DNA cleavage assay for the identification of topoisomerase I inhibitors
DNA cleavage assay for the identification of topoisomerase I inhibitors
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DOI：
10.1038/nprot.2008.174
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Pommier, Yves
中科院分区：
文献类型：
--
作者：
Dexheimer, Thomas S.;Pommier, Yves
抑制DNA拓扑异构酶I(Top1)已被证明是设计抗癌药物的一种成功方法。然而，尽管喜树碱衍生物在临床上取得了成功，但对毒性更低、化学更稳定的Top1抑制剂的需求仍然很大。在这里，我们描述了最常用的方案之一，以确定新的Top1抑制剂。这些方法使用独特的3‘-放射性标记的DNA底物和变性聚丙烯酰胺凝胶电泳法，为潜在的Top1抑制剂的Top1介导的DNA切割活性提供证据。这些检测方法可以比较不同药物在稳定Top1-DNA中间体或裂解(可切割)复合体方面的有效性。还提出了这些方法的一个变种，这为通过测量药物诱导的Top1-DNA切割复合体的可逆性来确定抑制剂是否阻止正向切割或宗教反应提供了一个合适的系统。整个协议可以在类似于2天的时间内完成。
The inhibition of DNA topoisomerase I (Top1) has proven to be a successful approach in the design of anticancer agents. However, despite the clinical successes of the camptothecin derivatives, a significant need for less toxic and more chemically stable Top1 inhibitors still persists. Here, we describe one of the most frequently used protocols to identify novel Top1 inhibitors. These methods use uniquely 3'-radiolabeled DNA substrates and denaturing polyacrylamide gel electrophoresis to provide evidence for the Top1-mediated DNA cleaving activity of potential Top1 inhibitors. These assays allow comparison of the effectiveness of different drugs in stabilizing the Top1-DNA intermediate or cleavage (cleavable) complex. A variation on these assays is also presented, which provides a suitable system for determining whether the inhibitor blocks the forward cleavage or religation reactions by measuring the reversibility of the drug-induced Top1-DNA cleavage complexes. This entire protocol can be completed in similar to 2 d.