Cellular Signaling in Drug-induced Toxicity

描述（由申请人提供）：Efavirenz （EFV）和nevirapine （NVP）是目前处方的具有肝毒性的同类抗逆转录病毒药物。最近，细胞信号通路在体外用原代人肝细胞调节EFV代谢下游毒性中的主要作用已被报道。氧化应激的增加和促凋亡蛋白BimEL （Bcl-2相互作用的细胞死亡超长介质）的上调已被证明是刺激efv诱导的细胞死亡的关键，NVP似乎通过相同的机制调节肝细胞死亡。本提案的目的是确定EFV和NVP上调BimEL的方式，同时也获得对BimEL下游介导肝细胞死亡事件的机制理解。实验目的如下：(1)测试促凋亡基因BimEL的缺乏是否对EFV和nvp介导的细胞死亡具有保护作用：BimEL缺失小鼠将被用来确定BimEL缺失是否使小鼠对这些化合物刺激的肝毒性产生抗性；肝细胞中的RNA干扰研究将用于确定BimEL下游效应蛋白Bax和Bak在调节EFV和nvp诱导的肝细胞死亡中的作用；siRNA和报告基因检测将用于确定EFV和NVP调节BimEL转录的机制；(2)确定在原代人肝细胞中被抗逆转录病毒激活的促凋亡通路是否在接受抗逆转录病毒治疗的猕猴中也被激活；(3)探讨谷胱甘肽通路改变在EFV和nvp诱导的肝毒性中的作用；这些化合物对谷胱甘肽途径的影响将使用基于质谱的方法进行检测，该方法将精确定量该途径的中间体/产物；RNA干扰将用于确定谷胱甘肽途径中酶在EFV和nvp诱导的肝毒性中的作用。预计完成拟议的研究将导致确定新的治疗靶点，以潜在地预防和治疗EFV和nvp介导的肝毒性。

DESCRIPTION (provided by applicant): Efavirenz (EFV) and nevirapine (NVP) are currently prescribed antiretroviral drugs of the same class that are hepatotoxic. Recently, a major role for cellular signaling pathways in modulating toxicity downstream of EFV metabolism has been reported in vitro using primary human hepatocytes. Increased oxidative stress and the upregulation of the proapoptotic protein BimEL (Bcl-2 interacting mediator of cell death extra long) have been demonstrated to be crucial in the stimulation of EFV-induced cell death, and NVP appears to modulate hepatocyte death via the same mechanism. The goal of this proposal is to determine the manner in which EFV and NVP upregulate BimEL, while also gaining a mechanistic understanding of the events downstream of BimEL that mediate the hepatocyte death. The aims are as follows: (1) Test whether genetic deficiency of proapoptotic BimEL results in protection against EFV- and NVP-mediated cell death in vivo: BimEL null mice will be used to determine whether the absence of BimEL renders mice resistant to hepatotoxicity stimulated by these compounds; RNA interference studies in hepatocytes will be used to determine the role of effector proteins, Bax and Bak, that are downstream of BimEL in modulating EFV- and NVP-induced hepatocyte death; siRNA and reporter gene assays will be used to define the mechanism by which EFV and NVP regulate the transcription of BimEL; (2) determine if proapoptotic pathways that are activated by antiretrovirals in primary human hepatocytes are also activated in macaques receiving antiretroviral therapy; (3) probe a role for alteration of the glutathione pathway in EFV- and NVP-induced hepatotoxicity; the effects of these compounds on the glutathione pathway will be examined using a mass spectrometry-based approach that will precisely quantitate the intermediates/products of this pathway; RNA interference will be used to define the role of enzymes in the glutathione pathway in EFV- and NVP-induced hepatotoxicity. It is expected that completion of the proposed studies will lead to the identification of novel therapeutic targets for the potential prevention and treatment of EFV- and NVP-mediated hepatotoxicity.