A whole-animal small molecule screen to identify and characterize modifiers of Apolipoprotein B

项目总结 代谢综合征，包括2型糖尿病、非酒精性脂肪肝病和心血管疾病 这种疾病影响着全世界10多亿人。虽然其病因复杂，但最好的生物标记物 代谢综合征的主要症状是载脂蛋白B(ApoB)-含脂蛋白(B-LPs)水平升高。B-LP 甘油三酯和胆固醇通过血浆输送到周围组织，过量的血浆B-脂蛋白是 导致新陈代谢综合症。一个单独的载脂蛋白B分子装饰每个B-LP，是其功能所必需的。 然而，最终调节载脂蛋白B和B-脂蛋白的产生、分泌、运输和 退化仍有待充分定义。拟议的研究旨在确定改变B-LP的新分子 生理学，希望不仅能产生新的治疗方法，而且能阐明新的细胞生物学机制 对载脂蛋白B的监管。人类的B-LP生物学在斑马鱼中非常保守。此外，斑马鱼还生产 大量的后代，幼虫是光学透明的，幼虫不需要外源食物来源。 因此，斑马鱼是识别载脂蛋白B调节新机制的理想模型。因此，法伯人 实验室产生了一种不破坏载脂蛋白B功能的体内化学发光报告。因此，我 假设我可以找到调节载脂蛋白B调节、周转和纠正功能的新药 代谢功能障碍使用载脂蛋白B的这位全动物记者。我已经开发了一种高通量的检测方法来 从全斑马鱼中筛选化学发光。每种从药物再利用中降低载脂蛋白B的化合物 文库将通过几种测定载脂蛋白B和B-脂蛋白产量、大小和周转的检测来进一步验证。这就做 并评价各化合物对全动物生理的影响。我的筛选工作已经确定了25个 降低载脂蛋白B的化合物。一种名为马来酸己甲酯的化合物，在一定剂量内特别降低载脂蛋白B水平- 依赖的态度。先前的研究表明，Pimthixene是一种5-羟色胺受体拮抗剂。研究表明 5-羟色胺通过调节哺乳动物的雷帕霉素靶标(MTOR)来影响B-LPs水平。因此， 我推测，5-HT2受体拮抗剂介导了依赖于Pimthixene的载脂蛋白B的降低。这就做 确定Pimethixene是否直接改变了这一途径。此外，我将检查这一化合物是否 利用一系列已建立的转基因报告改善与代谢性疾病相关的几个风险因素 线条和生物信息学方法。最终，本研究的目的不仅仅是寻找新的载脂蛋白B调节因子 治疗学将改善代谢性疾病的结果，但也将提供对 载脂蛋白B的调节和功能。总之，法伯实验室和卡内基研究所的研究环境 学院是这个项目成功的理想选择，也是我未来成功的理想选择，因为我将成为一个独立的 从事新陈代谢研究。

PROJECT SUMMARY Metabolic syndrome, encompassing type 2 diabetes, non-alcoholic fatty-liver disease, and cardiovascular disease, affects more than one billion people worldwide. While its etiology is complex, the best biological marker of metabolic syndrome is increased levels of apolipoprotein B (ApoB)-containing lipoproteins (B-lps). B-lps transport triglycerides and cholesterol through the plasma to peripheral tissues, and excess plasma B-lps are causative to metabolic syndrome. A single ApoB molecule decorates each B-lp and is essential for its function. However, the cellular mechanisms that ultimately regulate ApoB and B-lp production, secretion, transport, and degradation remains to be fully defined. The proposed studies aim to identify new molecules that alter B-lp physiology with the hope of not only generating new therapeutics but to elucidate new cell biological mechanisms of ApoB regulation. Human B-lp biology is remarkably conserved in the zebrafish. Further, zebrafish produce large numbers of progeny, larvae are optically transparent, and larvae do not require an exogenous food source. Thus, the zebrafish is the ideal model to identify novel mechanisms of ApoB modulation. Therefore, the Farber lab generated an in vivo chemiluminescent reporter of ApoB that does not disrupt ApoB function. Thus, I hypothesize that I can identify novel drugs that modulate ApoB regulation, turnover, and function to rectify metabolic dysfunction using this whole-animal reporter of ApoB. I have developed a high-throughput assay to screen chemiluminescence from whole zebrafish. Each compound that reduces ApoB from a drug repurposing library will be further validated by several assays measuring ApoB and B-lps production, size, and turnover. I will also evaluate the effects of each compound on whole-animal physiology. My screening efforts have identified 25 ApoB-lowering compounds. One compound, pimethixene maleate, specifically reduces ApoB levels in a dose- dependent manner. Prior research suggests pimethixene is a serotonin receptor antagonist. Studies suggest that serotonin influence B-lps levels through regulation of the mammalian Target of Rapamycin (mTOR). Thus, I hypothesize that pimethixene-dependent ApoB reduction is mediated by 5-HT2 receptor antagonism. I will determine whether pimethixene directly alters this pathway. Further, I will examine whether this compound improves several risk factors associated with metabolic disease using a series of established transgenic reporter lines and bioinformatic approaches. Ultimately, this research aims not only to identify novel ApoB-modulating therapeutics that would improve outcomes of metabolic disease but would also provide fundamental insights into the regulation and function of ApoB. Together, the research environment of the Farber lab and the Carnegie Institute are ideal for the success of this project and my success in the future as I grow towards an independent career in metabolism research.