1,2,4-Oxadiazole derivatives, a class of Nrf2-ARE activators, exert an extensive therapeutic effect on inflammation, cancer, neurodegeneration, and microbial infection. Among these analogues, DDO-7263 is the most potent Nrf2 activator and used as the core structure for bioactive probes to explore the precise mechanism. In this work, we obtained compound 7, a mimic of DDO-7263, and biotin-labeled and fluorescein-based probes, which exhibited homologous biological activities to DDO-7263, including activating Nrf2 and its downstream target genes, anti-oxidative stress, and anti-inflammatory effects. Affinity chromatography and mass analysis techniques revealed Rpn6 as the potential target protein regulating the Nrf2 signaling pathway. In vitro affinity experiments further confirmed that DDO-7263 upregulated Nrf2 through binding to Rpn6 to block the assembly of 26S proteasome and the subsequent degradation of ubiquitinated Nrf2. These results indicated that Rpn6 is a promising candidate target to activate the Nrf2 pathway for protecting cells and tissues from oxidative, electrophilic, and exogenous microbial stimulation.

中文翻译：

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Target Fishing 揭示了 1,2,4-恶二唑衍生物靶向 26S 蛋白酶体亚基 Rpn6 的新机制

1,2,4-恶二唑衍生物是一类 Nrf2-ARE 激活剂，对炎症、癌症、神经变性和微生物感染具有广泛的治疗作用。在这些类似物中，DDO-7263是最有效的 Nrf2 激活剂，用作生物活性探针的核心结构以探索其精确机制。在这项工作中，我们获得了化合物7 ， DDO-7263的模拟物，以及生物素标记和基于荧光素的探针，它们表现出与DDO-7263同源的生物活性，包括激活 Nrf2 及其下游靶基因、抗氧化应激和抗炎作用。亲和层析和质谱分析技术表明 Rpn6 是调节 Nrf2 信号通路的潜在靶蛋白。体外亲和实验进一步证实，DDO-7263通过与 Rpn6 结合来上调 Nrf2，从而阻断 26S 蛋白酶体的组装和随后泛素化 Nrf2 的降解。这些结果表明，Rpn6 是激活 Nrf2 通路以保护细胞和组织免受氧化、亲电和外源微生物刺激的有前途的候选靶标。