RNA Targeted Screens of the Prion 5'UTR

朊病毒 5UTR 的 RNA 靶向筛选

• 批准号: 7617517
• 负责人: JACK T ROGERS
• 金额: $ 17.59万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7617517
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Actins; Alzheimer' s Disease; Aminoglycosides; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Amyotrophic Lateral Sclerosis; Animal Model; Animals; Antibodies; Appendix; Base Pairing; Binding; Biological Assay; Bovine Spongiform Encephalopathy; Cell Line; Cells; Commit; Condition; DNA; Data; Disease; Disease Progression; Dose; Drug Delivery Systems; Elements; Enzyme-Linked Immunosorbent Assay; Ferritin; Frequencies; Gene Expression; Genetic Translation; H ferritin; Hepatitis C; Homeostasis; Housing; Human; Infection; Intervention; Iron; Iron-Regulatory Proteins; Lead; Left; Letters; Libraries; Luciferases; Macrolide Antibiotics; Meat; Mediating; Messenger RNA; Metalloproteins; Modeling; Molecular Bank; Molecular Probes; Mus; Nerve Degeneration; Neuroblastoma; Neurodegenerative Disorders; Neurons; Oxidative Stress; Parkinson Disease; Pharmaceutical Chemistry; Pharmaceutical Preparations; PrPSc Proteins; Prion Diseases; Prions; Production; Proteins; Public Health; RNA; Reagent; Regulation; Relative (related person); Reporter; Reporting; Resources; Risk; Running; Scrapie; Screening procedure; Sequence Alignment; Sheep; Small RNA; Source; Specificity; Synaptic Transmission; Syndrome; Testing; Therapeutic; Transcript; Transfection; Transferrin; Transferrin Receptor; Translations; Universities; Untranslated Regions; Variant; Viral; Western Blotting; alpha synuclein; base; beef; design; dopaminergic neuron; experience; high throughput screening; in vivo; infectious disease treatment; inhibitor/antagonist; neurotoxic; neurotoxicity; novel; novel therapeutics; prevent; receptor; relating to nervous system; small molecule; stem; synuclein; therapeutic target; tissue culture; wasting

DESCRIPTION (provided by applicant): We aim to set up a novel RNA based therapeutic strategy to screen and identify agents that slow down the spread of scrapies diseases to humans (Crueutzfeld-Jacob syndrome (CJD)) by limiting translation of the endogenous human cellular prion protein (PrPc). Limiting intracellular levels of endogenous PrPc should prevent this endogenous Cu/Zn metaloprotein from being a viable target for protein conversion by infectious PrP (PrPsc) from meat sources (i.e. Scapie from Sheep), and from beef (mad cow disease). Binding of small molecules to the 5' end of the PrPc transcript reduces its translational efficiency. This project is to set up the screening conditions to enable the use of the 5'untranslated region in front of the PrP (variant 2) transcript as a drug target so that we can identify novel and highly selective inhibitors that prevent intracellular prion (PrP) translation. We will use our prior expertise to high throughput screen the Alzheimer's APP 5'UTR, now to screen a chosen MLSCN drug library (HTS) and identify small molecule PrP UTR directed leads to be developed into effective prion translation inhibitors. Neurodegenerative diseases often occur due to the altered expression or aberrant folding of specific proteins, for example amyloid from APP in Alzheimer's disease, Prion protein in CJD and 1-synuclein in Parkinson's disease. However, our strategy is to prevent the translation of their key endogenous gene products in humans. Specificity of leads screened against the human PrP-vt2 5'UTR target will be assured after counter- screens with in-house stably transfected neural cell lines that each express the APP 5'UTR-luciferase and 1-syn 5'UTR-luciferase as key counter targets. This RNA based screening strategy will provide greater specificity to identify useful inhibitors of intracellular prion levels as a therapeutic strategy to offset this terrible neurodegenerative wasting disorder. Protein and DNA based approaches would be predicted to generate more off-target hits. PUBLIC HEALTH RELEVANCE: The data to be accumulated through this R21 molecular libraries screening cooperative mechanism will permit a high throughput transfection based screen of an important RNA target, the 5'untranslated region of the Parkinson's alpha synuclein (ASYN) transcript. This 5'UTR is an attractive therapeutic target for Parkinson's disease (PD), and newly identified ASYN 5'UTR specific leads may be developed by medicinal chemistry potentially to limit neurotoxic ASYN production in dopaminergic neurons. Use of 5'UTR specific MLSCN hits will probe the mechanism of translation of ASYN mRNA relevant to PD. Compounds directed to 5'UTRs of other mRNAs will probe mechanism of translation of the A2-amyloid precursor protein mRNA in Alzheimer's disease (SOD-1 mRNA in ALS, and PrP mRNA in Cruetzsfeld- Jacob Syndrome).

描述（由申请人提供）：我们的目的是建立一种新的基于RNA的治疗策略，以筛选和鉴定通过限制内源性人细胞朊病毒蛋白（PrPc）的翻译来减缓瘙痒病向人类（克-雅二氏综合征（CJD））传播的药物。限制内源性PrPc的细胞内水平应防止该内源性Cu/Zn金属蛋白成为来自肉类来源（即来自绵羊的Scapie）和来自牛肉（疯牛病）的感染性PrP（PrPsc）的蛋白质转化的可行靶标。小分子与PrPc转录物的5'末端的结合降低了其翻译效率。本项目旨在建立筛选条件，使PrP（变体2）转录本前的5 '非翻译区作为药物靶点，以便我们能够鉴定新型和高选择性的抑制剂，以阻止细胞内朊病毒（PrP）翻译。我们将利用我们先前的专业知识高通量筛选阿尔茨海默氏症APP 5 'UTR，现在筛选选定的MLSCN药物库（HTS）并鉴定小分子PrP UTR定向的先导物，以开发成有效的朊病毒翻译抑制剂。神经退行性疾病通常由于特定蛋白质的改变的表达或异常折叠而发生，例如阿尔茨海默病中来自APP的淀粉样蛋白、CJD中的朊病毒蛋白和帕金森病中的1-突触核蛋白。然而，我们的策略是阻止它们的关键内源性基因产物在人类中的翻译。在用内部稳定转染的神经细胞系进行反筛选后，将确保针对人PrP-vt 2 5 'UTR靶标筛选的先导物的特异性，所述细胞系各自表达APP 5' UTR-荧光素酶和1-syn 5 'UTR-荧光素酶作为关键反靶标。这种基于RNA的筛选策略将提供更大的特异性来鉴定细胞内朊病毒水平的有用抑制剂，作为抵消这种可怕的神经退行性消耗性疾病的治疗策略。预计基于蛋白质和DNA的方法将产生更多的脱靶命中。 公共卫生关系：通过该R21分子文库筛选合作机制积累的数据将允许基于高通量转染筛选重要的RNA靶标，帕金森氏α突触核蛋白（ASYN）转录物的5 '非翻译区。该5 'UTR是帕金森病（PD）的有吸引力的治疗靶点，并且新鉴定的ASYN 5' UTR特异性先导物可以通过药物化学开发，从而潜在地限制多巴胺能神经元中的神经毒性ASYN产生。使用5 'UTR特异性MLSCN命中将探测与PD相关的ASYN mRNA的翻译机制。针对其它mRNA的5 ′ UTR的化合物将探测阿尔茨海默病中A2-淀粉样前体蛋白mRNA（ALS中的SOD-1 mRNA和克鲁茨菲尔德-雅各布综合征中的PrP mRNA）的翻译机制。