The Kelch-like ECH-associated protein 1 (KEAP1) - nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway senses reactive oxygen species and regulates cellular oxidative stress. Inhibiting KEAP1 to activate the NRF2 antioxidant response has been proposed as a promising strategy to treat chronic diseases caused by oxidative stress. Here, we developed a proteolysis targeting chimera (PROTAC) that depletes KEAP1 from cells through the ubiquitin-proteasome pathway. A previously developed KEAP1 inhibitor and thalidomide were incorporated in the heterobifunctional design of the PROTAC as ligands for KEAP1 and CRBN recruitment, respectively. Optimization of the chemical composition and linker length resulted in PROTAC 14 which exhibited potent KEAP1 degradation with low nanomolar DC₅₀ in HEK293T (11 nM) and BEAS-2B (<1 nM) cell lines. Furthermore, PROTAC 14 increased the expression of NRF2 regulated antioxidant proteins and prevented cell death induced by reactive oxygen species. Together, these results established a blueprint for further development of KEAP1-targeted heterobifunctional degraders and will facilitate the study of the biological consequences of KEAP1 removal from cells. This approach represents an alternative therapeutic strategy to existing treatments for diseases caused by oxidative stress.

Kelch 样 ECH 相关蛋白 1 (KEAP1) - 核因子红细胞 2 相关因子 2 (NRF2) 信号通路可感知活性氧并调节细胞氧化应激。抑制 KEAP1 以激活 NRF2 抗氧化反应已被提议作为治疗由氧化应激引起的慢性疾病的有前途的策略。在这里，我们开发了一种蛋白水解靶向嵌合体 (PROTAC)，它通过遍在蛋白-蛋白酶体途径耗尽细胞中的 KEAP1。先前开发的 KEAP1 抑制剂和沙利度胺被纳入 PROTAC 的异双功能设计中，分别作为 KEAP1 和 CRBN 募集的配体。化学组成和接头长度的优化导致 PROTAC 14表现出有效的 KEAP1 降解和低纳摩尔 DC ₅₀在 HEK293T (11 nM) 和 BEAS-2B (<1 nM) 细胞系中。此外，PROTAC 14增加了 NRF2 调节的抗氧化蛋白的表达，并防止了活性氧诱导的细胞死亡。总之，这些结果为进一步开发以 KEAP1 为目标的异双功能降解剂建立了蓝图，并将促进研究从细胞中去除 KEAP1 的生物学后果。这种方法代表了对氧化应激引起的疾病的现有治疗方法的替代治疗策略。