A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells
A Multiplexed High-Content Screening Approach Using the Chromobody Technology to Identify Cell Cycle Modulators in Living Cells
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DOI：
10.1177/1087057116641935
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发表时间：
2016-10-01
影响因子：
--
通讯作者：
Hadian, Kamyar
中科院分区：
文献类型：
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作者：
Schorpp, Kenji;Rothenaigner, Ina;Hadian, Kamyar
许多筛选命中显示出关于后期疗效和安全性的相对较差的质量。因此，小分子筛选工作转向提供更详细信息的高含量分析。在这里，我们描述了一种新的筛选方法，以确定细胞周期调节剂与低毒性相结合的细胞周期染色体（CCC）技术与CytoTox-Glo（CTG）细胞毒性试验。CCC技术采用细胞内功能性单结构域抗体偶联到荧光蛋白（染色体），以可视化活细胞中增殖细胞核抗原（PCNA）的细胞周期依赖性再分布。将这种基于图像的细胞周期分析与通过CTG测定法测定细胞培养上清液中的死细胞蛋白酶活性相结合。我们采用这种多重方法来高通量格式，并筛选了960种食品和药物管理局（FDA）批准的药物。由此，我们鉴定了调节不同细胞周期阶段的无毒化合物，并在稳定表达CCC的不同细胞系中验证了选定的命中。此外，我们通过流式细胞术独立地验证了这些命中，作为当前最先进的细胞周期分析格式。这项研究表明，CCC成像是一种多功能的高内容筛选方法，以确定细胞周期调节剂，它可以与细胞毒性试验在筛选过程中早期消除有毒化合物的多路复用。
Many screening hits show relatively poor quality regarding later efficacy and safety. Therefore, small-molecule screening efforts shift toward high-content analysis providing more detailed information. Here, we describe a novel screening approach to identify cell cycle modulators with low toxicity by combining the Cell Cycle Chromobody (CCC) technology with the CytoTox-Glo (CTG) cytotoxicity assay. The CCC technology employs intracellularly functional single-domain antibodies coupled to a fluorescent protein (chromobodies) to visualize the cell cycle-dependent redistribution of the proliferating cell nuclear antigen (PCNA) in living cells. This image-based cell cycle analysis was combined with determination of dead-cell protease activity in cell culture supernatants by the CTG assay. We adopted this multiplex approach to high-throughput format and screened 960 Food and Drug Administration (FDA)-approved drugs. By this, we identified nontoxic compounds, which modulate different cell cycle stages, and validated selected hits in diverse cell lines stably expressing CCC. Additionally, we independently validated these hits by flow cytometry as the current state-of-the-art format for cell cycle analysis. This study demonstrates that CCC imaging is a versatile high-content screening approach to identify cell cycle modulators, which can be multiplexed with cytotoxicity assays for early elimination of toxic compounds during screening.