title: High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.
abstract: To discover original inhibitors of the ubiquitin-proteasome pathway, the authors have developed a cell-based bioluminescent assay and used it to screen collections of plant extracts and chemical compounds. They first established a DLD-1 human colon cancer cell line that stably expresses a 4Ubiquitin-Luciferase (4Ub-Luc) reporter protein, efficiently targeted to the ubiquitin-proteasome degradation pathway. The assay was then adapted to 96- and 384-well plate formats and calibrated with reference proteasome inhibitors. Assay robustness was carefully assessed, particularly cell toxicity, and the statistical Z factor value was calculated to 0.83, demonstrating a good performance level of the assay. A total of 18,239 molecules and 15,744 plant extracts and fractions thereof were screened for their capacity to increase the luciferase activity in DLD-1 4Ub-Luc cells, and 21 molecules and 66 extracts inhibiting the ubiquitin-proteasome pathway were identified. The fractionation of an active methanol extract of Physalis angulata L. aerial parts was performed to isolate 2 secosteroids known as physalin B and C. In a cell-based Western blot assay, the ubiquitinated protein accumulation was confirmed after a physalin treatment confirming the accuracy of the screening process. The method reported here thus provides a robust approach to identify novel ubiquitin-proteasome pathway inhibitors in large collections of chemical compounds and natural products.
authors.affiliation: Centre de Criblage Pharmacologique, CNRS-Pierre Fabre Joint Service Unit #2646, Toulouse, France. frederic.ausseil@pierre-fabre.com
authors.forename: Frederic
authors.initials: F
authors.lastname: Ausseil
authors.forename: Arnaud
authors.initials: A
authors.lastname: Samson
authors.forename: Yannick
authors.initials: Y
authors.lastname: Aussagues
authors.forename: Isabelle
authors.initials: I
authors.lastname: Vandenberghe
authors.forename: Laurent
authors.initials: L
authors.lastname: Creancier
authors.forename: Isabelle
authors.initials: I
authors.lastname: Pouny
authors.forename: Anna
authors.initials: A
authors.lastname: Kruczynski
authors.forename: Georges
authors.initials: G
authors.lastname: Massiot
authors.forename: Christian
authors.initials: C
authors.lastname: Bailly
mesh_terms: D002452:Cell Count; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005591:Chemical Fractionation; D064420:Drug-Related Side Effects and Adverse Reactions; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007783:Lactones / Q000737:chemistry / Q000494:pharmacology; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010936:Plant Extracts / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D061988:Proteasome Inhibitors; D015203:Reproducibility of Results; D012632:Secosteroids; D013256:Steroids / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000037:antagonists & inhibitors*
chemical_list: D004791:Enzyme Inhibitors; D007783:Lactones; D009363:Neoplasm Proteins; D009842:Oligopeptides; D010936:Plant Extracts; D061988:Proteasome Inhibitors; D012632:Secosteroids; D013256:Steroids; D025801:Ubiquitin; C033270:physalin B; D008156:Luciferases; C078846:epoxomicin