High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources.
J Biomol Screen
; 12(1): 106-16, 2007 Feb.
Article
en En
| MEDLINE
| ID: mdl-17175525
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.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Extractos Vegetales
/
Ubiquitina
/
Inhibidores Enzimáticos
/
Inhibidores de Proteasoma
/
Luciferasas
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
/
Screening_studies
Límite:
Humans
Idioma:
En
Revista:
J Biomol Screen
Asunto de la revista:
BIOLOGIA MOLECULAR
Año:
2007
Tipo del documento:
Article
País de afiliación:
Francia