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

• 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
• 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

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.

5-HT2CR(5-HT2CR)蛋白是一种有效的减肥药靶标，可与厌食物质芬芬(芬氟拉明和芬太明)结合。由于它们与其他受体的交叉反应，这些药物不再使用。5-HT2CR蛋白的活性受其前体mRNA的不同加工途径的调节。只有当选择性外显子VB包含在mRNA中时，才能形成功能性受体。这种包涵体可以通过两种机制实现：(I)RNA编辑，它产生活性较低的受体；(Ii)由特定的小核RNA促进，它产生最活跃的受体。Prader-Willi综合征患者不表达小核RNA，这很可能是导致他们肥胖的原因。我们建议在不改变RNA编辑的情况下，鉴定促进包含替代外显子VB的化合物。这种前信使核糖核酸加工的变化将产生最活跃的5-HT2CR蛋白，可能起到反胃口的作用。我们生成了包含基于荧光的剪接报告构建的细胞系。这些细胞系在一个包含1692种化合物的中试筛选中得到了成功的验证。该筛查是可靠的(Z因子6.1)，并产生了一种化合物，在已建立的基于PCR的二级和三级筛查中，该化合物的IC50为65M。我们对甲酸吡喃和作用于5-羟色胺前mRNA处理的小核RNA的初步分析表明，外显子VB的功能类似于细菌核糖开关。低分子化合物引发的构象变化导致其结构的变化，使其剪接位点被识别，导致其被包含。因此，为了深入了解构效关系，我们进一步建议测试帕莫酸吡喃和其他有效的HITS对5-羟色胺受体2C前mRNA结构的影响。除了常规的结构探测实验外，我们还将使用碱基经过修饰的RNA寡核苷酸，通过荧光变化来反映结构的变化。目前还没有尝试选择性地修改结构前mRNA的加工路径，这是该提案的主要创新之处。通过改变5-羟色胺受体2C的前信使核糖核酸的加工过程来促进其活性的化合物的鉴定将具有非常重要的意义，因为它们有望具有抗食欲的特性。识别一种抗肥胖的化合物将具有重大的治疗效果，而第一个哺乳动物核糖开关的可能识别将具有强大的科学影响。 与公共健康相关：肥胖是一个主要的健康问题，影响着超过26%的美国成年人。该项目将识别能够改变人体制造5-羟色胺受体蛋白方式的化合物。由于这种蛋白质是饥饿的主要控制点，这些化合物预计会降低食欲，这可能有助于对抗肥胖症流行。