High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources
High-throughput bioluminescence screening of ubiquitin-proteasome pathway inhibitors from chemical and natural sources
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DOI：
10.1177/1087057106296494
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发表时间：
2007-02-01
影响因子：
--
通讯作者：
Bailly, Christian
中科院分区：
文献类型：
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作者：
Ausseil, Frederic;Samson, Arnaud;Bailly, Christian
为了发现泛素-蛋白酶体途径的原始抑制剂，作者开发了一种基于细胞的生物发光测定法，并用它来筛选植物提取物和化合物的集合。他们首先建立了DLD-1人结肠癌细胞系，该细胞系稳定表达4Ub-Luc报告蛋白，有效靶向泛素-蛋白酶体降解途径。然后将该测定法适应于96孔和384孔板形式，并用参比蛋白酶体抑制剂校准。仔细评估了试验耐用性，特别是细胞毒性，并将统计Z'因子值计算为0.83，证明试验性能水平良好。筛选了总共18，239种分子和15，744种植物提取物及其级分，以确定它们在DLD-1 4Ub-Luc细胞中增加荧光素酶活性的能力，并鉴定了21种分子和66种提取物抑制泛素-蛋白酶体途径。对苦酸浆甲醇提取物的活性成分进行了分级分离。进行地上部分分离，以分离2种开环甾类化合物，称为酸浆素B和C。在基于细胞的蛋白质印迹分析中，在酸浆素处理后证实了泛素化蛋白质的积累，从而证实了筛选过程的准确性。因此，这里报道的方法提供了一个强大的方法来确定新的泛素-蛋白酶体途径抑制剂的化学化合物和天然产物的大集合。
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.