Discovery of Novel Proteasome Inhibitors Using a High-Content Cell-Based Screening System

The regulated degradation of damaged or misfolded proteins, as well as down-regulation of key signaling proteins, within eukaryotic and bacterial cells is catalyzed primarily by large, ATP-dependent multimeric proteolytic complexes, termed proteasomes. Inhibition of proteasomal activity affects a wide variety of physiological and pathological processes, and was found to be particularly effective for cancer therapy. We report here on the development of a novel high throughput assay for proteasome inhibition using a unique, highly sensitive live-cell screening, based on the cytoplasm-to-nucleus translocation of a fluorescent proteasome inhibition reporter (PIR) protein, consisting of nuclear localization signal-deficient p53 derivative. We further show here that mdm2, a key negative regulator of p53 plays a key role in the accumulation of PIR in the nucleus upon proteasome inhibition. Using this assay, we have screened the NCI Diversity Set library, containing 1,992 low molecular weight synthetic compounds, and identified four proteasome inhibitors. The special features of the current screen, compared to those of other approaches are discussed.

在真核与细菌细胞中，受损或错误折叠蛋白质的受控降解，以及关键信号蛋白的下调过程，主要由大型ATP依赖型多聚蛋白水解复合物催化，这类复合物被称为蛋白酶体（proteasome）。蛋白酶体活性的抑制可影响诸多生理与病理进程，且已被证实对癌症治疗具有显著疗效。本研究报道了一种全新的高通量蛋白酶体抑制活性检测方法：该技术依托荧光标记的蛋白酶体抑制报告蛋白（proteasome inhibition reporter, PIR）从细胞质向细胞核的转位，采用独特且高灵敏度的活细胞筛选体系，该报告蛋白由缺失核定位信号的p53突变体构成。本研究进一步证实，作为p53关键负调控因子的mdm2，在蛋白酶体受抑制时，可介导PIR在细胞核内的积累。利用该检测方法，我们对包含1992种低分子量合成化合物的NCI多样性集（NCI Diversity Set）文库进行了筛选，成功鉴定出4种蛋白酶体抑制剂。本研究还讨论了本筛选方法相较于其他同类方法的独特优势。