HTS Molecules Targeting APP 5'untranslated Region(RMI)

靶向 APP 5非翻译区 (RMI) 的 HTS 分子

• 批准号: 7021329
• 负责人: JACK T ROGERS
• 金额: $ 8.75万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7021329
• 关键词: Alzheimer' s disease; RNA binding protein; amyloid proteins; biotechnology; cell line; drug discovery /isolation; gene induction /repression; genetic regulatory element; genetic translation; green fluorescent proteins; high throughput technology; iron sulfur protein; messenger RNA; neuroblastoma; small molecule; technology /technique development; transfection

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.

描述（由申请人提供）：阿尔茨海默氏淀粉样前体蛋白mRNA的5'非翻译区（APP 5' UTR）是一个关键的翻译调节元件，其设定任何给定细胞中APP产生的量。IL-1通过铁反应元件（IRE）增强铁调节蛋白（IRP）与APP 5 'UTR RNA二级结构之间的相互作用。这种调控提示APP 5' 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预批准的药物库，并使用瞬时转染的神经母细胞瘤细胞鉴定了17个限制APP 5' UTR驱动翻译的前导序列。在二级蛋白质印迹分析中，帕罗西汀（SSRI）和二巯基丙醇（螯合剂）选择性降低APP全蛋白表达。作为在APP 5 'UTR筛选期间实现的药物选择性的证据，这2个命中不改变SH-SY 5 Y细胞中的APLP-1和APLP-2表达（Payton等人，2003年）。一项初步研究表明，帕罗西汀降低了AD转基因小鼠模型（TgCRNDS小鼠）中的淀粉样蛋白负荷。通过R 03 RFA机制收集的数据将有助于我们开发FDA中试筛选，以建立基于高通量转染的重要RNA靶点筛选。通过我们与神经退行性疾病药物发现实验室（LDDN）的合作，现在可以使用自动化设备进行384孔筛选。 我们的策略将是优化LDDN药物库中110，000种化合物的高通量筛选，以鉴定选择性抑制APP 5 'UTR增强子驱动的翻译的新药物。这些先导物可以很好地提供下游治疗性抗淀粉样蛋白限制作用。 在基于RNA凝胶移位和APP表达的实验中使用我们的药物命中将探测APP翻译的途径。IRP-1和IRP-2通过调节铁蛋白翻译和转铁蛋白受体mRNA稳定性在转录后控制铁稳态。针对146 nt APP 5 'UTR的HTS命中将提供新的工具来精确评估IRP-1和IRP-2与编码APP（金属蛋白）的mRNA的5' UTR相对于其它关键铁蛋白的mRNA的相互作用的重要性。