High throughput assay development for Huntington?s Disease

亨廷顿病的高通量检测开发

• 批准号: 7826695
• 负责人: RONALD B WETZEL
• 金额: $ 18.67万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7826695
• 关键词: Address; Agreement; Alleles; Amino Acid Sequence; Amino Acids; Amyloid; Amyloid Fibrils; Animal Model; Appearance; Autopsy; Behavior; Benchmarking; Biological; Biological Assay; Brain; CAG repeat; Cell Culture Techniques; Cell Death; Cells; Clinical Trials; DNA; Data; Diffuse; Disease; Elements; Event; Exhibits; Failure; Fluorescence; Fluorescence Microscopy; Funding; Future; Gene Proteins; Genetic; Genetic Transcription; Growth; Health; Human; Huntington Disease; In Vitro; Indium; Inherited; Kinetics; Knowledge; Lead; Length; Lesion; Link; Mammalian Cell; Modeling; Molecular; Molecular Conformation; Mutate; N-terminal; PC12 Cells; Pathology; Pathway interactions; Peptide Sequence Determination; Peptides; Physiological; Play; Proteins; Reaction; Recombinants; Recruitment Activity; Research Project Grants; Riluzole; Role; Running; Screening procedure; Sorting - Cell Movement; Staining method; Structure; Testing; Therapeutic; Time; Toxic effect; Translating; Translations; Triplet Multiple Birth; United States National Institutes of Health; Validation; Work; aggregation pathway; assay development; base; drug discovery; high throughput screening; human Huntingtin protein; inhibitor/antagonist; monomer; mutant; novel; polyglutamine; polymerization; preclinical study; prevent; protein aggregation; protein misfolding; public health relevance; research study; response; small molecule; small molecule libraries

DESCRIPTION (provided by applicant): The immediate cause of Huntington's disease is a triplet expansion in the DNA leading to an inherited gene for the protein huntingtin (htt) with an equivalent expansion of a polyglutamine (polyQ) repeat. How this mutated protein sequence triggers the molecular and cellular events leading to HD pathology continues to be debated. There is now general agreement that the large htt containing inclusions observed in HD brains on autopsy, and in cellular and animal models of HD, are probably not the toxic species. However, no evidence as been presented against the possible involvement of smaller aggregates not visible in fluorescence microscopy. This is significant since the only widely accepted known consequence of polyQ expansion to the behavior of polyQ disease proteins like htt is an enhancement of protein aggregation. A number of screening assays for small molecules capable of blocking aggregation have been conducted leading to compounds that block aggregation and suppress toxicity in animal models. One such compound, riluzole, failed to induce a response in human clinical trials, but then it also failed in a animal model preclinical trial, perhaps because of insufficient concentration in the brain. Recently our group has elucidated a new aggregation mechanism for the exon1 fragment of huntingtin containing the polyQ sequence, which depends on a triggering protein unfolding event in the exon1 N-terminus just before the polyQ. Characterization of exon1 aggregation in vitro led to the discovery that there are two aggregation pathways competing for exon1 molecules, each of which produces different aggregation intermediates and/or products. Using a new staining method specific for amyloid-like aggregates of polyQ, we identified a new aggregate in mammalian cells producing exon1 that have been thought to only produce large inclusions of exon1. In this application we propose to develop new high throughput screening assays for identifying aggregation inhibitors in large libraries of small molecules. The premise of this proposal is that we do not know which of the known huntingtin exon1 aggregated species is the most toxic and most likely to contribute to HD. We propose two new screening assays based on our preliminary data. One focuses on finding molecules that will prevent, in vitro, the protein misfolding event in the exon1 N-terminus that we believe triggers a portion of htt exon1 aggregation. The other focuses on preventing the formation in mammalian cell culture of the amyloid-like aggregates of exon1 that are different from the large inclusions previously focused on in screening and molecular mechanism studies. We propose to develop these assays and fine-tune them to a state ready for high throughput screening. We will also develop a variety of secondary screening assays that will be required to eliminate false positives from future high throughput screens. We believe that these assays could lead to the discovery of new classes of inhibitors capable of slowing the progression of HD. PUBLIC HEALTH RELEVANCE: This research project is immediately relevant to human health in that it proposes to develop new screening assays for drug discovery of potential Huntington's disease therapeutics. If successful, these assays could very quickly be consigned to high throughput screens to identify new candidate molecules.

描述（由申请人提供）：亨廷顿氏病的直接原因是DNA中的三重扩增导致亨廷顿蛋白（htt）的遗传基因与聚谷氨酰胺（polyQ）重复序列的等效扩增。这种突变的蛋白质序列如何触发导致HD病理的分子和细胞事件仍在争论中。现在有一个普遍的共识，在HD的大脑解剖中，在HD的细胞和动物模型中观察到的含有包涵体的大htt可能不是有毒的物种。然而，没有证据表明荧光显微镜中不可见的较小聚集体可能参与其中。这是重要的，因为唯一被广泛接受的polyQ扩增对多q疾病蛋白（如htt）行为的已知后果是蛋白质聚集的增强。对能够阻断聚集的小分子进行了许多筛选试验，从而在动物模型中获得了阻断聚集和抑制毒性的化合物。其中一种化合物利鲁唑在人体临床试验中未能引起反应，但随后在动物模型临床前试验中也失败了，可能是因为大脑中的浓度不足。最近，我们的研究小组已经阐明了包含polyQ序列的亨廷顿蛋白外显子1片段的一种新的聚集机制，这种聚集机制依赖于在polyQ之前的外显子1 n端触发蛋白展开事件。体外外显子1聚集的表征导致发现有两种聚集途径竞争外显子1分子，每一种都产生不同的聚集中间体和/或产物。利用一种新的特异性染色方法，我们在哺乳动物细胞中发现了一种产生外显子1的新聚集体，该聚集体被认为只产生外显子1的大内含物。在这个应用中，我们建议开发新的高通量筛选方法来鉴定小分子文库中的聚集抑制剂。这一建议的前提是，我们不知道哪个已知的亨廷顿外显子1聚集物种是最有毒的，最有可能导致HD。基于我们的初步数据，我们提出了两种新的筛选方法。其中一个重点是寻找能够在体外防止外显子1 n端蛋白质错误折叠事件的分子，我们认为这触发了部分htt外显子1聚集。另一个重点是防止哺乳动物细胞培养中外显子1淀粉样聚集体的形成，这与之前在筛选和分子机制研究中关注的大内含物不同。我们建议开发这些检测方法，并对其进行微调，使其达到高通量筛选的状态。我们还将开发各种二级筛选分析，以消除未来高通量筛选中的假阳性。我们相信这些试验可能会导致发现能够减缓HD进展的新型抑制剂。公共卫生相关性：该研究项目与人类健康直接相关，因为它建议为潜在的亨廷顿病治疗药物的药物发现开发新的筛选试验。如果成功，这些试验可以很快地进行高通量筛选，以鉴定新的候选分子。