A cell-based screen for small molecule binders of mutant huntingtin messenger RNA

基于细胞的突变亨廷顿信使 RNA 小分子结合物筛选

• 批准号: 7169387
• 负责人: ROBERT E HUGHES
• 金额: $ 21.9万
• 依托单位: BUCK INSTITUTE FOR RESEARCH ON AGING
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169387
• 关键词: Huntington' s disease; NIH Roadmap Initiative tag; drug discovery /isolation; genetic translation; glutamine; inhibitor /antagonist; messenger RNA; protein binding; protein folding; protein structure; protein structure function; small molecule

DESCRIPTION (provided by applicant): Huntington's Disease (HD) is a member of a family of dominantly inherited neurodegenerative diseases caused by expansion in a glutamine-encoding CAG tract. Mutant huntingtin (Htt) protein containing an expanded polyglutamine (polyQ) tract acquires novel properties that are believed to directly cause cellular dysfunction and disease. Proteins containing expanded polyQ tracts have been shown to be toxic when expressed in a wide range of experimental transgenic systems including yeast, cultured mammalian cells, C. elegans, Drosophila and mouse. The pathologic phenotype in HD is therefore thought to be driven primarily by a toxic gain of function mechanism. The overwhelming majority of HD patients are heterozygous for the mutation and therefore have one expanded and one wild-type copy of the HD gene. Studies in HD knock- out mice have shown that HD is an essential gene, but heterozygous null animals have no apparent phenotype. These observations taken together indicate that specifically inhibiting expression of the expanded HD gene would be of therapeutic benefit in a heterozygous HD patient. Secondary-structure prediction algorithms indicate that the 5' regions of the mutant and wild-type HD messenger RNAs have different folds. This suggests that the CAG-expanded message may contain a mutant-specific RNA epitope that could exploited as a drug target. We are developing a human cell based assay to screen for small molecules that bind to the mutant HD mRNA in such a way as to slow or prevent its translation into protein. This cell-based assay uses a translational reporter gene consisting of the 5' region of a mutant HD gene (with 48 CAG codons) fused to the luciferase gene. Since the HD part of the fusion contains no start codon, it serves as a 5' untranslated region (UTR) for luciferase, and the ribosome must read through the HD message to produce luciferase protein. The reporter construct also contains a GFP reporter to monitor for non-specific inhibitors of gene-expression. This assay will be used to screen small molecules libraries to identify compounds that down-regulate expression of the HD-UTR-luciferase fusion without affecting GFP expression. Hits will be counter screened against control reporter genes including non expanded HD fused to luciferase as a UTR. The ultimate goal of this project is to identify compounds that bind specifically to the mutant HD messenger RNA so as to inhibit its translation into mutant HD protein.

描述（由申请人提供）：亨廷顿氏病（HD）是由谷氨酰胺编码的CAG区扩张引起的主要遗传神经退行性疾病家族的成员。含有扩展的聚谷氨酰胺（Polyq）的突变亨廷汀（HTT）蛋白具有据信直接引起细胞功能障碍和疾病的新型特性。当在各种实验性转基因系统中表达，包括酵母，培养的哺乳动物细胞，秀丽隐杆线虫，果蝇和小鼠，含有含有扩展的Polyq块的蛋白质已被证明是有毒的。因此，HD中的病理表型被认为主要由功能机制的有毒毒性驱动。绝大多数HD患者对于突变而言是杂合子，因此有一个HD基因的扩展和一份野生型副本。在高清敲除小鼠中的研究表明，HD是必不可少的基因，但是杂合无效动物没有明显的表型。这些观察结果共同表明，在杂合HD患者中特别抑制扩展的HD基因表达将具有治疗益处。二级结构预测算法表明，突变体和野生型HD Messenger RNA的5'区域具有不同的折叠。这表明，CAG扩展的消息可能包含一个可能被用作药物靶标的突变特异性RNA表位。我们正在开发一种基于人类细胞的测定法，以筛选与突变体HD mRNA结合的小分子，以减慢或防止其转化为蛋白质。该基于细胞的测定方法使用了一个翻译报告基因，该基因由与荧光素酶基因融合的突变HD基因（具有48个CAG密码子）的5'区域组成。由于融合的HD部分不包含起始密码子，因此它是荧光素酶的5'未翻译区（UTR），核糖体必须通过HD消息读取以产生荧光素酶蛋白。记者构造还包含一个GFP报告基因，以监测基因表达的非特异性抑制剂。该测定法将用于筛选小分子库，以鉴定下调节HD-UTR-荧光素酶融合表达的化合物，而不会影响GFP表达。将对控制报告基因（包括非扩展的HD融合至荧光素酶作为UTR）进行筛选。该项目的最终目标是识别特异性结合的化合物与突变HD Messenger RNA结合，以抑制其转化为突变HD蛋白。