title: An AlphaScreen®-based assay for high-throughput screening for specific inhibitors of nuclear import.
abstract: Specific viral proteins enter the nucleus of infected cells to perform essential functions, as part of the viral life cycle. The integrase (IN) molecule of human immunodeficiency virus (HIV)-1 is of particular interest in this context due to its integral role in integrating the HIV genome into that of the infected host cell. Most IN-based antiviral compounds target the IN/DNA interaction, but since IN must first enter the nucleus before it can perform these critical functions, nuclear transport of IN is also an attractive target for therapeutic intervention. Here the authors describe a novel high-throughput screening assay for identifying inhibitors of nuclear import, particularly IN, based on amplified luminescent proximity homogeneous assay (AlphaScreen(®)) technology, which is high throughput, requires low amounts of material, and is efficient and cost-effective. The authors use the assay to screen for specific inhibitors of the interaction between IN and its nuclear transport receptor importin α/β, successfully identifying several inhibitors of the IN/importin α/β interaction. Importantly, they demonstrate that one of the identified compounds, mifepristone, is effective in preventing active nuclear transport of IN in transfected cells and hence may represent a useful anti-HIV therapeutic. The screen also identified broad-spectrum importin α/β inhibitors such as ivermectin, which may represent useful tools for nuclear transport research in the future. The authors validate the activity and specificity of mifepristone and ivermectin in inhibiting nuclear protein import in living cells, underlining the utility of the screening approach.
authors.affiliation: Nuclear Signalling Laboratory, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
authors.forename: Kylie M
authors.initials: KM
authors.lastname: Wagstaff
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Rawlinson
authors.forename: Anna C
authors.initials: AC
authors.lastname: Hearps
authors.forename: David A
authors.initials: DA
authors.lastname: Jans
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000977:Antiparasitic Agents / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism*; D055808:Drug Discovery / Q000379:methods*; D019427:HIV Integrase / Q000378:metabolism; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006727:Hormone Antagonists / Q000494:pharmacology; D006801:Humans; D007559:Ivermectin / Q000494:pharmacology; D015735:Mifepristone / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D028901:alpha Karyopherins / Q000378:metabolism; D028961:beta Karyopherins / Q000378:metabolism
chemical_list: D000977:Antiparasitic Agents; D006727:Hormone Antagonists; D028901:alpha Karyopherins; D028961:beta Karyopherins; D015735:Mifepristone; D007559:Ivermectin; D019427:HIV Integrase