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MLSN Screen of the PD Alpha Synuclein 5'UTR

PD Alpha 突触核蛋白 5UTR 的 MLSN 筛选

基本信息

批准号：
7680767
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
项目类别：
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/7680767
关键词：
5&apos Untranslated Regions Alzheimer&apos 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

项目摘要

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（由申请人提供）：帕金森氏α-突触核蛋白mRNA的5'非翻译区（ASYN 5'UTR）是一个关键的翻译调节元件，有助于设定神经细胞中α-突触核蛋白的产生量，特别是响应Fe流入（铁积累是黑质中PD的关键标志之一）。我们的初步数据显示，神经母细胞瘤（SH-SY5Y 细胞）中 ASYN 基因表达在信息翻译水平上受铁控制，而不会改变 ASYN mRNA 的转录或稳态水平。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错误代谢和/或相关的氧化事件有关。这些发现以及我们在 APP mRNA 翻译调节方面的经验表明，ASYN 5'UTR 将是一个极好的药物靶点。我们已经生成了 ASYN 和 APP 5'UTR 构建体以及稳定细胞系，可用于从包含 500,000 种化合物的 MLSCN 分子库中筛选小分子。我们用于识别 APP 5'UTR 药物命中的筛选策略之前是与 FDA 和 LDDN 分子库一起进行的，现在已针对本次 NIMH 征集进行了调整，用于筛选 α-突触核蛋白 5'UTR。作为我们筛选神经退行性疾病转录本 5'UTR 的方法的先前验证，我们的 APP 5'UTR 筛选产生了药物选择性，因为帕罗西汀是一种 APP 5'UTR 导向的 FDA 抑制剂，不会改变 SH-SY5Y 细胞中的 APLP-1 和 APLP-2 表达（Payton 等人，2003）。帕罗西汀随后减少了 AD 转基因小鼠模型（TgCRND8 小鼠）中的淀粉样蛋白负担（Tucker 等人，(2005/2006)。本次拨款申请的目的是与 MLSCN 合作，筛选 5'UTR 定向的 α 突触核蛋白 mRNA 翻译的小分子抑制剂，用于 PD 治疗。我们的实验室拥有稳定的细胞系，可表达荧光素酶报告基因由目标神经退行性蛋白的 5'UTR 翻译驱动，包括 ASYN (PD)、APP (AD) 和朊病毒蛋白（PrP、克雅氏病）。 Ippolita Cantuti-Castelvetri 博士（coPI、MIND、MGH）将提供正确的试剂、细胞系和经验，以成功确保 ASYN 5'UTR 特异性线索维持神经元活力，并且具有足够的特异性来抑制有毒的 α-突触核蛋白表达，但保持补偿性 β-突触核蛋白和 γ-突触核蛋白表达不变。通过该 R21 分子库筛选合作机制积累的数据将允许对重要的 RNA 靶标（帕金森氏α的 5'非翻译区）进行基于高通量转染的筛选。突触核蛋白 (ASYN) 转录本。该 5'UTR 是帕金森病 (PD) 的一个有吸引力的治疗靶点，新发现的 ASYN 5'UTR 特异性先导化合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神经毒性 ASYN 的产生。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。针对其他 mRNA 的 5'UTR 的化合物将探索阿尔茨海默氏病中 Aβ-淀粉样蛋白前体蛋白 mRNA（ALS 中的 SOD-1 mRNA，以及克鲁茨菲尔德-雅各布氏综合症中的 PrP mRNA）的翻译机制。