High throughput assay development for Huntington?s Disease

亨廷顿病的高通量检测开发

基本信息

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
项目类别：
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

项目摘要

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中的三联体扩展，导致蛋白质亨廷顿蛋白（HTT）的遗传基因，并具有等效的聚谷氨酰胺（PolyQ）重复的膨胀。这种突变的蛋白序列如何触发导致HD病理的分子和细胞事件继续进行辩论。现在普遍同意，在尸检中观察到的含有HD大脑中的大型HTT，在HD的细胞和动物模型中，可能不是有毒物种。但是，没有证据表明可能参与荧光显微镜中不可见的较小聚集体。这很重要，因为PolyQ扩展对Polyq疾病蛋白（如HTT）的行为的唯一已知的已知结果是蛋白质聚集的增强。已经对能够阻断聚集的小分子进行了许多筛选测定，导致化合物阻断聚集并抑制动物模型中的毒性。一种这样的化合物Riluzole未能在人类临床试验中引起反应，但随后在动物模型临床前试验中也失败了，这也许是因为大脑浓度不足。最近，我们的小组阐明了一种新的聚集机制，用于含有polyQ序列的亨廷顿蛋白的外部1片段，该机制取决于在polyq之前的exon1 n-terminus中触发蛋白质展开的事件。在体外表征外观1聚集的表征导致发现，有两种竞争Exon1分子的聚集途径，每个途径都会产生不同的聚集中间体和/或产品。使用针对Polyq的淀粉样蛋白样聚集体的新染色方法，我们确定了产生Exon1的哺乳动物细胞中的一个新聚集体，该骨料被认为仅产生大量外exon1。在此应用中，我们建议开发新的高吞吐量筛选测定法，以识别大分子大型库中的聚集抑制剂。该提议的前提是，我们不知道哪个已知的Huntingtin Exon1汇总物种是毒性最大，最有可能促成HD的物种。我们根据我们的初步数据提出了两个新的筛选分析。人们专注于寻找可以预防exon1 n末端中蛋白质错误折叠事件的分子，我们认为这会触发HTT Exon1聚集的一部分。另一个侧重于防止外显1淀粉样蛋白样骨料的哺乳动物细胞培养物中的形成，这些淀粉样蛋白样骨料与以前针对筛选和分子机理研究的大型夹杂物不同。我们建议开发这些测定法，然后将它们微调到准备进行高吞吐量筛查的状态。我们还将开发各种辅助筛选测定法，以消除将来的高吞吐量屏幕中的误报。我们认为，这些测定可能会导致发现能够减慢HD进展的新类别抑制剂。公共卫生相关性：该研究项目与人类健康有关，因为它建议开发新的筛查测定法，以发现潜在的亨廷顿疾病疗法。如果成功的话，这些测定可能很快将其托运到高吞吐量屏幕上，以识别新的候选分子。