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This version was published on February 1, 2007
Journal of Biomolecular Screening, Vol. 12, No. 1, 106-116 (2007)
DOI: 10.1177/1087057106296494

High-Throughput Bioluminescence Screening of Ubiquitin-Proteasome Pathway Inhibitors from Chemical and Natural Sources

Frederic Ausseil

Arnaud Samson

Yannick Aussagues

Centre de Criblage Pharmacologique, CNRS—Pierre Fabre Joint Service Unit #2646, Toulouse, France

Isabelle Vandenberghe

Laurent Creancier

Centre de Recherche en Oncologie Expérimentale—Institut de Recherche Pierre Fabre, Toulouse, France

Isabelle Pouny

Chimie des Substances Naturelles Bio-Actives, CNRS—Pierre Fabre Joint Service Unit #2597, Toulouse, France

Anna Kruczynski

Centre de Recherche en Oncologie Expérimentale—Institut de Recherche Pierre Fabre, Toulouse, France

Georges Massiot

Chimie des Substances Naturelles Bio-Actives, CNRS—Pierre Fabre Joint Service Unit #2597, Toulouse, France

Christian Bailly

Centre de Recherche en Oncologie Expérimentale—Institut de Recherche Pierre Fabre, Toulouse, France

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 ubiquitinproteasome 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.

Key Words: proteasome • ubiquitin • screening • natural product • bioluminescence • Physalis angulata L • physalin


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