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).

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).我们的初步数据显示，ASYN基因表达受到消息翻译水平的铁控制，而没有变为神经母细胞瘤（SH-SY5Y细胞）中ASYN mRNA的转录或稳态水平。该调节类似于另一种金属蛋白，即阿尔茨海默氏病的淀粉样蛋白前体蛋白，这意味着神经退行性疾病基因可能与铁和/或相关氧化事件的非代谢有关。这些发现，以及我们对APP mRNA翻译的调节经验，表明Asyn 5'utr将是一个极好的药物靶标。我们已经生成了ASYN和APP 5'UTR构建体和稳定的细胞系，可用于从MLSCN分子库中筛选500,000种化合物的小分子。我们以前使用FDA和LDDN分子文库进行了识别App 5'UTR药物打击的筛选策略，现在已改编成alpha-synnuclein 5'utr，以筛选此NIMH招标。作为我们筛选神经退行性疾病转录物5'UTRS的方法的事先验证，我们的APP 5'UTR筛选产生了药物选择性，因为Paroxetine是APP 5'UTR指示的FDA抑制剂，在SH-Sy5Y细胞中没有改变APLP-1和APLP-2的表达（Payton等，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萤光素酶报告基因在靶神经退行性蛋白的5'UTR上翻译，包括ASYN（PD）（PD），APP（AD）和Prion蛋白（PRP，Creutzfeldt-Jacob病），copi和Coptiut tementr and Suceelly and coce tongents tongents tongents tongents and coce tongents and copsection tongents and coce tongents in coce and coce and coce tongent保持神经元的生存力，并且足以抑制有毒α-核蛋白表达，但留下代偿性β-核蛋白和γ-突触核蛋白表达不变，可以通过这种R21分子筛选的cooperation cooperative cooperation cops of the the the the the the Interpation targe the the the the the the the the the the the the the the the the the ryntra coolly tarte coop targe的r21分子库。突触核蛋白（ASYN）转录本。这5'UTR是帕金森氏病（PD）的有吸引力的治疗靶标，新近鉴定的ASYN 5'UTR特异性铅可能是由药物化学可能开发的，可能会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翻译机理。针对其他mRNA的5'UTRS的化合物将探测阿尔茨海默氏病（ALS中的SOD-1 mRNA和cruetzsfeld- Jacob综合征中的prp mRNA）中Abeta-淀粉样蛋白前体蛋白mRNA翻译的机理。