Cellular Signaling in Drug Induced Toxicity

药物引起的毒性中的细胞信号转导

• 批准号: 10227081
• 负责人: Namandje N Bumpus
• 金额: $ 36.03万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10227081
• 关键词: Acute Liver Failure; Address; Adverse event; Anti-Retroviral Agents; Antiepileptic Agents; Antiviral Agents; Apoptosis; Area; BIM Bcl-2-binding protein; Biological Assay; CRISPR/Cas technology; Carbamazepine; Cell Death; Cells; Cellular Stress; Cessation of life; Chemicals; Clinical; Clinical Data; Clinical Markers; Complex; Cyclic AMP-Dependent Protein Kinases; Data; Diclofenac; Endoribonucleases; Enzymes; Event; Foundations; Genes; Genetic; Genetic Transcription; Genetic Variation; Genomic DNA; Genotype; Goals; Hepatic; Hepatocyte; Hepatotoxicity; Histology; Human; Image; Individual; Inositol; Knockout Mice; Knowledge; Lead; Life; Liver; MAPK8 gene; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Medicine; Mind; Mitochondria; Modeling; Molecular; Mus; Mutagenesis; Non-Steroidal Anti-Inflammatory Agents; Pathogenesis; Patients; Pharmaceutical Preparations; Play; Prevention; Process; Protein Kinase; Proteins; Regulation; Reporter Genes; Reporting; Resistance; Role; Severities; Signal Pathway; Signal Transduction; Signaling Molecule; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stimulus; Structure-Activity Relationship; System; TRAF2 gene; Testing; Therapeutic; Toxic effect; United States; Up-Regulation; Withdrawal; Work; World Health Organization; analog; drug development; drug distribution; drug market; efavirenz; genetic variant; insight; isoniazid; knock-down; liver injury; prevent; prototype; response; sensor; stress kinase; tuberculosis drugs

Drug-induced hepatotoxicity is a leading cause of both the withdrawal of approved drugs from the market and the attrition of new chemical entities during the drug development process; however, the mechanisms underlying drug-induced hepatotoxicity are not fully understood. We have used efavirenz, an antiretroviral drug that is hepatotoxic in certain patients, as a model compound to investigate cellular signaling mechanisms that may play a causal role in drug- induced hepatocyte death. Previously, using primary human hepatocytes, we demonstrated that efavirenz and the major oxidative metabolite of efavirenz, denoted as 8-hydroxyefavirenz (8- OHefavirenz), stimulate cell death in a manner that is dependent upon activation of the stress kinase c-Jun N-terminal kinase and upregulation of the proapoptotic protein BimEL (Bcl-2 interacting mediator of cell death extra long). Subsequently, we have reported that efavirenz can also activate inositol requiring enzyme 1α (IRE1α), a key regulator of cell stress that lies upstream of JNK and BimEL. The goal of this proposal is to determine the mechanism by which efavirenz and 8-OHefavirenz activate BimEL and IRE1α, while also gaining a mechanistic understanding of how genetic variation in IRE1α might impact efavirenz and 8-OHefavirenz-induced cell death. Importantly, we will leverage the insights we have gained through using efavirenz as a model compound and employ prototypic hepatotoxic drugs beyond efavirenz, namely carbamazepine, diclofenac and isoniazid, in order to establish BimEL and IRE1α as central regulators of drug- induced hepatotoxicity across a range of drug classes. The aims are as follows: (1) to test the hypothesis that BimEL acts as an executioner of cell death in response to efavirenz and other prototypic hepatotoxic drugs: BimEL null mice will be used to determine whether the absence of BimEL prevents hepatotoxicity stimulated by the hepatotoxic drugs being investigated here; CRISPR/Cas9 systems will be used to determine the role of effector proteins, Bax and Bak, that are downstream of BimEL in modulating hepatocyte death; CRISPR/Cas9 and reporter gene assays will be used to define the mechanism by which efavirenz, 8-OHefavirenz and other hepatotoxic drugs regulate the transcription of BimEL; efavirenz analogs will be employed in order to elucidate the structure-activity relationship of BimEL activation by efavirenz; (2) to test the hypothesis that IRE1α is a central upstream regulator of drug-induced hepatotoxicity that is stimulated by several classes of drugs: we will determine whether efavirenz, 8-OHefavirenz, and other hepatotoxic drugs stimulate formation of the IRE1α/TRAF2/ASK1/JNK complex that results in IRE1α-dependent activation of JNK; we will test the impact of naturally occurring genetic variants of IRE1α on activity and cell death. It is expected that these studies will define BimEL and IRE1α activation as important molecular mechanisms by which a range of drugs induce- hepatotoxicity.

药物引起的肝毒性是批准药物撤回的主要原因 药物开发过程中新化学实体的市场和消耗； 然而，药物引起的肝毒性的机制尚不完全清楚。 使用依非韦伦（efavirenz）作为模型，依非韦伦是一种抗逆转录病毒药物，对某些患者具有肝毒性 化合物来研究可能在药物中发挥因果作用的细胞信号传导机制 此前，我们使用原代人肝细胞证明了这一点。 依非韦伦和依非韦伦的主要氧化代谢产物，表示为 8-羟基依非韦伦 (8- OHefavirenz），以依赖于应激激活的方式刺激细胞死亡 激酶 c-Jun N 末端激酶和促凋亡蛋白 BimEL (Bcl-2) 的上调 随后，我们报道了依非韦伦可以 还激活肌醇需要酶 1α (IRE1α)，这是上游细胞应激的关键调节因子 JNK 和 BimEL 的目标是确定依非韦伦的作用机制。 和 8-OHefavirenz 激活 BimEL 和 IRE1α，同时还获得了以下机制的理解 IRE1α 的遗传变异如何影响依非韦伦和 8-OH 依非韦伦诱导的细胞死亡。 重要的是，我们将利用通过使用依非韦伦作为模型获得的见解 合成并使用依非韦伦以外的原型肝毒性药物，即卡马西平， 双氯芬酸和异烟肼，以便将 BimEL 和 IRE1α 确立为药物的中央调节剂 一系列药物类别的诱导肝毒性目的如下：（1）测试 假设 BimEL 作为细胞死亡的刽子手来响应依非韦伦和其他药物 原型肝毒性药物：BimEL 无效小鼠将用于确定是否缺乏 BimEL 可防止此处正在研究的肝毒性药物刺激的肝毒性； CRISPR/Cas9 系统将用于确定效应蛋白 Bax 和 Bak 的作用， 是 BimEL 调节肝细胞死亡的下游； 测定将用于确定依非韦伦、8-OH依非韦伦和其他药物的作用机制 肝毒性药物可调节 BimEL 类似物的转录； (2) 测试 假设 IRE1α 是药物引起的肝毒性的中央上游调节因子 受到几类药物的刺激：我们将确定依非韦伦、8-OH依非韦伦和 其他肝毒性药物刺激 IRE1α/TRAF2/ASK1/JNK 复合物的形成，从而导致 在 IRE1α 依赖性 JNK 激活中，我们将测试自然发生的遗传的影响； IRE1α 的变体对活性和细胞死亡的影响预计这些研究将定义 BimEL。 IRE1α 激活是一系列药物诱导的重要分子机制 肝毒性。