Identification of substances that change alternative pre-mRNA splicing of the ser

鉴定改变序列选择性前 mRNA 剪接的物质

基本信息

批准号：
8207127
负责人：
Stefan Stamm
金额：
$ 3.71万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8207127
关键词：
Adult Adverse effects Affect Agonist Alternative Splicing American Amino Acids Anti-Obesity Agents Appetite Depressants Binding Biological Assay Cell Line Chemicals Data Desire for food Drug Binding Site Drug Delivery Systems Drug Receptors Eating Epidemic Exons Fenfluramine Fluorescence Gene Expression Profile Health Hormonal Human Hunger Inhibitory Concentration 50 Intervention Libraries Marketing Messenger RNA Modeling Modification Molecular Bank Molecular Weight Neurons Non-Insulin-Dependent Diabetes Mellitus Obesity Oligonucleotides Outcome Pathway interactions Patients Pharmaceutical Preparations Population Prader-Willi Syndrome Prevalence Process Production Property Protein Isoforms Proteins Public Sector Pyrvinium pamoate RNA RNA Editing RNA Splicing Regulation Reporter Research Personnel Role Screening procedure Serotonin Serotonin Receptor 5-HT2C Site Small Nuclear RNA Small Nucleolar RNA Structure Structure of nucleus infundibularis hypothalami Structure-Activity Relationship Testing Therapeutic United States National Institutes of Health Work base combat cost cross reactivity fighting gene function high throughput screening innovation insight mRNA Precursor mouse model receptor research study response serotonin 5 receptor serotonin receptor small molecule

项目摘要

DESCRIPTION (provided by applicant): Identification of substances that change alternative pre-mRNA splicing of the serotonin 2C receptor the serotonin receptor 2C (5-HT2CR) protein is a validated anti-obesity drug target that binds to the anorectic substances fen-phen (fenfluramine and phentermin). Due to their cross-reactivity with other receptors, these drugs are no longer in use. The activity of the 5-HT2CR protein is regulated by alternative processing pathways of its pre- mRNA. Only when the alternative exon Vb is included in the mRNA, a functional receptor can be formed. This inclusion can be achieved by two mechanisms: (i) RNA editing, which generates less active receptors and (ii) promotion by specific small nuclear RNAs which generates the most active receptors. Patients with Prader-Willi syndrome do not express the small nuclear RNAs, which most likely contributes to their obesity. We propose to identify compounds that promote inclusion of the alternative exon Vb, without a change on RNA editing. This change in pre-mRNA processing will generate the most active 5-HT2CR protein that is likely to exert an anti-appetite effect. We generated cell lines containing a fluorescent-based splicing reporter construct. These cell lines were successfully validated in a pilot screen using a library of 1692 compounds. The screen was robust (Z factor 6.1) and resulted in one compound, pyrvinium pamoate that showed an IC50 of 65M in established secondary and tertiary PCR-based screens. Our preliminary analysis of pyrvinium pamoate and small nuclear RNAs acting on serotonin pre-mRNA processing suggests that exon Vb functions similar to a bacterial riboswitch. A conformational change triggered by low molecular weight compounds causes a change in its structure that allows its splice site to be recognized, leading to its inclusion. To gain insight into structure activity relationships, we therefore further propose to test the influence of pyrvinium pamoate and other validated hits on serotonin receptor 2C pre-mRNA structure. In addition to conventional structure probing experiments, we will use RNA oligonucleotides with modified bases that reflect an alteration in structure by a change in fluorescence the selective modification of the processing pathways of a structured pre-mRNA has not been attempted yet and is the major innovation of the proposal. The identification of compounds that promote the activity of the serotonin receptor 2C by changing its pre-mRNA processing would be highly significant, as they are expected to have anti-appetite properties. Identifying a compound that combats obesity would have a major therapeutic impact and the possible identification of the first mammalian riboswitch will have a strong scientific impact. PUBLIC HEALTH RELEVANCE: Obesity is a major health problem affecting more than 26% of adult Americans. This project will identify compounds that change the way the body makes the serotonin receptor protein. Since this protein is a major control point of hunger, the compounds are expected to decrease appetite, which could help fighting the obesity epidemic.

描述（由申请人提供）：鉴定5-羟色胺2C受体的替代性mRNA剪接的物质羟色胺受体2C（5-HT2CR）蛋白是一种经过验证的抗肥胖药物，与厌食药物结合（芬氟拉明和芬特明）。由于它们与其他受体的交叉反应性，这些药物不再使用。 5-HT2CR蛋白的活性通过其前mRNA的替代加工途径来调节。只有当替代外显子Vb包含在mRNA中时，才能形成功能受体。可以通过两种机制来实现此包含：（i）RNA编辑，它会产生较少的活性受体，并且（ii）通过特定的小核RNA促进产生最活跃的受体。 Prader-Willi综合征患者不会表达小核RNA，这很可能有助于其肥胖症。我们建议鉴定促进替代外显子VB的化合物，而无需更改RNA编辑。前MRNA加工的这种变化将产生最活跃的5-HT2CR蛋白，该蛋白可能发挥抗苹果岩效应。我们生成了包含基于荧光的剪接报告基因结构的细胞系。使用1692种化合物的库在试点屏幕中成功验证了这些细胞系。屏幕非常健壮（Z因子6.1），并导致一种化合物，pamoate，在既定的次级和第三级PCR的筛选中显示IC50为65m。我们对作用于5-羟色胺前MRNA加工的小吡啶甲酸吡啶酸盐和小的核RNA的初步分析表明，外显子Vb的功能与细菌核糖开关相似。低分子量化合物触发的构象变化会导致其结构的变化，从而允许其剪接位点识别，从而导致其包含。为了深入了解结构活性关系，我们进一步提议测试pamoate吡格酸盐的影响和其他经过验证的命中对5-羟色胺受体2C前MRNA结构的影响。除了传统的结构探测实验外，我们还将使用带有修饰碱基的RNA寡核苷酸，这些寡核苷酸通过荧光的变化反映了结构的改变，尚未尝试选择性修改结构化Pre-MRNA的加工途径，并且是主要的创新。提案。通过更改其前MRNA加工来促进5-羟色胺受体2c活性的化合物的鉴定将非常重要，因为预计它们具有抗苹果岩性能。鉴定与肥胖作斗争的化合物将产生重大的治疗影响，并且可能鉴定出第一个哺乳动物的核糖开关将产生强烈的科学影响。公共卫生相关性：肥胖是影响超过26％的成年美国人的主要健康问题。该项目将确定改变人体制造5-羟色胺受体蛋白的化合物。由于该蛋白质是饥饿的主要控制点，因此预期这些化合物会降低食欲，这可能有助于抵抗肥胖症的流行。