Department of Medicinal Chemistry, University of Florida, Gainesville, Florida 32610, United States.
ACS Chem Biol. 2013 Aug 16;8(8):1764-74. doi: 10.1021/cb4000103. Epub 2013 Jun 17.
The cell utilizes the Keap1/Nrf2-ARE signaling pathway to detoxify harmful chemicals in order to protect itself from oxidative stress and to maintain its reducing environment. When exposed to oxidative stress and xenobiotic inducers, the redox sensitive Keap1 is covalently modified at specific cysteine residues. Consequently, the latent transcription factor Nrf2 is stabilized and translocates into the nucleus, where it transactivates the expression of detoxification genes through binding to the antioxidant response element (ARE). In the pursuit of potent and bioavailable activators of the ARE, we validated hits from a pathway-directed high-throughput screening campaign by testing them in cell culture and a reporter strain of a whole animal model, Caenorhabditis elegans. These studies allowed us to identify AI-3 as an ARE activator that induces cytoprotective genes in human cells and in worms, which also translated into in vivo activity in mice. AI-3 is an electrophilic ARE activator with two thiol sensitive sites toward a nucleophilic aromatic substitution, and SAR studies indicated the tunability of the system. Tandem LC-MS analysis revealed that AI-3 alkylates Keap1 primarily at Cys151, while AI-3 is reactive toward additional cysteine residues at higher doses in vitro and in vivo. The immediate effects of such alkylation included the disruption of Keap1-Cul3 (low [AI-3]) and/or Keap1-Nrf2 (high [AI-3]) interactions that both led to the stabilization of Nrf2. This further translated into the downstream Nrf2-ARE regulated cytoprotective gene activation. Collectively, AI-3 may become a valuable biological tool and may even provide therapeutic benefits in oxidative stress related diseases.
细胞利用 Keap1/Nrf2-ARE 信号通路来解毒有害化学物质，以保护自身免受氧化应激，并维持其还原环境。当暴露于氧化应激和外源诱导物时，氧化还原敏感的 Keap1 在特定半胱氨酸残基上发生共价修饰。结果，潜伏转录因子 Nrf2 被稳定下来并易位到细胞核内，在那里它通过与抗氧化反应元件（ARE）结合来转录激活解毒基因的表达。在寻求有效的和可生物利用的 ARE 激活剂的过程中，我们通过在细胞培养和整个动物模型（秀丽隐杆线虫）的报告菌株中测试高内涵筛选活动的命中物，验证了这些命中物。这些研究使我们能够鉴定 AI-3 作为一种 ARE 激活剂，它可以在人类细胞和蠕虫中诱导细胞保护基因，并且在小鼠中也具有体内活性。AI-3 是一种亲电 ARE 激活剂，具有两个对亲核芳香取代敏感的巯基敏感位点，并且 SAR 研究表明该系统具有可调节性。串联 LC-MS 分析表明，AI-3 主要在 Cys151 处烷基化 Keap1，而在体外和体内更高剂量下，AI-3 对其他半胱氨酸残基也具有反应性。这种烷基化的直接影响包括破坏 Keap1-Cul3（低 [AI-3]）和/或 Keap1-Nrf2（高 [AI-3]）相互作用，这两者都导致 Nrf2 的稳定。这进一步转化为下游 Nrf2-ARE 调节的细胞保护基因激活。总的来说，AI-3 可能成为一种有价值的生物学工具，甚至可能在与氧化应激相关的疾病中提供治疗益处。
