RNA Targeted Screens of the Prion 5'UTR

朊病毒 5UTR 的 RNA 靶向筛选

• 批准号: 8112177
• 负责人: JACK T ROGERS
• 金额: $ 5万
• 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/8112177
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Actins; Alzheimer' s Disease; Aminoglycosides; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Animal Model; Animals; Antibodies; Base Pairing; Binding; Biological Assay; Bovine Spongiform Encephalopathy; Cell Line; Cells; Commit; 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; RNA; Reagent; Regulation; Relative (related person); Reporter; Reporter Genes; 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 Genes; Western Blotting; alpha synuclein; base; beef; design; dopaminergic neuron; experience; high throughput screening; in vivo; infectious disease treatment; inhibitor/antagonist; neurotoxic; neurotoxicity; novel; novel therapeutic intervention; novel therapeutics; prevent; public health relevance; 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)：我们的目标是建立一种新的基于RNA的治疗策略，通过限制内源性人类细胞普恩蛋白(PrPc)的翻译来筛选和识别减缓瘙痒病向人类传播(Crueutzfeld-Jacob综合征(CJD))的药物。限制细胞内内源性PrPc的水平应该可以防止这种内源性铜/锌金属蛋白成为来自肉类(即绵羊的头盖骨)和牛肉(疯牛病)的感染性PrP(PrPsc)进行蛋白质转换的有效靶点。小分子与PrPc转录本5‘端的结合降低了其翻译效率。该项目旨在建立筛选条件，使PrP(变体2)转录本前面的5‘非翻译区能够用作药物靶点，以便我们能够识别新型和高选择性的抑制物，以防止PrP(PrP)翻译。我们将利用我们以前的专业知识来高通量筛选阿尔茨海默氏症APP 5‘UTR，现在将筛选选定的MLSCN药库(HTS)，并识别小分子PrP UTR导向的线索，以开发成有效的Prion翻译抑制剂。神经退行性疾病通常是由于特定蛋白质的表达改变或异常折叠所致，例如阿尔茨海默病中APP的淀粉样蛋白，CJD中的Prion蛋白，以及帕金森病中的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身上翻译。在用内部稳定转染的神经细胞株进行反筛选后，针对人PrP-VT2 5‘UTR靶标筛选的导联的特异性将得到确保，每个神经细胞系都表达APP 5’UTR-荧光素酶和1-SYN 5‘UTR-荧光素酶作为关键的反靶标。这种基于RNA的筛选策略将提供更高的特异性，以确定细胞内Pron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。基于蛋白质和DNA的方法预计会产生更多偏离目标的命中率。 公共卫生相关性：通过这种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基因筛选重要的RNA靶标，即帕金森病α突触核蛋白(ASYN)转录本的5‘非翻译区。这种5‘非编码区是治疗帕金森病(PD)的一个有吸引力的靶点，新近发现的ASYN 5’非编码区特异性导联可能通过药物化学来限制多巴胺能神经元中神经毒性ASYN的产生。利用5‘端非编码区特异性MLSCN HITS将探索与帕金森病相关的ASYN mRNA翻译机制。针对其他mRNAs的5‘UTRs的化合物将探索A2-淀粉样前体蛋白mRNA在阿尔茨海默病中的翻译机制(在ALS中为SOD-1mRNA，在Cruetzsfeld-Jacob综合征中为PrP mRNA)。