PDF(Pubmed)
Abstract：
Activation of nuclear factor erythroid 2-related factor 2 (Nrf2) by Kelch-like ECH-associated protein 1 (Keap1) alkylation plays a central role in anti-inflammatory therapy. However, activators of Nrf2 through alkylation of Keap1-Kelch domain have not been identified. Deoxynyboquinone (DNQ) is a natural small molecule discovered from marine actinomycetes. The current study was designed to investigate the anti-inflammatory effects and molecular mechanisms of DNQ via alkylation of Keap1. DNQ exhibited significant anti-inflammatory properties both in vitro and in vivo. The pharmacophore responsible for the anti-inflammatory properties of DNQ was determined to be the α, β-unsaturated amides moieties by a chemical reaction between DNQ and N-acetylcysteine. DNQ exerted anti-inflammatory effects through activation of Nrf2/ARE pathway. Keap1 was demonstrated to be the direct target of DNQ and bound with DNQ through conjugate addition reaction involving alkylation. The specific alkylation site of DNQ on Keap1 for Nrf2 activation was elucidated with a synthesized probe in conjunction with liquid chromatography-tandem mass spectrometry. DNQ triggered the ubiquitination and subsequent degradation of Keap1 by alkylation of the cysteine residue 489 (Cys489) on Keap1-Kelch domain, ultimately enabling the activation of Nrf2. Our findings revealed that DNQ exhibited potent anti-inflammatory capacity through α, β-unsaturated amides moieties active group which specifically activated Nrf2 signal pathway via alkylation/ubiquitination of Keap1-Kelch domain, suggesting the potential values of targeting Cys489 on Keap1-Kelch domain by DNQ-like small molecules in inflammatory therapies.
摘要：
通过Kelch样ECH相关蛋白1（Keap1）烷基化激活核因子类2相关因子2（Nrf2）在抗炎治疗中起着重要作用。然而,尚未鉴定通过Keap1-Kelch结构域烷基化的Nrf2活化剂。脱氧醌(DNQ)是从海洋放线菌中发现的一种天然小分子。本研究旨在研究DNQ通过Keap1烷基化的抗炎作用及其分子机制。DNQ在体外和体内均表现出显著的抗炎特性。负责DNQ抗炎特性的药效团被确定为α，β-不饱和酰胺部分通过DNQ和N-乙酰半胱氨酸之间的化学反应。DNQ通过激活Nrf2/ARE途径发挥抗炎作用。Keap1被证明是DNQ的直接靶标，并通过涉及烷基化的缀合加成反应与DNQ结合。用合成探针结合液相色谱-串联质谱法阐明了DNQ在Keap1上用于Nrf2活化的特定烷基化位点。DNQ通过Keap1-Kelch结构域上的半胱氨酸残基489（Cys489）的烷基化触发了Keap1的泛素化和随后的降解，最终激活Nrf2。我们的研究结果表明，DNQ通过α表现出有效的抗炎能力，β-不饱和酰胺部分活性基团通过Keap1-Kelch结构域的烷基化/泛素化特异性激活Nrf2信号通路，提示在炎症治疗中DNQ样小分子靶向Keap1-Kelch结构域Cys489的潜在价值。
