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）：亨廷顿病（HD）是显性遗传性神经退行性疾病家族的一员，由编码谷氨酰胺的 CAG 束扩张引起。含有扩展的聚谷氨酰胺 (polyQ) 束的突变亨廷顿 (Htt) 蛋白获得了新的特性，被认为可直接导致细胞功能障碍和疾病。含有扩展的polyQ束的蛋白质在广泛的实验转基因系统（包括酵母、培养的哺乳动物细胞、线虫、果蝇和小鼠）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 患者的突变是杂合的，因此具有 HD 基因的一个扩展拷贝和一个野生型拷贝。对 HD 敲除小鼠的研究表明，HD 是一种必需基因，但杂合无效动物没有明显的表型。这些观察结果共同表明，特异性抑制扩增的 HD 基因的表达对于杂合 HD 患者将具有治疗益处。二级结构预测算法表明突变型和野生型 HD 信使 RNA 的 5' 区域具有不同的折叠。这表明 CAG 扩展的信息可能包含突变体特异性 RNA 表位，可用作药物靶点。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 HD mRNA 结合的小分子，从而减缓或阻止其翻译成蛋白质。这种基于细胞的检测使用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' 区域组成的翻译报告基因。由于融合体的 HD 部分不包含起始密码子，因此它充当荧光素酶的 5' 非翻译区 (UTR)，并且核糖体必须读取 HD 信息才能产生荧光素酶蛋白。报告基因构建体还包含一个 GFP 报告基因，用于监测基因表达的非特异性抑制剂。该测定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。将针对对照报告基因（包括作为 UTR 与荧光素酶融合的非扩展 HD）进行反筛选。该项目的最终目标是鉴定与突变型HD信使RNA特异性结合的化合物，从而抑制其翻译成突变型HD蛋白。