Implementation of a high-throughput screen for identifying small molecules to activate the Keap1-Nrf2-ARE pathway.
Implementation of a high-throughput screen for identifying small molecules to activate the Keap1-Nrf2-ARE pathway.
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DOI：
10.1371/journal.pone.0044686
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Klaassen CD
中科院分区：
文献类型：
--
作者：
Wu KC;McDonald PR;Liu JJ;Chaguturu R;Klaassen CD
核因子红细胞2相关因子2 （Nrf2）是一种转录因子，通过与位于这些基因启动子区域的抗氧化反应元件（ARE）结合，诱导一系列参与抗氧化防御的细胞保护基因。为了鉴定用于治疗氧化/亲电应激诱导疾病的Nrf2激活因子，本研究开发了一种高通量测定方法，利用ARE启动子控制下含有荧光素酶基因的AREc32细胞来评估Nrf2的激活。在筛选的47000种化合物中，238种（前0.5%）化学物质的发光信号增加了14.4倍以上，并在0.01-30µM范围内的11种浓度下重新测试。在这238种化合物中，231种（96%）以浓度依赖的方式增加了发光信号。化学结构关系分析表明，这231个化合物富集了四种化学支架（二芳酰酰胺和二芳酰脲、恶唑和噻唑、吡喃酮和噻喃酮、吡啶酮和吡啶嗪酮）。此外，这231种化合物中有30种对激活Nrf2非常有效和/或有效，发光增加超过80倍，EC50低于1.6µM。这前30个化合物也在Hepa1c1c7细胞中进行了筛选，以增加Nqo1 mRNA，这是典型的nrf2靶基因。在这30种化合物中，17种以浓度依赖的方式增加Nqo1 mRNA。总之，本研究记录了一种高通量筛选方法的开发、实施和验证，以识别Keap1-Nrf2-ARE通路的激活因子。筛选结果确定了Nrf2激活因子，并为化学支架提供了新的见解，这些化学支架可能阻止氧化/亲电应激诱导的毒性和致癌作用。
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01–30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.
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影响因子：
14.9
作者：
Malhotra D;Portales-Casamar E;Singh A;Srivastava S;Arenillas D;Happel C;Shyr C;Wakabayashi N;Kensler TW;Wasserman WW;Biswal S
通讯作者：
Biswal S
影响因子：
10.5
作者：
Itoh, K;Wakabayashi, N;Yamamoto, M
通讯作者：
Yamamoto, M
影响因子：
3.8
作者：
Reisman, Scott A.;Csanaky, Ivan L.;Klaassen, Curtis D.
通讯作者：
Klaassen, Curtis D.
DOI：
10.1073/pnas.172398899
发表时间：
2002-09-03
影响因子：
11.1
作者：
Dinkova-Kostova, AT;Holtzclaw, WD;Talalay, P
通讯作者：
Talalay, P
DOI：
10.1073/pnas.0500815102
发表时间：
2005-03-22
影响因子：
11.1
作者：
Dinkova-Kostova, AT;Liby, KT;Talalay, P
通讯作者：
Talalay, P