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CAG通道扩张引起的显性遗传性神经退行性疾病家族的一员。含有扩展的聚谷氨酰胺（polyQ）通道的突变亨廷顿蛋白（Htt）获得了被认为直接导致细胞功能障碍和疾病的新特性。含有扩增多q束的蛋白质在酵母、培养的哺乳动物细胞、秀丽隐杆线虫、果蝇和小鼠等多种实验性转基因系统中表达时已被证明是有毒的。因此，HD的病理表型被认为主要是由毒性功能获得机制驱动的。绝大多数HD患者是突变的杂合子，因此有一个扩展型和一个野生型HD基因拷贝。对HD基因敲除小鼠的研究表明，HD是一种必需基因，但杂合缺失动物没有明显的表型。这些观察结果共同表明，特异性抑制扩增HD基因的表达将对杂合子HD患者的治疗有益。二级结构预测算法表明突变型和野生型HD信使rna的5′区具有不同的折叠。这表明cag扩展的信息可能包含一个突变特异性RNA表位，可以作为药物靶点利用。我们正在开发一种基于人类细胞的检测方法，以筛选与突变的HD mRNA结合的小分子，从而减缓或阻止其转化为蛋白质。这种基于细胞的检测方法使用翻译报告基因，该基因由突变HD基因的5'区（具有48个CAG密码子）融合到荧光素酶基因上。由于融合的HD部分不包含起始密码子，因此它作为荧光素酶的5'非翻译区（UTR），核糖体必须通过HD信息读取以产生荧光素酶蛋白。报告基因结构还包含一个GFP报告基因，用于监测基因表达的非特异性抑制剂。该试验将用于筛选小分子文库，以鉴定下调hd - utr -荧光素酶融合表达而不影响GFP表达的化合物。命中将对对照报告基因进行反筛选，包括融合到荧光素酶作为UTR的非扩增HD。该项目的最终目标是鉴定特异性结合突变HD信使RNA的化合物，从而抑制其翻译为突变HD蛋白。