated nuclear import. It is an effective inhibitor of the Ran/importin-β interaction in vitro.

Cell Assay: For all import and export experiments, HEK 293 cells stably expressing NFAT-GFP are grown on glass coverslips to approximately 50% confluency prior to drug treatment. In all cases, importazole is used at 40 μM and leptomycin B is used at 10 ng/ml. For controls, DMSO is used at a concentration of 0.4%. Ionomycin is added at 1.25 μM. Importazole and leptomycin B treatments are all for 1 hour. In all experiments cells are fixed with 4% formaldehyde prior to fluorescence microscopy. DNA is visualized with 1 μg/ml Hoechst dye. For quantification, 100 cells from each condition are analyzed and the percentage that shows nuclear accumulation of NFAT-GFP calculated.