Targeting Tau Splicing for Dementia

针对痴呆症的 Tau 剪接

• 批准号: 8351373
• 负责人: Michael S Wolfe
• 金额: $ 26.78万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8351373
• 关键词: A-Microtubule; Affect; Alternative Splicing; Alzheimer' s Disease; Amyloid; Antisense Oligonucleotides; Binding; Biological Assay; Biology; Cell Culture Techniques; Cells; Complex; DNA; Dementia; Disease; Electrophoretic Mobility Shift Assay; Equilibrium; Event; Exons; Frontotemporal Dementia; Genes; Genomics; Human; Human Genome; Introns; Light; Link; Messenger RNA; Microtubules; Mitoxantrone; Modeling; Molecular; Mutation; Nerve Degeneration; Neurons; Oligonucleotides; Pathogenesis; Pathology; Pathway interactions; Peptides; Pharmaceutical Preparations; Play; Process; Protein Isoforms; Proteins; RNA; RNA Splicing; RNase protection assay; Ribonucleases; Role; Silent Mutation; Site; Structural Models; Structure; System; Tauopathies; Testing; Therapeutic; Therapeutic Agents; Variant; amyloid peptide; analog; base; design; high throughput screening; mRNA Precursor; mouse model; novel; novel therapeutics; peptide A; prototype; stem; success; tau Proteins; therapeutic target; tool

DESCRIPTION (provided by applicant): The amyloid-¿ peptide (A¿) and the microtubule-associated protein tau are both strongly implicated in the pathogenesis of Alzheimer's disease (AD). However, the role of tau in AD pathogenesis is not well understood, and tau is underdeveloped as a therapeutic target. Major clues to its pathogenic role are nearly 40 mutations in the tau gene that cause certain forms of frontotemporal dementia (FTD), a "tauopathy" related to AD. Many of these mutations are silent and alter the splicing of the tau pre-mRNA, leading to increased inclusion of exon 10 and thereby shifting the balance between tau proteins that contain 3 or 4 microtubule binding domains (3R and 4R tau, respectively). We have validated a postulated stem-loop at the boundary between exon 10 and intron 10 as a bona fide structure that regulates 3R versus 4R tau formation in cells. We have also carried out a high-throughput screen to identify small drug-like compounds that bind to and stabilize this stem-loop structure as a potential therapeutic strategy, determined the structure of one of these compounds bound to the tau mRNA stem-loop by NMR, and validated this structural model through analogue design. In addition, we have developed novel antisense molecules that recognize the discontinuous mRNA sequences that flank the tau pre-mRNA stem-loop structure and skew tau pre-mRNA splicing away from the 4R isoforms. These antisense molecules are bipartite, connected via a linker region, with one part complementary to the sequence just upstream from the stem-loop and the other complementary to the sequence immediately downstream. Such bipartite antisense molecules could be said to be both sequence- and structure-specific, as simultaneous binding to the two flanking regions requires stem-loop formation. In light of these findings, we now propose to combine these two approaches and develop antisense-MTX conjugates that stabilize the tau stem-loop structure with high potency and selectivity. Because MTX binds near the bottom of the stem, close to the sites where antisense molecules bind, linking MTX to such antisense molecules is expected to result in highly potent and specific "molecular clasps" that simultaneously target the tau pre-mRNA stem-loop structure, sequences upstream of this structure, and sequences downstream of this structure. The result of such tripartite binding by these molecular clasps would be potent and selective alteration of tau splicing in a therapeutically beneficial way. Specifically, we propose o: (1) generate antisense-MTX conjugates, (2) test these conjugates for their ability to bind to and stabilize the tau pre-mRNA stem-loop structure, and (3) determine the ability of these conjugates to shift splicing of the tau pre-mRNA away from the 4R isoforms. PUBLIC HEALTH RELEVANCE: Targeting Tau Splicing for Dementia RNA is a critical intermediate in the pathway from DNA (genes) to their encoded proteins. These RNA intermediates undergo complex alternative processing that ultimately leads to variations of the proteins. Such complex processing occurs with an important RNA molecule involved in Alzheimer's disease and related dementias, an RNA encoding a protein called tau. In this project, we aim to develop a strategy for controlling the alternative processing of the tau RNA with small drug-like molecules linked to larger polymeric molecules, agents we have dubbed "molecular clasps".

描述（由应用提供）：淀粉样蛋白肽（A a。）和与微管相关的蛋白质Tau在阿尔茨海默氏病（AD）的发病机理中都强烈隐含。然而，tau在AD发病机理中的作用尚不清楚，而Tau作为治疗靶标不发达。其致病作用的主要线索是在tau基因中几乎40个突变，这些突变引起某些形式的额颞痴呆（FTD），这是与AD相关的“ tauopathy”。这些突变中的许多是静音的，并改变了tau前mRNA的剪接，从而增加了外显子10的纳入，从而改变了包含3或4个微管结合域（分别为3R和4R TAU）的Tau蛋白之间的平衡。我们已经在外显子10和内含子10之间的边界上验证了一个假定的茎环，作为调节3R与4R tau形成的真正结构。我们还进行了一个高通量筛选，以鉴定与该词干结构结合并稳定为潜在理论策略的小型药物样化合物，并通过NMR确定了与Tau mRNA茎环结合的一种结构，并通过模拟设计验证了这种结构模型。此外，我们开发了新型的反义分子，这些分子识别出tau前mRNA茎环结构的不连续的mRNA序列，并偏向于从4R同工型中脱离的tau前mRNA。这些反义分子是双分的，通过接头区域连接，一个部分完善到序列的序列，正好从茎环的上游，另一个完整的序列到下游的序列。这种两分的反义分子可以说是序列和结构特异性的，因为与两个侧翼区域的简单结合需要茎环的形成。鉴于这些发现，我们现在建议将这两种方法结合起来，并开发反义的MTX偶联物，以高效力和选择性稳定Tau Stem-loop结构。由于MTX结合茎的底部附近，靠近反义分子结合的位点，因此将MTX与这种反义分子联系起来会导致高度潜在和特定的“分子clasps”，这些分子clasps仅针对Tau Pre-mRNA Pre-mRNA茎环结构，该结构的序列和该结构下游的序列。这些分子扣结合这种三方结合的结果将是潜在的，并且以治疗性有益的方式对tau剪接的选择性改变。具体而言，我们提出o：（1）生成反义的MTX结合物，（2）测试这些结合物的结合和稳定的能力，并稳定tau pre-mRNA茎环结构，（3）确定这些结合物可以移动tau prain pre-mRNA的能力，从4R同型中移出tau prapla。 公共卫生相关性：将tau剪接用于痴呆RNA是从DNA（基因）到其编码蛋白的途径中的关键中间体。这些RNA中间体经历了复杂的替代加工，最终导致蛋白质的变化。这种复杂的加工发生在参与阿尔茨海默氏病和相关痴呆症的重要RNA分子中，这是一种编码称为tau的蛋白质的RNA。在这个项目中，我们旨在制定一种策略，以控制tau RNA的替代加工，该策略用与较大的聚合物分子相关的小药物样分子，我们称为“分子clasps”。