Compounds regulating BACE 1 splicing and activity

阿尔茨海默病（Alzheimer's disease，AD）的主要特征之一是淀粉样斑块的存款 由淀粉样前体蛋白（APP）被称为γ和β的两种分泌酶裂解产生的A-β肽组成。人们已经做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，BACE 1， β-分泌酶在其外显子3和4内进行选择性剪接，产生活性大大降低的同种型，这表明BACE 1剪接的调节可能是AD治疗的靶点。本提案的目标是开发一种基于细胞的测定方法，并使用该测定方法鉴定特异性和有效的小分子，这些小分子通过调节外显子3和4剪接产生更少的501-aa形式的BACE 1（活性β分泌酶）来减少A-β的产生。本项目的第一个目标是建立基于细胞的筛选方法。我们将产生用于BACE 1剪接研究的带有荧光素酶报告基因的微型基因构建体。将通过荧光素酶活性监测BACE 1前体mRNA外显子3和4内内部剪接位点的选择性使用效率。通过RT-PCR验证瞬时转染细胞中的微小基因后，将建立稳定的神经元细胞系。该项目的第二个目的是使用这些稳定的细胞系来鉴定减少501-aa BACE 1活性同种型产生的小分子。这些小分子将进一步测试其减少A-β生成的有效性。这些研究可能会导致新的药物开发的AD和理解的信号转导途径调节mRNA剪接。我们将从使用我们收集的化合物库的小规模筛选开始。高通量筛选和作用机制将是我们的长期目标。

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.