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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）是一种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（铁蓄积是黑质中PD的关键标志之一）。我们的初步数据显示，在神经母细胞瘤（SH-SY 5 Y细胞）中，铁在信息翻译水平上控制ASYN基因表达，而不改变ASYN mRNA的转录或稳态水平。这种调节类似于另一种金属蛋白，阿尔茨海默氏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。我们已经产生了ASYN和APP 5 'UTR构建体和稳定的细胞系，可用于从500，000种化合物的MLSCN分子文库中筛选小分子。我们鉴定APP 5 'UTR药物命中的筛选策略先前是用FDA和LDDN分子文库进行的，并且现在已经被调整以筛选用于该NIMH请求的α-突触核蛋白5' UTR。作为我们筛选神经变性疾病转录物的5 'UTR的方法的先前验证，我们的APP 5' UTR筛选产生药物选择性，因为帕罗西汀是APP 5 'UTR指导的FDA抑制剂，其不改变SH-SY 5 Y细胞中的APLP-1和APLP-2表达（Payton et al.，2003年）。帕罗西汀随后降低了AD转基因小鼠模型（TgCRND 8小鼠）中的淀粉样蛋白负荷（Tucker等人，(2005/2006）。该资助申请的目的是与MLSCN合作，筛选用于PD治疗的α突触核蛋白mRNA翻译的5 'UTR定向小分子抑制剂。我们的实验室具有稳定的细胞系，其表达由靶神经退行性蛋白（包括ASYN（PD）、APP（AD）和朊病毒蛋白（PrP，Creutzfeldt-Jacob病））的5 'UTR自发驱动的荧光素酶报告基因。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特异性导联保持神经元活力，并具有足够的特异性抑制毒性α-突触核蛋白表达，但保持代偿性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A β-淀粉样前体蛋白mRNA（ALS中的SOD-1 mRNA和Cruetzsfeld-Jacob综合征中的PrP mRNA）的翻译机制。