MLSN Screen of the PD Alpha Synuclein 5'UTR

PD Alpha 突触核蛋白 5UTR 的 MLSN 筛选

• 批准号: 7290548
• 负责人: JACK T ROGERS
• 金额: $ 21.88万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7290548
• 关键词: 5' Untranslated Regions; Alzheimer' s Disease; Aminoglycosides; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Amyotrophic Lateral Sclerosis; Animal Model; Antibodies; Appendix; Applications Grants; Base Pairing; Binding; Biological Assay; Brain Pathology; Cell Line; Cells; Chelating Agents; Collaborations; Condition; Corpus striatum structure; Creutzfeldt-Jakob Syndrome; Cultured Cells; Data; Deferoxamine; Development; Disease Progression; Dose; Drug Delivery Systems; Elements; Ensure; Event; Ferritin; Figs - dietary; Gamma synuclein; Gene Duplication; Gene Expression; Genes; Genetic; Genetic Transcription; Genetic Translation; H ferritin; HIV; Hepatitis C; Homeostasis; Iron; Iron-Regulatory Proteins; Lead; Left; Letters; Lewy Body Disease; Link; Luciferases; Macrolide Antibiotics; Mediating; Messenger RNA; Metalloproteins; Mind; Modeling; Molecular Bank; Molecular Probes; Mus; National Institute of Mental Health; Nerve Degeneration; Neuroblastoma; Neurodegenerative Disorders; Neurons; Nucleotides; Oxidative Stress; Parkinson Disease; Paroxetine; Patients; Pharmaceutical Chemistry; Pharmaceutical Preparations; PrP amyloid; PrP sequence; Prions; Production; Proteins; RNA; Reagent; Regulation; Regulatory Element; Relative (related person); Reporter; Reporter Genes; Reporting; Running; SNCA gene; Screening procedure; Sequence Alignment; Shapes; Small RNA; Substantia nigra structure; Syndrome; Testing; Therapeutic; Therapeutic Agents; Transcript; Transfection; Transferrin; Transferrin Receptor; Transgenic Organisms; Translations; United States Food and Drug Administration; Untranslated Regions; Validation; Variant; Viral; Western Blotting; alpha synuclein; base; design; dopaminergic neuron; experience; in vivo; infectious disease treatment; inhibitor/antagonist; iron metabolism; mouse model; neurotoxic; neurotoxicity; novel therapeutics; phosphoneuroprotein 14; receptor; response; small molecule; stable cell line; stem; synuclein; therapeutic target; tissue culture

DESCRIPTION (provided by applicant): The 5'untranslated region of the mRNA for the Parkinson's a-synuclein (ASYN 5'UTR) is a key translational regulatory element that contributes to setting the amount of alpha-synuclein production in neural cells, particularly in response to Fe influx (iron accumulation is one of the key hallmarks of PD in the Substantia nigra). Our preliminary data showed ASYN gene expression is controlled by iron at the level of message translation without change to transcription or steady state levels of ASYN mRNA in neuroblastoma (SH-SY5Y cells). This regulation is similar to another metalloprotein, the Amyloid Precursor Protein of Alzheimer's disease, implying that neurodegenerative disease genes may be linked to the mis-metabolism of iron and/or associated oxidative events. These findings, and our experience with the regulation of APP mRNA translation, suggested the ASYN 5'UTR would be an excellent drug target. We already generated both ASYN and APP 5'UTR constructs and stable cell lines available for screening small molecules from the MLSCN molecular library of 500,000 compounds. Our screening strategy to identify APP 5'UTR drug hits was previously conducted with FDA and LDDN molecular libraries, and has now been adapted to screen the alpha-synuclein 5'UTR for this NIMH solicitation. As prior validation of our approach to screen the 5'UTRs of neurodegenerative disease transcripts, our APP 5'UTR screens generated drug selectivity since paroxetine was an APP 5'UTR directed FDA inhibitor that did not change APLP-1 and APLP-2 expression in SH-SY5Y cells (Payton et al., 2003). Paroxetine subsequently reduced the amyloid burden in a transgenic mouse model for AD (TgCRND8 mice) (Tucker et al., (2005/2006). The aim of this grant application is to collaborate with the MLSCN to screen for 5'UTR directed small molecule inhibitors of translation of alpha synuclein mRNA for PD therapeutics. Our lab has stable cell lines that express luciferase reporter genes translationally driven by the 5'UTRs of target neurodegenerative proteins including ASYN (PD), APP (AD) and Prion protein (PrP, Creutzfeldt-Jacob disease). Dr. Ippolita Cantuti-Castelvetri (coPI, MIND, MGH) will provide the correct reagents, cell lines and experience to successfully ensure ASYN 5'UTR specific leads maintain neuronal viability and are sufficiently specific to inhibit toxic alpha-synuclein expression but leave compensatory beta-synuclein and gamma- synuclein expression unchanged. 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 Abeta-amyloid precursor protein mRNA in Alzheimer's disease (SOD-1 mRNA in ALS, and PrP mRNA in Cruetzsfeld- Jacob Syndrome).

描述(申请人提供)：帕金森病α-突触核蛋白的5‘非翻译区(ASYN 5’UTR)是一个关键的翻译调控元件，它有助于设定神经细胞中α-突触核蛋白的产生量，特别是对铁内流的反应(铁的积累是黑质帕金森病的关键标志之一)。我们的初步数据表明，在神经母细胞瘤(SH-SY5Y细胞)中，ASYN基因的表达是由铁在消息翻译水平上控制的，而不改变ASYN基因的转录或稳定水平。这种调节类似于另一种金属蛋白，即阿尔茨海默病的淀粉样前体蛋白，这意味着神经退行性疾病基因可能与铁的代谢失调和/或相关的氧化事件有关。这些发现，以及我们在APP mRNA翻译调控方面的经验，表明ASYN 5‘UTR将是一个很好的药物靶点。我们已经生成了ASYN和APP 5‘UTR结构和稳定的细胞系，可用于从包含500,000种化合物的MLSCN分子库中筛选小分子。我们识别APP 5‘非编码区药物点击的筛选策略以前是与FDA和LDDN分子库一起进行的，现在已经被适应于为这次NIMH征集筛选α-突触核蛋白5’非编码区。作为我们筛选神经退行性疾病转录本5‘端非编码区的方法的预先验证，我们的APP 5’非编码区筛选产生了药物选择性，因为帕罗西汀是一种APP 5‘非编码区指导的FDA抑制剂，不会改变SH-SY5Y细胞中APLP-1和APLP-2的表达(Payton等人，2003年)。帕罗西汀随后降低了阿尔茨海默病转基因小鼠模型(TgCRND8小鼠)的淀粉样蛋白负担(Tucker等人，(2005/2006))。这项拨款申请的目的是与MLSCN合作，筛选用于帕金森病治疗的5‘UTR导向的α突触核蛋白mRNA翻译的小分子抑制物。我们实验室有稳定的细胞系，其表达的荧光素酶报告基因是由靶神经退行性蛋白的5‘UTRs驱动的，包括ASYN(PD)、APP(AD)和Prion蛋白(PrP，CreutzFeldt-Jacob病)。Ippolita Cantuti-Castelvetri博士(COPI，Mind，MGH)将提供正确的试剂、细胞系和经验，成功地确保ASYN 5‘UTR特异性导联维持神经元活性，并具有足够的特异性，以抑制毒性α-突触核蛋白的表达，但保持代偿性β-突触核蛋白和伽马-突触核蛋白的表达不变。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的筛选重要的RNA靶标，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的化合物将探索阿尔茨海默病(ALS中的SOD-1mRNA和Cruetzsfeld-Jacob综合征中的PrP mRNA)中Abeta-淀粉样前体蛋白mRNA的翻译机制。