An HTS approach to screen for antagonists of the nuclear export machinery using high content cell-based assays.

Intracellular localization is essential for the regulated activity of many signaling molecules associated with disease-relevant pathways. High content screening is a powerful technology to monitor the impact of small molecules or interfering RNAs on translocation of proteins within intact cells. Several assays have been developed to measure the nucleocytoplasmic shuttling of proteins like nuclear factor kappaB, FoxO, or nuclear factor of activated T-cells involved in distinct signaling networks. However, since all these proteins bear a leucine-rich nuclear export signal (NES), modulators of the NES-dependent export machinery can lead to misinterpretation of the assay readout. Here we report the generation of U2nesRELOC, a cell-based system for the identification of nuclear export inhibitors and specific silencers of the nuclear export machinery, and its adaptation to high throughput screening. The assay is based on mammalian cells stably expressing green fluorescent protein (GFP)-labeled Rev protein, which contains a strong heterologous NES. The fluorescent signal of untreated U2nesRELOC cells localizes exclusively to the cytoplasm. Upon treatment with the nuclear export inhibitor leptomycin B the GFP-labeled reporter protein accumulates rapidly in the cell nucleus. The assay has been adapted to 96-multiwell format and fully automated. Pilot experiments with a panel of 50 test compounds using three different concentrations per compound resulted in very consistent data sets with excellent reproducibility and an average Z' value of 0.76. In summary, U2nesRELOC is a cell-based nuclear export assay suitable for high throughput screening, providing counterscreens for pathway deconvolution.