RNA Targeted Screens of the Prion 5'UTR

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，通过限制内源性人细胞朊蛋白（PrPc）的翻译来筛选和鉴定减缓痒病向人类（克鲁茨feld- jacob综合征（CJD））传播的药物。限制细胞内内源性PrPc的水平可以防止这种内源性Cu/Zn金属蛋白成为肉源（即羊皮）和牛肉（疯牛病）感染性PrP （PrPsc）转化蛋白质的可行靶点。小分子结合到PrPc转录物的5'端会降低其翻译效率。本项目旨在建立筛选条件，使PrP （variant 2）转录物前的5'非翻译区能够作为药物靶点，从而鉴定出阻止细胞内朊病毒（PrP）翻译的新型高选择性抑制剂。我们将利用我们之前的专业知识高通量筛选阿尔茨海默病APP 5'UTR，现在筛选选定的MLSCN药物库（HTS），并确定小分子PrP UTR导向的导联物，以开发成有效的朊病毒翻译抑制剂。神经退行性疾病通常是由于特定蛋白的表达改变或异常折叠而发生的，例如阿尔茨海默病的APP淀粉样蛋白，CJD的朊蛋白和帕金森病的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中的翻译。在用内部稳定转染的神经细胞系进行反筛选后，将确保针对人prp - vt25 5'UTR靶标筛选的导联的特异性，这些神经细胞系分别表达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大的特异性，以识别有用的细胞内朊病毒水平抑制剂，作为一种治疗策略，以抵消这种可怕的神经退行性萎缩疾病。基于蛋白质和DNA的方法预计会产生更多的脱靶。

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).