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Abstract：
Oxidative stress due to abnormal accumulation of reactive oxygen species (ROS) is an initiator of a large number of human diseases, and thus, the elimination and prevention of excessive ROS are important aspects of preventing the development of such diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is an essential transcription factor that defends against oxidative stress, and its function is negatively controlled by Kelch-like ECH-associated protein 1 (KEAP1). Therefore, activating NRF2 by inhibiting KEAP1 is viewed as a strategy for combating oxidative stress-related diseases. Here, we generated a cereblon (CRBN)-based proteolysis-targeting chimera (PROTAC), which we named SD2267, that induces the proteasomal degradation of KEAP1 and leads to NRF2 activation. As was intended, SD2267 bound to KEAP1, recruited CRBN, and induced the degradation of KEAP1. Furthermore, the KEAP1 degradation efficacy of SD2267 was diminished by MG132 (a proteasomal degradation inhibitor) but not by chloroquine (an autophagy inhibitor), which suggested that KEAP1 degradation by SD2267 was proteasomal degradation-dependent and autophagy-independent. Following KEAP1 degradation, SD2267 induced the nuclear translocation of NRF2, which led to the expression of NRF2 target genes and attenuated ROS accumulation induced by acetaminophen (APAP) in hepatocytes. Based on in vivo pharmacokinetic study, SD2267 was injected intraperitoneally at 1 or 3 mg/kg in APAP-induced liver injury mouse model. We observed that SD2267 degraded hepatic KEAP1 and attenuated APAP-induced liver damage. Summarizing, we described the synthesis of a KEAP1-targeting PROTAC (SD2267) and its efficacy and mode of action in vitro and in vivo. The results obtained suggest that SD2267 could be used to treat hepatic diseases related to oxidative stress.
摘要：
由于活性氧(ROS)的异常积累而引起的氧化应激是人类大量疾病的引发剂,因此，消除和预防过量ROS是预防此类疾病发展的重要方面。核因子红系2相关因子2（NRF2）是防御氧化应激的必需转录因子，其功能受Kelch样ECH相关蛋白1（KEAP1）的负控制。因此,通过抑制KEAP1激活NRF2被视为对抗氧化应激相关疾病的策略。这里,我们产生了一个基于cereblon（CRBN）的蛋白水解靶向嵌合体（PROTAC），我们将其命名为SD2267，它诱导KEAP1的蛋白酶体降解并导致NRF2激活。按照预期，SD2267绑定到KEAP1，招募CRBN，并诱导KEAP1的降解。此外，MG132（一种蛋白酶体降解抑制剂）降低了SD2267的KEAP1降解功效，但氯喹（一种自噬抑制剂）没有降低，这表明SD2267对KEAP1的降解是蛋白酶体降解依赖性和自噬依赖性。KEAP1降解后，SD2267诱导NRF2的核易位，导致NRF2靶基因的表达，并减弱对乙酰氨基酚（APAP）在肝细胞中诱导的ROS积累。基于体内药代动力学研究，在APAP诱导的肝损伤小鼠模型中，以1或3mg/kg腹膜内注射SD2267。我们观察到SD2267降解肝KEAP1并减轻APAP诱导的肝损伤。总结，我们描述了KEAP1靶向PROTAC(SD2267)的合成及其在体外和体内的功效和作用方式.结果表明，SD2267可用于治疗与氧化应激相关的肝脏疾病。
