Cellular Signaling in Drug Induced Toxicity

药物诱导的肝毒性是导致批准的药物从药物治疗中撤回的主要原因。 药物开发过程中新化学实体的市场和消耗; 然而，药物诱导的肝毒性的潜在机制尚未完全了解。我们 我用依非韦伦作为模型，依非韦伦是一种抗逆转录病毒药物，在某些患者中具有肝毒性 化合物，以研究细胞信号传导机制，可能发挥因果作用的药物- 诱导肝细胞死亡。以前，使用原代人肝细胞，我们证明， 依法韦仑和依法韦仑的主要氧化代谢物，表示为8-羟基依法韦仑（8-hydroxyefavirenz）， 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 细胞死亡的相互作用介质（interacting mediator of cell death extra long）随后，我们报道了依法韦仑可以 还激活肌醇需要酶1α（IRE 1 α），一种位于上游的细胞应激的关键调节因子 JNK和Bimel的。本提案的目的是确定依法韦仑 和8-OHefavirenz激活BimEL和IRE 1 α，同时也获得了 IRE 1 α的遗传变异如何影响依法韦仑和8-OHefavirenz诱导的细胞死亡。 重要的是，我们将利用我们通过使用依法韦仑作为模型获得的见解 复合应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，以确定BimEL和IRE 1 α作为药物的中心调节剂， 在一系列药物类别中诱导肝毒性。目的如下：（1）测试 假设BimEL作为响应于依法韦仑和其他药物细胞死亡的刽子手 原型肝毒性药物：将使用BimEL无效小鼠来确定是否不存在 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和巴克的作用， 在调节肝细胞死亡中位于BimEL的下游; CRISPR/Cas9和报告基因 将使用测定来确定依法韦仑、8-OHefavirenz和其他 肝毒性药物调节BimEL的转录;依非韦伦类似物将按顺序使用 阐明依非韦伦激活BimEL的构效关系;（2）测试依非韦伦激活BimEL的构效关系。 假设IRE 1 α是药物诱导肝毒性的中心上游调节因子， 通过几类药物的刺激：我们将确定是否依法韦仑，8-OHefavirenz， 其他肝毒性药物刺激IRE 1 α/TRAF 2/ASK 1/JNK复合物的形成， 在IRE 1 α依赖的JNK激活中，我们将测试自然发生的遗传因素对JNK的影响。 IRE 1 α变体对活性和细胞死亡的影响。预计这些研究将定义BimEL 和IRE 1 α激活是一系列药物诱导- 肝毒性

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.