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

用于识别和表征载脂蛋白 B 修饰物的全动物小分子筛选

• 批准号: 10460567
• 负责人: Daniel Kelpsch
• 金额: $ 6.98万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-03 至 2023-09-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10460567
• 关键词: Affect; Affinity; Animal Model; Animals; Apolipoproteins B; Binding; Bioinformatics; Biological; Biological Assay; Biological Markers; Biology; Blood Circulation; Cardiovascular Diseases; Cells; Cholesterol; Chronic; Collaborations; Complex; Data; Dietary Fats; Disease Progression; Dose; Drug Screening; Environment; Etiology; FRAP1 gene; Female; Food; Future; Gene Expression; Homeostasis; Human; Imaging Techniques; Inflammation; Innovative Therapy; Institutes; Insulin Resistance; Intention; Intestines; Larva; Libraries; Lipids; Lipoproteins; Liver; Maleates; Measures; Mediating; Metabolic Diseases; Metabolic dysfunction; Metabolic syndrome; Metabolism; Modeling; Non-Insulin-Dependent Diabetes Mellitus; Optics; Other Genetics; Pathway interactions; Peripheral; Persons; Pharmacologic Substance; Phenotype; Physiology; Plasma; Production; Regulation; Reporter; Research; Risk Factors; Series; Serotonin; Serotonin Antagonists; Source; Syndrome; System; Testing; Therapeutic; Tissues; Transgenic Organisms; Triglycerides; Zebrafish; antagonist; base; career; design; drug repurposing; endoplasmic reticulum stress; experimental study; high throughput screening; high-throughput drug screening; hormonal signals; improved; improved outcome; in vivo; inhibitor; insight; interest; lipid metabolism; lipid transport; microsomal triglyceride transfer protein; nanoluciferase; non-alcoholic fatty liver disease; novel; novel therapeutics; receptor; screening; serotonin receptor; side effect; small molecule; success; transcriptome sequencing

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.

项目概要 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 疾病，影响着全世界超过十亿人。虽然其病因复杂，但最好的生物标志物 代谢综合征的一个重要原因是载脂蛋白 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至外周组织，过量的血浆 B-lps 被 代谢综合征的病因。每个 B-lp 上都有一个 ApoB 分子，并且对其功能至关重要。 然而，最终调节 ApoB 和 B-lp 产生、分泌、运输和 退化仍有待完全定义。拟议的研究旨在鉴定改变 B-lp 的新分子 生理学不仅希望产生新的治疗方法，而且能够阐明新的细胞生物学机制 ApoB 调节。人类 B-lp 生物学在斑马鱼中非常保守。此外，斑马鱼产生 大量后代，幼虫光学透明，并且幼虫不需要外源食物源。 因此，斑马鱼是识别 ApoB 调节新机制的理想模型。因此，法伯 实验室生成了 ApoB 的体内化学发光报告基因，不会破坏 ApoB 功能。于是，我 假设我可以找到调节 ApoB 调节、周转和纠正功能的新药物 使用 ApoB 的整个动物报告基因进行代谢功能障碍。我开发了一种高通量检测方法 筛选整个斑马鱼的化学发光。每种通过药物再利用而减少 ApoB 的化合物 文库将通过测量 ApoB 和 B-lps 产量、大小和周转率的多项测定进一步验证。我会 还评估每种化合物对整个动物生理学的影响。我的筛选工作已确定 25 降低 ApoB 的化合物。一种化合物马来酸哌美辛在一定剂量下可特异性降低 ApoB 水平 依赖方式。先前的研究表明吡美噻烯是一种血清素受体拮抗剂。研究表明 血清素通过调节哺乳动物雷帕霉素靶点 (mTOR) 影响 B-lps 水平。因此， 我推测吡美噻烯依赖性 ApoB 减少是由 5-HT2 受体拮抗作用介导的。我会 确定吡美噻烯是否直接改变该途径。此外，我将检查该化合物是否 使用一系列已建立的转基因报告基因改善与代谢疾病相关的几个危险因素 线和生物信息学方法。最终，这项研究的目的不仅是确定新型 ApoB 调节剂 可以改善代谢疾病的治疗结果，但也可以提供基本见解 ApoB 的调节和功能。法伯实验室和卡内基实验室的研究环境一起 学院对于这个项目的成功和我未来的成功是理想的，因为我成长为一个独立的人 从事新陈代谢研究。