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High-Throughput Bioluminescence Screening of Ubiquitin-Proteasome Pathway Inhibitors from Chemical and Natural Sources
1 Centre de Criblage Pharmacologique, CNRS--Pierre Fabre Joint Service Unit #2646, Toulouse, France.
* To whom correspondence should be addressed. E-mail: frederic.ausseil{at}pierre-fabre.com.
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. Key Words: proteasome, ubiquitin, screening, natural product, bioluminescence, Physalis angulata L., physalin
First published on December 14, 2006, doi:10.1177/1087057106296494 |
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