title: Discovery of novel proteasome inhibitors using a high-content cell-based screening system.
abstract: The regulated degradation of damaged or misfolded proteins, as well as down-regulation of key signaling proteins, within eukaryotic and bacterial cells is catalyzed primarily by large, ATP-dependent multimeric proteolytic complexes, termed proteasomes. Inhibition of proteasomal activity affects a wide variety of physiological and pathological processes, and was found to be particularly effective for cancer therapy. We report here on the development of a novel high throughput assay for proteasome inhibition using a unique, highly sensitive live-cell screening, based on the cytoplasm-to-nucleus translocation of a fluorescent proteasome inhibition reporter (PIR) protein, consisting of nuclear localization signal-deficient p53 derivative. We further show here that mdm2, a key negative regulator of p53 plays a key role in the accumulation of PIR in the nucleus upon proteasome inhibition. Using this assay, we have screened the NCI Diversity Set library, containing 1,992 low molecular weight synthetic compounds, and identified four proteasome inhibitors. The special features of the current screen, compared to those of other approaches are discussed.
authors.affiliation: Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, Israel.
authors.forename: Irena
authors.initials: I
authors.lastname: Lavelin
authors.forename: Avital
authors.initials: A
authors.lastname: Beer
authors.forename: Zvi
authors.initials: Z
authors.lastname: Kam
authors.forename: Varda
authors.initials: V
authors.lastname: Rotter
authors.forename: Moshe
authors.initials: M
authors.lastname: Oren
authors.forename: Ami
authors.initials: A
authors.lastname: Navon
authors.forename: Benjamin
authors.initials: B
authors.lastname: Geiger
mesh_terms: D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D055808:Drug Discovery / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D054547:National Cancer Institute (U.S.); D061988:Proteasome Inhibitors; D015202:Protein Engineering; D021381:Protein Transport / Q000187:drug effects; D051736:Proto-Oncogene Proteins c-mdm2 / Q000378:metabolism; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis / Q000494:pharmacology; D014481:United States
chemical_list: D004791:Enzyme Inhibitors; D061988:Proteasome Inhibitors; D054852:Small Molecule Libraries; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2