HTS Molecules Targeting APP 5'untranslated Region(RMI)

描述（由申请人提供）：阿尔茨海默氏淀粉样蛋白前体蛋白（APP 5‘ UTR） mRNA的5’非翻译区是一个关键的翻译调控元件，它决定了任何给定细胞中APP的产量。IL-1通过铁响应元件（IRE）增强了铁调控蛋白（IRPs）与APP 5'UTR RNA二级结构之间的相互作用。这表明app5’UTR是一个很好的药物靶点。我们的实验室在药物发现领域获得了宝贵的经验，我们筛选了FDA预先批准的药物库，并确定了17个利用瞬时转染的神经母细胞瘤细胞限制APP 5' UTR驱动翻译的线索。在二次western blot检测中，帕罗西汀（SSRI）和二巯基丙醇（螯合剂）选择性地降低了APP全蛋白的表达。作为APP 5'UTR筛选过程中获得的药物选择性的证据，这2次命中并未改变SH-SY5Y细胞中APLP-1和APLP-2的表达（Payton et al., 2003）。一项初步研究表明，帕罗西汀减少了AD转基因小鼠模型（TgCRNDS小鼠）的淀粉样蛋白负荷。通过R03 RFA机制收集的数据将帮助我们开发FDA试点筛选，以建立基于重要RNA靶点的高通量转染筛选。通过我们与神经退行性疾病药物发现实验室（LDDN）的合作，现在可以使用384个基于孔的筛选自动化设备。我们的策略将是优化LDDN药物库中11万种化合物的高通量筛选，以确定选择性抑制APP 5'UTR增强子驱动的翻译的新药物。这些导联可能很好地提供下游抗淀粉样蛋白限制治疗作用。在RNA凝胶转移和基于APP表达的实验中使用我们的药物命中将探索APP翻译的途径。IRP-1和IRP-2转录后通过调节铁蛋白翻译和转铁蛋白受体mRNA的稳定性来控制铁稳态。针对146nt APP 5'UTR的HTS击中将提供新的工具，以准确评估IRP-1和IRP-2与编码APP（金属蛋白）的mRNA的5'UTR相互作用的重要性，相对于其他关键铁蛋白的mRNA。

DESCRIPTION (provided by applicant): The 5' untranslated region of the mRNA for the Alzheimer's Amyloid Precursor Protein (APP 5' UTR) is a key translational regulatory element that sets the amount of APP production in any given cell. IL-1 enhanced the interaction between Iron-regulatory Proteins (IRPs) and APP 5'UTR RNA secondary structure via an Iron responsive Element (IRE). This regulation suggested the APP 5' UTR to be an excellent drug target. Our laboratory gained valuable experience in the field of drug discovery when we screened a library of FDA pre-approved drugs and identified 17 leads that limited APP 5' UTR driven translation using transiently transfected neuroblastoma cells. In secondary western blot-based assays paroxetine (SSRI) and dimercaptopropanol (chelator) selectively reduced APP holoprotein expression. As proof of drug selectivity achieved during the APP 5'UTR screen these 2 hits did not change APLP-1 and APLP-2 expression in SH-SY5Y cells (Payton et al., 2003). A pilot study indicated that Paroxetine reduced the amyloid burden in a transgenic mouse model for AD (TgCRNDS mice). The data to be gathered through this R03 RFA mechanism will assist us to develop our FDA pilot screen to set up a high throughput transfection based screen of an important RNA target. Use of automated equipment for 384 well based screens is now available through our collaboration with the Laboratory of Drug Discovery for Neurodegeneration (LDDN). Our strategy will be to optimize a high throughput screen of the110,000 compounds in the LDDN drug library to identify new agents that selectively inhibit translation driven by the APP 5'UTR enhancer. These leads may well provide downstream therapeutic anti-amyloid limiting action. Use of our drug hits in RNA gel-shift and APP expression based experiments will probe the pathway of APP translation. IRP-1 and IRP-2 post-transcriptionally control iron homeostasis by modulating ferritin translation and transferrin receptor mRNA stability. HTS hits directed to the 146 nt APP 5'UTR will provide new tools to assess precisely the importance of the interaction of IRP-1 and IRP-2 with 5'UTR of the mRNA encodiing APP (metalloprotein), relative to the mRNAs of other key iron proteins.