HTS Molecules Targeting APP 5'untranslated Region(RMI)

靶向 APP 5非翻译区 (RMI) 的 HTS 分子

• 批准号: 7407177
• 负责人: JACK T ROGERS
• 金额: $ 4.38万
• 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/7407177
• 关键词: 5' Untranslated Regions; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Animals; Antibiotics; Azithromycin; Binding; Biological; Biological Assay; Brain; Cell Line; Cells; Chelating Agents; Collaborations; Condition; Data; Deferoxamine; Development; Down Syndrome; Drug Delivery Systems; Drug Design; Elements; Enhancers; Equipment; Erythromycin; Ferritin; Figs - dietary; Gel; Genes; Genome; HIV; HIV Infections; Homeostasis; Hour; Initiator Codon; Interleukin-1; Iron; Iron-Regulatory Proteins; Laboratories; Libraries; Luciferases; Messenger RNA; Metalloproteins; Modeling; Mus; Nerve Degeneration; Neuroblastoma; New Agents; Output; Paroxetine; Pathology; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pilot Projects; Preclinical Drug Evaluation; Production; Protein Biosynthesis; Proteins; RNA; RNA Sequences; RNA-Protein Interaction; Regulation; Regulatory Element; Relative (related person); Reporter; Reporting; Ribosomal RNA; Ribosomes; Screening procedure; Senile Plaques; Standards of Weights and Measures; Structure; Tars; Testing; Therapeutic; Time; Transcript; Transfection; Transferrin Receptor; Transgenic Organisms; Translations; United States Food and Drug Administration; Untranslated Regions; Western Blotting; base; concept; density; drug discovery; experience; high throughput screening; in vivo; inhibitor/antagonist; mRNA Stability; mouse model; prevent; protein expression; research study; small molecule; stable cell line; therapeutic target; tissue culture; tool

The 5'untranslated region of the mRNAforthe 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) selectivley reduced APP holoprotein expression. As proof of drug selectivity achieved during the APP 5'UTR screen these two 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 Neurodegenreration (LDDN). Our strategy will be to optimize a high throughput screen of the 110,000 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.

阿尔茨海默病淀粉样前体蛋白 (APP 5'UTR) mRNA 的 5' 非翻译区是关键 决定任何给定细胞中 APP 产量的翻译调节元件。 IL-1 增强 铁调节蛋白 (IRP) 和 APP 5'UTR RNA 二级结构之间通过铁调节蛋白相互作用 响应元件（IRE）。这一规定表明 APP 5'UTR 是一个极好的药物靶点。 当我们筛选 FDA 的库时，我们的实验室在药物发现领域获得了宝贵的经验- 预先批准的药物并确定了 17 个先导化合物，这些先导化合物使用瞬时限制了 APP 5'UTR 驱动的翻译 转染的神经母细胞瘤细胞。在基于蛋白质印迹的二次检测中，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。作为药物选择性的证明 在 APP 5'UTR 筛选过程中实现的这两个命中不会改变 APLP-1 和 APLP-2 的表达 SH-SY5Y 细胞（Payton 等，2003）。一项初步研究表明，帕罗西汀减少了淀粉样蛋白的负担 AD 转基因小鼠模型（TgCRNDS 小鼠）。 通过 R03 RFA 机制收集的数据将帮助我们开发 FDA 试点筛选 建立了基于高通量转染的重要 RNA 靶标筛选。使用自动化 我们与药物实验室合作现已提供 384 个孔筛选设备 神经退行性疾病发现 (LDDN)。我们的策略是优化高通量筛选 LDDN 药物库中的 110,000 种化合物可用于识别选择性抑制翻译驱动的新药物 通过 APP 5'UTR 增强子。这些先导化合物很可能提供下游治疗性抗淀粉样蛋白限制 行动。在 RNA 凝胶迁移和基于 APP 表达的实验中使用我们的药物命中将探测 APP翻译途径。 IRP-1 和 IRP-2 通过调节转录后控制铁稳态 铁蛋白翻译和转铁蛋白受体 mRNA 稳定性。指向 146 nt APP 5'UTR 的 HTS 命中将 提供新工具来准确评估 IRP-1 和 IRP-2 与 5'UTR 相互作用的重要性 相对于其他关键铁蛋白的 mRNA，编码 APP（金属蛋白）的 mRNA。