Li Min, Wei Jun, Zhu Guofeng, Fu Shufang, He Xiaoyan, Hu Xinqian, Yu Yajie, Mou Yan, Wang Jia, You Xiaoling, Xiao Xin, Gu Tanrong, Ye Zhi, Zha Yunhong
Institute of Neural Regeneration and Repair, The First College of Clinical Medical Science, China Three Gorges University, Yichang, China.
Department of Neurology, The First College of Clinical Medical Science, China Three Gorges University, Yichang, China.
FASEB J. 2023 Oct;37(10):e23178. doi: 10.1096/fj.202300600RR.
Systemic inflammatory response syndrome (SIRS), at least in part driven by necroptosis, is characterized by life-threatening multiple organ failure. Blocking the progression of SIRS and consequent multiple organ dysfunction is challenging. Receptor-interacting serine/threonine protein kinase 1 (RIPK1) is an important cell death and inflammatory mediator, making it a potential treatment target in several diseases. Here, using a drug repurposing approach, we show that inhibiting RIPK1 is also an effective treatment for SIRS. We performed cell-based high-throughput drug screening of an US Food and Drug Administration (FDA)-approved drug library that contains 1953 drugs to identify effective inhibitors of necroptotic cell death by SYTOX green staining. Dose-response validation of the top candidate, quizartinib, was conducted in two cell lines of HT-22 and MEFs. The effect of quizartinib on necroptosis-related proteins was evaluated using western blotting, immunoprecipitation, and an in vitro RIPK1 kinase assay. The in vivo effects of quizartinib were assessed in a murine tumor necrosis factor α (TNFα)-induced SIRS model. High-throughput screening identified quizartinib as the top "hit" in the compound library that rescued cells from necroptosis in vitro. Quizartinib inhibited necroptosis by directly inhibiting RIPK1 kinase activity and blocking downstream complex IIb formation. Furthermore, quizartinib protected mice against TNFα-induced SIRS. Quizartinib, as an FDA-approved drug with proven safety and efficacy, was repurposed for targeted inhibition of RIPK1. This work provides essential preclinical data for transferring quizartinib to the treatment of RIPK1-dependent necroptosis-induced inflammatory diseases, including SIRS.
全身炎症反应综合征（SIRS）至少部分由坏死性凋亡驱动，其特征为危及生命的多器官功能衰竭。阻断SIRS的进展及随之而来的多器官功能障碍具有挑战性。受体相互作用丝氨酸/苏氨酸蛋白激酶1（RIPK1）是一种重要的细胞死亡和炎症介质，使其成为多种疾病的潜在治疗靶点。在此，我们采用药物重新利用方法，表明抑制RIPK1也是治疗SIRS的有效方法。我们对一个包含1953种药物的美国食品药品监督管理局（FDA）批准的药物库进行了基于细胞的高通量药物筛选，通过SYTOX绿染色来鉴定坏死性凋亡细胞死亡的有效抑制剂。对顶级候选药物quizartinib在HT - 22和MEF两种细胞系中进行了剂量反应验证。使用蛋白质印迹法、免疫沉淀法和体外RIPK1激酶测定法评估了quizartinib对坏死性凋亡相关蛋白的影响。在小鼠肿瘤坏死因子α（TNFα）诱导的SIRS模型中评估了quizartinib的体内作用。高通量筛选确定quizartinib为化合物库中的顶级“命中”药物，可在体外使细胞从坏死性凋亡中获救。Quizartinib通过直接抑制RIPK1激酶活性和阻断下游复合物IIb的形成来抑制坏死性凋亡。此外，quizartinib保护小鼠免受TNFα诱导的SIRS。Quizartinib作为一种已证明具有安全性和有效性的FDA批准药物，被重新用于靶向抑制RIPK1。这项工作为将quizartinib转移用于治疗包括SIRS在内的RIPK1依赖性坏死性凋亡诱导的炎症性疾病提供了重要的临床前数据。
