High throughput assay development for Huntington?s Disease

描述（由申请人提供）：亨廷顿病的直接原因是DNA中的三联体扩增，导致蛋白亨廷顿（htt）的遗传基因与多聚谷氨酰胺（polyQ）重复的等效扩增。这种突变的蛋白质序列如何触发导致HD病理学的分子和细胞事件仍有争议。现在普遍认为，在HD尸检大脑中以及HD的细胞和动物模型中观察到的含有大htt的内含物可能不是有毒物质。然而，没有证据表明可能涉及荧光显微镜下不可见的较小聚集体。这是重要的，因为polyQ扩增对polyQ疾病蛋白如htt的行为的唯一广泛接受的已知后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，从而产生了在动物模型中阻断聚集并抑制毒性的化合物。其中一种化合物利鲁唑在人体临床试验中未能诱导反应，但随后在动物模型临床前试验中也失败了，可能是因为在大脑中的浓度不足。最近，我们的小组已经阐明了一个新的聚集机制的亨廷顿蛋白的外显子1片段含有polyQ序列，这取决于一个触发蛋白展开事件的外显子1的N-末端之前的polyQ。外显子1聚集的体外表征导致发现存在两种竞争外显子1分子的聚集途径，每种途径产生不同的聚集中间体和/或产物。使用一种新的染色方法特异性淀粉样聚合物的polyQ，我们确定了一种新的聚合物在哺乳动物细胞生产外显子1，已被认为只产生大的夹杂物的外显子1。在本申请中，我们提出开发新的高通量筛选测定，用于在大的小分子文库中鉴定聚集抑制剂。这一建议的前提是，我们不知道已知的亨廷顿蛋白外显子1聚集的物种是最有毒的，最有可能有助于HD。我们提出了两个新的筛选试验的基础上，我们的初步数据。其中一个重点是寻找能够在体外预防外显子1 N末端蛋白质错误折叠事件的分子，我们认为这种事件会引发部分htt外显子1聚集。另一个重点是防止在哺乳动物细胞培养的外显子1的淀粉样蛋白样聚集体的形成，这是不同于以前集中在筛选和分子机制研究的大夹杂物。我们建议开发这些检测方法，并将其微调到适合高通量筛选的状态。我们还将开发各种二次筛选试验，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新的抑制剂能够减缓HD的进展。公共卫生相关性：该研究项目与人类健康密切相关，因为它提出开发新的筛选测定法，用于发现潜在的亨廷顿病治疗药物。如果成功的话，这些检测可以很快地进行高通量筛选，以识别新的候选分子。

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.