Compounds regulating BACE 1 splicing and activity

调节 BACE 1 剪接和活性的化合物

• 批准号: 6727375
• 负责人: JIANHUA ZHOU
• 金额: $ 7.95万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-15 至 2005-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727375
• 关键词: Alzheimer' s disease; RNA splicing; amyloid proteins; biological signal transduction; biotechnology; cell line; chemical genetics; chemical registry /resource; drug discovery /isolation; drug screening /evaluation; endopeptidases; enzyme activity; genetic regulation; high throughput technology; luciferin monooxygenase; polymerase chain reaction; protein biosynthesis; protein isoforms; reporter genes; small molecule

One of the major characteristics for Alzheimer's disease (AD) is deposit of amyloid plaques composed of A-beta peptides generated from cleavage of amyloid precursor protein (APP) by two secretases termed gamma and beta. Great efforts have been taken to identify compounds that inhibit the activities of these two enzymes. Recent studies have shown that BACE1, the beta-secretase undergoes alternative splicing within its exons 3 and 4 that produces isoforms with greatly reduced activity, suggesting that regulation of BACE1 splicing may be targeted for AD therapy. The goal of this proposal is to develop a cell-based assay and to use this assay to identify specific and potent small molecules that reduce A-beta generation by modulating exons 3 and 4 splicing to produce less 501-aa form of BACE1(active beta-secretase). The first aim of this project is to establish the cell-based assay for the screen. We will generate mini-gene constructs for BACE1 splicing studies with the luciferase reporter gene attached. The efficiency of alternative usage of the internal splicing sites within exons 3 and 4 of BACE1 pre-mRNA will be monitored by luciferase activities. After validation of the mini-genes in transiently transfected cells by RT-PCR, stable neuronal cell lines will be established. The second aim of this project is to use these stable cell lines to identify small molecules that reduce the production of the active isoform of 501-aa BACE1. These small molecules will be further tested for their effectiveness on reduction of A-beta generation. The investigations may lead to novel drug development for AD and understanding of signal transduction pathways regulating mRNA splicing. We will start with small scale screens using our collected compound libraries. High-throughput screens and mechanisms of action will be our long-term goals.

阿尔茨海默病(AD)的主要特征之一是淀粉样斑块沉积 由淀粉样前体蛋白(APP)被称为伽马和β的两种分泌酶裂解产生的A-β多肽组成。人们已经做出了很大的努力来鉴定抑制这两种酶活性的化合物。最近的研究表明，BACE1，即 β-分泌酶在其外显子3和4内经历选择性剪接，产生活性大大降低的异构体，这表明调控BACE1剪接可能是AD治疗的靶点。这项建议的目标是发展一种基于细胞的分析方法，并使用这种方法来识别特定和有效的小分子，通过调节外显子3和4的剪接来减少A-β的产生，从而产生较少的501-AA形式的BACE1(活性β-分泌酶)。该项目的第一个目标是建立基于细胞的筛查方法。我们将生成微型基因构建物，用于BACE1剪接研究，并附上荧光素酶报告基因。BACE1前-mRNA外显子3和4内的内部剪接位点的替代使用效率将通过荧光素酶活性进行监测。在通过RT-PCR验证瞬时转染细胞中的微小基因后，将建立稳定的神经细胞系。该项目的第二个目标是利用这些稳定的细胞系来识别减少501-AA BACE1活性异构体产生的小分子。这些小分子将进一步测试它们在减少A-β生成方面的有效性。这些研究可能有助于开发治疗阿尔茨海默病的新药，并有助于了解调控mRNA剪接的信号转导途径。我们将从使用我们收集的复合库的小屏幕开始。高通量的屏幕和作用机制将是我们的长期目标。