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.

药物诱导的肝毒性是撤出批准药物的主要原因 在药物开发过程中，新化学实体的市场和属性； 但是，尚未完全了解药物诱导的肝毒性的机制。我们 已经使用Efavirenz是一种在某些患者中是肝毒性的抗逆转录病毒药物，作为模型 化合物研究可能在药物中起因果作用的细胞信号传导机制 诱导肝细胞死亡。以前，使用原代人肝细胞，我们证明了 efavirenz和Efavirenz的主要氧化代谢产物，称为8-羟基甲韦伦兹（8-- ohefavirenz），以依赖压力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 相互作用的细胞死亡介质超长）。随后，我们报告了efavirenz可以 还激活需要酶1α（IRE1α）的肌醇，这是一个在上游的细胞应激的关键调节剂 JNK和Bimel的著作。该提案的目的是确定efavirenz的机制 8-ohefavirenz激活Bimel和IRE1α，同时也获得了对 IRE1α的遗传变异如何影响Efavirenz和8- ohefavirenz诱导的细胞死亡。 重要的是，我们将利用通过使用efavirenz作为模型获得的见解 复合和员工原型肝毒性药物以外的efavirenz，即卡马西平， 双氯芬酸和异烟肼，以建立BIMEL和IRE1α作为药物的中心调节剂 在各种药物类别中诱导的肝毒性。目的如下：（1）测试 假设Bimel在响应Efavirenz和其他 原型肝毒性药物：Bimel Null小鼠将用于确定是否不存在 BIMEL可防止此处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， 在调节肝细胞死亡时，是Bimel的下游； CRISPR/CAS9和记者基因 测定将用于定义Efavirenz，8-Ohefavirenz等的机制 肝毒性药物调节Bimel的转录； Efavirenz类似物将按顺序雇用 阐明efavirenz Bimel激活的结构活性关系； （2）测试 假设IRE1α是药物诱导的肝毒性的中央上游调节剂 受到几类药物的刺激：我们将确定efavirenz，8-ohefavirenz和 其他肝毒性药物刺激IRE1α/TRAF2/Ask1/JNK复合物的形成 在IRE1α依赖性激活中；我们将测试自然存在的遗传的影响 IRE1α对活性和细胞死亡的变体。预计这些研究将定义Bimel IRE1α激活是重要的分子机制，通过这些机制，一系列药物诱导 肝毒性。