Cellular Signaling in Drug Induced Toxicity

药物引起的肝毒性是两种已批准药物从 药物开发过程中新化学物质的市场和消耗情况； 然而，药物所致肝毒性的机制尚不完全清楚。我们 已经使用了Eefavirenz作为模型，这是一种在某些患者中具有肝毒性的抗逆转录病毒药物 化合物研究细胞信号机制可能在药物- 诱导肝细胞死亡。之前，我们使用原代人类肝细胞证明了 Eefavirenz和Eefavirenz的主要氧化代谢物，记为8-羟基法韦林(8-HYF)。 OHefavirenz)，以依赖于应激激活的方式刺激细胞死亡 激酶c-jun氨基末端激酶与促凋亡蛋白BimEL(Bcl2)的上调 细胞死亡的相互作用介体超长)。随后，我们报道了Eefavirenz可以 也激活肌醇需要酶1α(IRE1α)，这是位于上游的细胞应激的关键调节因子 JNK和BimEL的。这项提议的目标是确定依法韦仑的机制 和8-OHefavirenz激活BimEL和IRE1α，同时也获得了对 IRE1α中的遗传变异如何影响依法韦仑和8-羟基法韦仑诱导的细胞死亡。 重要的是，我们将利用我们通过使用efavirenz作为模型而获得的见解 合成和使用除依法韦仑以外的典型肝毒性药物，即卡马西平， 双氯芬酸和异烟肼，以建立BimEL和IRE1α作为药物- 在一系列药物类别中引起肝毒性。目标如下：(1)测试 假设BimEL作为细胞死亡的刽子手对Eefavirenz和其他 典型肝毒性药物：将BimEL基因缺失的小鼠用于确定是否缺乏 BimEL可预防所研究的肝毒性药物所致的肝毒性； CRISPR/Cas9系统将被用来确定效应蛋白Bax和Bak的作用，即 是BimEL下游调控肝细胞死亡的基因；CRISPR/Cas9和报告基因 检测将被用来定义Eefavirenz、8-OHefavirenz和其他 肝毒性药物调节BimEL的转录；依法韦仑类似物将按顺序使用 阐明Eefavirenz激活BimEL的构效关系；(2)检测 假设Ire 1α是药物所致肝毒性的中心上游调节因子，即 在几类药物的刺激下：我们将确定Eefavirenz、8-OHefavirenz和 其他肝毒性药物可刺激IRE1TRAF2/ASK1/JNK复合体的形成，从而导致α 在IRE1JNK依赖的α激活中，我们将测试自然发生的遗传基因的影响 IRE1α的变种对活性和细胞死亡的影响。预计这些研究将定义BimEL 和IRE1α的激活是一系列药物诱导- 肝脏毒性。

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.