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: 10261421
• 负责人: Daniel Kelpsch
• 金额: $ 6.64万
• 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/10261421
• 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; 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; 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/antagonist; 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.

项目摘要 代谢综合征，包括2型糖尿病，非酒精性脂肪肝病和心血管 疾病在全球影响着超过十亿人。虽然其病因很复杂，但最好的生物标记物 代谢综合征的是载脂蛋白B（APOB）含脂蛋白（B-LPS）的水平增加。 B-LPS 通过血浆传输甘油三酸酯和胆固醇到周围组织，血浆B-LPS过多 代谢综合征的病因。单个APOB分子装饰每个B-LP，对于其功能至关重要。 但是，最终调节APOB和B-LP产生，分泌，运输和的细胞机制 退化仍然有待充分定义。拟议的研究旨在确定改变B-LP的新分子 生理学不仅希望产生新的治疗剂，还希望阐明新的细胞生物学机制 APOB调节。人B-LP生物学在斑马鱼中非常保守。此外，斑马鱼农产品 大量后代，幼虫是光学透明的，幼虫不需要外源性食物来源。 因此，斑马鱼是识别APOB调制的新机制的理想模型。因此，法伯 LAB生成了APOB的体内化学发光报告基因，该报告不会破坏APOB函数。因此，我 假设我可以识别调节APOB调节，周转和功能以纠正的新型药物 使用APOB的整个动物记者使用代谢功能障碍。我已经开发了一个高通量测定法 全斑马鱼的筛选化学发光。每种化合物都会从药物重新利用中降低APOB 库将通过测量APOB和B-LPS生产，大小和营业额的几种测定法进一步验证。我会 还评估每种化合物对全动物生理学的影响。我的筛查工作已经确定了25 降低APOB的化合物。一种化合物，彼赛烯酸酯，特别降低了剂量中的APOB水平 依赖方式。先前的研究表明，偶聚甲基二烯是一种5-羟色胺受体拮抗剂。研究表明 5-羟色胺通过调节雷帕霉素（MTOR）的哺乳动物靶标影响B-LPS水平。因此， 我假设偶氮依赖性的APOB还原是由5-HT2受体拮抗作用介导的。我会 确定彼蒂替烯是否直接改变了这一途径。此外，我将检查这种化合物是否 使用一系列已建立的转基因记者改善与代谢疾病相关的几种危险因素 线条和生物信息学方法。最终，这项研究不仅旨在确定新颖的APOB调制 可以改善代谢疾病结果的治疗剂，但也将提供基本的见解 APOB的调节和功能。一起，Farber Lab和Carnegie的研究环境 研究所是该项目成功的理想之选，以及我将来的成功，随着我向独立发展 新陈代谢研究的职业。