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.

描述（由申请人提供）：改变5-羟色胺2C受体的前体mRNA可变剪接的物质的鉴定5-羟色胺受体2C（5-HT 2CR）蛋白是一种经验证的抗肥胖药物靶标，可与厌食物质芬氟拉明和芬特明（fenfluramine and phentermin）结合。由于它们与其他受体的交叉反应性，这些药物不再使用。 5-HT 2CR蛋白的活性受其前体mRNA的替代加工途径调节。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。这种包含可以通过两种机制实现：（i）RNA编辑，其产生活性较低的受体;以及（ii）通过产生活性最高的受体的特定小核RNA的促进。Prader-Willi综合征患者不表达小的核RNA，这很可能导致他们的肥胖。 我们建议鉴定促进包含选择性外显子Vb的化合物，而不改变RNA编辑。这种前mRNA加工的变化将产生最活跃的5-HT 2CR蛋白，可能发挥抗食欲作用。 我们产生了含有基于荧光的剪接报告构建体的细胞系。这些细胞系在使用1692种化合物的文库的中试筛选中成功验证。筛选是稳健的（Z因子6.1），并产生了一种化合物，双羟萘酸吡维铵，其在已建立的二级和三级基于PCR的筛选中显示出65 M的IC 50。 我们对pyrvinium pamoate和作用于5-羟色胺前体mRNA加工的小核RNA的初步分析表明，外显子Vb的功能类似于细菌核糖开关。由低分子量化合物引发的构象变化导致其结构的变化，从而允许其剪接位点被识别，导致其包含。 为了深入了解结构活性关系，我们因此进一步提出测试双羟萘酸吡维铵和其他经验证的命中物对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，我们将使用具有修饰碱基的RNA寡核苷酸，所述修饰碱基通过荧光的变化反映结构的改变。尚未尝试结构化前mRNA的加工途径的选择性修饰，这是该提议的主要创新。通过改变5-羟色胺受体2C的前mRNA加工来促进其活性的化合物的鉴定将是非常重要的，因为它们预期具有抗食欲特性。确定一种对抗肥胖的化合物将产生重大的治疗影响，而可能确定的第一个哺乳动物核糖开关将产生强大的科学影响。 公共卫生相关性：肥胖是一个主要的健康问题，影响超过26%的美国成年人。这个项目将确定化合物，改变身体的方式，使血清素受体蛋白。由于这种蛋白质是饥饿的主要控制点，这些化合物有望降低食欲，这可能有助于对抗肥胖流行病。