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.

[1]  Jing Zhang,et al.  High-content screening moves to the front of the line. , 2006, Drug discovery today.

[2]  B. K. Lundholt,et al.  Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen , 2006, Journal of biomolecular screening.

[3]  K. Yeow,et al.  Cellular imaging in drug discovery , 2006, Nature Reviews Drug Discovery.

[4]  A. Sanfridson,et al.  Inflammatory pathway analysis using a high content screening platform. , 2005, Assay and drug development technologies.

[5]  B. Harrison,et al.  The CRM1 Nuclear Export Receptor Controls Pathological Cardiac Gene Expression , 2004, Molecular and Cellular Biology.

[6]  S. Møller,et al.  Nuclear export inhibitors and kinase inhibitors identified using a MAPK-activated protein kinase 2 redistribution screen. , 2004, Assay and drug development technologies.

[7]  Pamela A. Silver,et al.  A chemical genetic screen identifies inhibitors of regulated nuclear export of a Forkhead transcription factor in PTEN-deficient tumor cells. , 2003, Cancer cell.

[8]  Peter Washausen,et al.  Ratjadones inhibit nuclear export by blocking CRM1/exportin 1. , 2003, Experimental cell research.

[9]  R. Seger,et al.  Non-regulated and stimulated mechanisms cooperate in the nuclear accumulation of MEK1 , 2001, Oncogene.

[10]  Roger L. Williams,et al.  Structural determinants of phosphoinositide 3-kinase inhibition by wortmannin, LY294002, quercetin, myricetin, and staurosporine. , 2000, Molecular cell.

[11]  B. Henderson,et al.  A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals. , 2000, Experimental cell research.

[12]  P. Silver,et al.  Regulated nuclear localization of stress-responsive factors: how the nuclear trafficking of protein kinases and transcription factors contributes to cell survival , 1999, Oncogene.

[13]  N. Kudo,et al.  Leptomycin B inactivates CRM1/exportin 1 by covalent modification at a cysteine residue in the central conserved region. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[15]  C. Marshall,et al.  Nuclear export of the stress-activated protein kinase p38 mediated by its substrate MAPKAP kinase-2 , 1998, Current Biology.

[16]  M. Yanagida,et al.  Leptomycin B inhibition of signal-mediated nuclear export by direct binding to CRM1. , 1998, Experimental cell research.

[17]  Minoru Yoshida,et al.  CRM1 is responsible for intracellular transport mediated by the nuclear export signal , 1997, Nature.

[18]  Minoru Yoshida,et al.  CRM1 Is an Export Receptor for Leucine-Rich Nuclear Export Signals , 1997, Cell.

[19]  U. Kutay,et al.  Transport between the cell nucleus and the cytoplasm. , 1999, Annual review of cell and developmental biology.

[20]  Y. Wang,et al.  Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA. , 1997, Chemistry & biology.