Huang Zhengwan, Peng Zhengang, Huang Dandan, Zhou Zhongyu
Bloomage Biotechnology Corporation Limited, Shanghai 200100, China.
Guangdong Provincial Key Laboratory of Applied Botany & Key Laboratory of National Forestry and Grassland Administration on Plant Conservation and Utilization in Southern China, South China Botanical Garden, Guangzhou 510650, China.
Molecules. 2025 Apr 17;30(8):1815. doi: 10.3390/molecules30081815.
The transcription factor erythroid 2-related factor 2 (Nrf2) is a central regulator of cellular defense mechanisms against oxidative stress and inflammation. Keap1 (Kelch-like ECH-associated protein 1) regulates Nrf2 activity by ubiquitination-mediated cytoplasmic retention, thereby suppressing its nuclear translocation and subsequent transcriptional activation of genes encoding phase II detoxifying enzymes. Using a structure-based virtual screening approach, we screened ~16,000 natural compounds to identify Keap1-Nrf2 PPI inhibitors. Nine compounds were identified based on their high binding affinities and favorable interactions with Keap1, primarily through non-covalent interactions. To validate the binding stability of these inhibitors, molecular dynamics (MD) simulations were performed, confirming the robustness of the Keap1-inhibitor complexes over time. Subsequent in vitro assays on human epithelial keratinocyte cells (HaCaT) revealed that six of these compounds notably upregulated Nrf2 mRNA expression, regis tering increases from 23% to 50% in comparison to the control. Notably, chebulinic acid emerged as the most potent compound, demonstrating the greatest elevation in Nrf2 expression. Penetration studies further showed that chebulinic acid, when encapsulated in silk fibroin, achieved a 0.14% penetration rate after 24 h though it could not penetrate into the stratum corneum alone. This result highlighted the potential of chebulinic acid in the use of anti-aging skincare formulations. Collectively, our findings affirmed that molecular docking is a reliable and effective approach for the identification of novel anti-aging agents targeting the Keap1-Nrf2 pathway.
转录因子红细胞2相关因子2（Nrf2）是细胞对抗氧化应激和炎症防御机制的核心调节因子。Keap1（类 Kelch 样 ECH 相关蛋白1）通过泛素化介导的细胞质滞留来调节 Nrf2 活性，从而抑制其核转位以及随后对编码 II 期解毒酶基因的转录激活。我们采用基于结构的虚拟筛选方法，筛选了约16,000种天然化合物以鉴定 Keap1-Nrf2 蛋白-蛋白相互作用抑制剂。基于其高结合亲和力以及与 Keap1 的良好相互作用（主要通过非共价相互作用），鉴定出了九种化合物。为验证这些抑制剂的结合稳定性，进行了分子动力学（MD）模拟，证实了 Keap1-抑制剂复合物随时间的稳定性。随后对人上皮角质形成细胞（HaCaT）进行的体外试验表明，其中六种化合物显著上调了 Nrf2 mRNA 表达，与对照相比增加了23%至50%。值得注意的是，诃子次酸是最有效的化合物，其 Nrf2 表达升高幅度最大。渗透研究进一步表明，诃子次酸包裹在丝素蛋白中时，24小时后渗透率达到0.14%，不过它单独无法穿透角质层。这一结果突出了诃子次酸在抗衰老护肤品配方中的应用潜力。总体而言，我们的研究结果证实分子对接是一种可靠且有效的方法，可用于鉴定靶向 Keap1-Nrf2 途径的新型抗衰老剂。
