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

项目摘要 代谢综合征，包括2型糖尿病、非酒精性脂肪肝和心血管疾病 这种疾病影响着全世界10亿多人。虽然其病因复杂，但最好的生物学标志物 代谢综合征的一个重要特征是含载脂蛋白B（ApoB）的脂蛋白（B-lps）水平升高。B-LPS 通过血浆将甘油三酯和胆固醇转运到外周组织， 导致代谢综合征。单个ApoB分子装饰每个B-lp，并且对其功能至关重要。 然而，最终调节ApoB和B-lp产生、分泌、转运和代谢的细胞机制尚不清楚。 退化仍有待充分界定。这项研究旨在确定改变B-lp的新分子 不仅希望产生新的治疗方法，而且希望阐明新的细胞生物学机制 ApoB调节人类B-lp生物学在斑马鱼中是非常保守的。此外，斑马鱼产生 大量的后代，幼虫是光学透明的，幼虫不需要外源性食物来源。 因此，斑马鱼是鉴定ApoB调节的新机制的理想模型。因此，法伯 实验室产生了一种不破坏ApoB功能的ApoB体内荧光报告基因。所以我 假设我可以鉴定出调节ApoB调节、周转和纠正ApoB功能的新药， 使用ApoB的这种全动物报告物来研究代谢功能障碍。我开发了一种高通量检测方法 从整条斑马鱼中筛选化学发光。每种化合物都能减少药物再利用中的ApoB 将通过测量ApoB和B-lps产生、大小和周转的几种测定进一步验证文库。我会 还评估每种化合物对整个动物生理学的影响。我的筛选工作已经确定了25个 降低ApoB的化合物。一种化合物，pimethixene maleate，在一定剂量下特异性降低ApoB水平- 依赖的方式。先前的研究表明pimethixene是一种血清素受体拮抗剂。研究表明 5-羟色胺通过调节哺乳动物雷帕霉素靶蛋白（mTOR）影响B-LPS水平。因此，在本发明中， 我推测，匹美塞烯依赖性ApoB减少是由5-HT 2受体拮抗作用介导的。我会 确定pimethixene是否直接改变了这一途径。此外，我将研究这种化合物是否 使用一系列已建立的转基因报告基因改善了与代谢疾病相关的几个风险因素 线和生物信息学方法。最终，这项研究的目的不仅是确定新的ApoB调节 这些治疗方法将改善代谢性疾病的结果，但也将为以下方面提供基本的见解： ApoB的调节和功能。法伯实验室和卡内基实验室的研究环境 研究所是这个项目的成功和我在未来的成功，因为我朝着一个独立的理想 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.