Department of Pharmacology and Molecular Sciences (C.J.S.H., N.N.B.) and Department of Medicine, Division of Clinical Pharmacology (A.N.H., N.N.B.), Johns Hopkins University School of Medicine, Baltimore, Maryland.
Department of Pharmacology and Molecular Sciences (C.J.S.H., N.N.B.) and Department of Medicine, Division of Clinical Pharmacology (A.N.H., N.N.B.), Johns Hopkins University School of Medicine, Baltimore, Maryland
Mol Pharmacol. 2019 Feb;95(2):183-195. doi: 10.1124/mol.118.113647. Epub 2018 Nov 15.
Efavirenz (EFV), a widely used antiretroviral drug, is associated with idiosyncratic hepatotoxicity and dyslipidemia. Here we demonstrate that EFV stimulates the activation in primary hepatocytes of key cell stress regulators: inositol-requiring 1 (IRE1) and X-box binding protein 1 (XBP1). Following EFV exposure, XBP1 splicing (indicating activation) was increased 35.7-fold in primary human hepatocytes. In parallel, XBP1 splicing and IRE1 phosphorylation (p-IRE1, active IRE1) were elevated 36.4-fold and 4.9-fold, respectively, in primary mouse hepatocytes. Of note, with EFV treatment, 47.2% of mouse hepatocytes were apoptotic; which was decreased to 23.9% in the presence of STF 083010, an inhibitor of XBP1 splicing. Experiments performed using pregnane X receptor (PXR)-null mouse hepatocytes revealed that EFV-mediated XBP1 splicing and hepatocyte death were not dependent on PXR, which is a nuclear receptor transcription factor that plays a crucial role in the cellular response to xenobiotics. Interestingly, incubation with the primary metabolite of EFV, 8-hydroxyefavirenz (8-OHEFV), only resulted in 10.3- and 2.9-fold increased XBP1 splicing in human and mouse hepatocytes and no change in levels of p-IRE1 in mouse hepatocytes. To further probe the structure-activity relationship of IRE1-XBP1 activation by EFV, 16 EFV analogs were employed. Of these, an analog in which the EFV alkyne is replaced with an alkene and an analog in which the oxazinone oxygen is replaced by a carbon stimulated XBP1 splicing in human, mouse, and macaque hepatocytes. These data demonstrate that EFV and compounds sharing the EFV scaffold can activate IRE1-XBP1 across human, mouse, and macaque species.
依非韦伦（EFV）是一种广泛使用的抗逆转录病毒药物，与特异质肝毒性和血脂异常有关。在这里，我们证明 EFV 刺激原代肝细胞中关键细胞应激调节剂的激活：肌醇需求 1（IRE1）和 X 盒结合蛋白 1（XBP1）。EFV 暴露后，原代人肝细胞中 XBP1 剪接（表示激活）增加了 35.7 倍。平行地，XBP1 剪接和 IRE1 磷酸化（p-IRE1，活性 IRE1）分别升高了 36.4 倍和 4.9 倍。值得注意的是，EFV 处理后，47.2%的小鼠肝细胞发生凋亡；而在 XBP1 剪接抑制剂 STF 083010 的存在下，凋亡率降低至 23.9%。使用妊娠相关 X 受体（PXR）缺失型小鼠肝细胞进行的实验表明，EFV 介导的 XBP1 剪接和肝细胞死亡不依赖于 PXR，PXR 是一种核受体转录因子，在细胞对外源物的反应中起着至关重要的作用。有趣的是，与 EFV 的主要代谢物 8-羟基依非韦伦（8-OHEFV）孵育仅导致人源和鼠源肝细胞中 XBP1 剪接分别增加 10.3 倍和 2.9 倍，而鼠源肝细胞中 p-IRE1 水平没有变化。为了进一步探究 EFV 激活 IRE1-XBP1 的结构-活性关系，我们使用了 16 种 EFV 类似物。其中，一种将 EFV 炔烃替换为烯烃的类似物和一种将恶嗪酮氧替换为碳的类似物，在人、鼠和食蟹猴肝细胞中刺激 XBP1 剪接。这些数据表明，EFV 和具有 EFV 支架的化合物可以激活人类、小鼠和食蟹猴物种的 IRE1-XBP1。
