y Identification of compounds that inhibit the binding of Keap1a/Keap1b Kelch DGR domain with Nrf2 ETGE/DLG motifs in zebrafish
y Identification of compounds that inhibit the binding of Keap1a/Keap1b Kelch DGR domain with Nrf2 ETGE/DLG motifs in zebrafish
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DOI：
10.1111/bcpt.13222
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发表时间：
2019-09-01
影响因子：
3.1
通讯作者：
Perumal, Ekambaram
中科院分区：
文献类型：
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作者：
Raghunath, Azhwar;Nagarajan, Raju;Perumal, Ekambaram
Keap1-Nrf2-ARE系统是抑制氧化应激的主要防御机制，氧化应激是各种疾病衰老和发病的主要原因之一。Nrf2是细胞防御系统的主要调节因子，其激活仍然是治疗慢性疾病的前瞻性方法。最近的一种策略是通过化合物占据Keap1中阻断与Nrf2界面的位置来破坏Keap1-Nrf2蛋白-蛋白相互作用（PPI），这种相互作用改变了Keap1与Nrf2的对接。在本研究中，我们试图通过斑马鱼的硅分子对接，鉴定出具有抗癌、抗氧化和抗炎功能的化合物来破坏Keap1a/b-Nrf2 PPI。Keap1蛋白的系统发育分析表明，包括斑马鱼在内的低等脊椎动物中存在同源的Keap1- nrf2 - are系统。以小家鼠Keap1 （PDB ID:5CGJ）为模板，利用model9.19对斑马鱼Keap1a和Keap1b的DGR结构域进行建模。根据对接计算，确定了破坏Keap1a和Keap1b与Nrf2相互作用的顶级hit化合物，包括槲皮素3,4 '-二葡糖苷、黄素腺嘌呤二核苷酸二钠盐水合物、丹酚酸A、tunicamycin和esculin。esculin在3只dpf斑马鱼幼鱼中的LC50值为5 mmol/L， qRT-PCR结果显示esculin显著增加了Nrf2靶基因gstpi、Nqo1、Hmox1a和Prdx1在3只dpf斑马鱼幼鱼中的转录。这些潜在的靶点由于其对Keap1-Nrf2 PPI的非共价破坏，可以作为更安全的Nrf2激活剂，并被开发成各种疾病的有效预防/治疗药物。
The Keap1-Nrf2-ARE system serves as a premier defence mechanism to curb oxidative stress, which remains as one of the major causes of ageing and pathogenesis in various diseases. Nrf2 is the principal master regulator of the cellular defence system, and its activation remains the prospective therapeutic approach against chronic diseases. One of the recent strategies is to disrupt Keap1-Nrf2 protein-protein interaction (PPI) that alters the docking of Keap1 with Nrf2 by compounds occupying a position in the Keap1 blocking the interface with Nrf2. In this study, we made an attempt to identify the compounds with anticancer, antioxidant and anti-inflammatory properties to disrupt Keap1a/b-Nrf2 PPI through in silico molecular docking in zebrafish. The phylogenetic analysis of Keap1 proteins revealed the existence of orthologous Keap1-Nrf2-ARE system in lower vertebrates that includes zebrafish. The DGR domains of zebrafish Keap1a and Keap1b were modelled with Modeller 9.19 using Keap1 of Mus musculus (PDB ID:5CGJ) as template. Based on the docking calculations, top hit compounds were identified to disrupt both Keap1a and Keap1b interaction with Nrf2 which include quercetin 3,4 '-diglucoside, flavin adenine dinucleotide disodium salt hydrate, salvianolic acid A, tunicamycin and esculin. The LC50 of esculin in 3 dpf zebrafish larvae is 5 mmol/L, and the qRT-PCR results showed that esculin significantly increased the transcription of Nrf2 target genes-Gstpi, Nqo1, Hmox1a and Prdx1 in 3 dpf zebrafish larvae. These potential hits could serve as safer Nrf2 activators due to their non-covalent disruption of Keap1-Nrf2 PPI and be developed into efficacious preventive/therapeutic agents for various diseases.