Towards Therapeutics for Neurodegenerative Diseases

描述（由申请人提供）：神经退行性疾病包括阿尔茨海默病、帕金森病和朊病毒病，它们都是蛋白质加工的疾病。为了开发有效的治疗蛋白质加工疾病的方法，我们把重点放在朊病毒疾病作为模型系统上。朊病毒疾病的研究是有利的，因为健壮的细胞和转基因（Tg）小鼠疾病模型是可用的。这些细胞和小鼠系统有助于疾病进展和治疗干预的测量。由于蛋白质加工疾病之间的共性，人们认为在开发朊病毒疾病的治疗方法中吸取的经验教训可能对影响大量人群的阿尔茨海默氏症和帕金森病等疾病具有重要意义。朊病毒疾病的特征是将朊病毒蛋白的细胞异构体（称为PrPc）错误折叠为致病异构体（称为PrPSc）。我们已经确定了PrPSc复制的关键点，可能适合小分子治疗干预。这些包括底物PrPc的表达和定位；随后转换为PrPSc；以及PrPSc的细胞清除率。在这个应用中，我们建议产生新的筛选程序，可能作为治疗朊病毒疾病的化合物。我们的目标是为PrPc、PrPSc积累和PrPSc清除开发稳健的高通量系统（HTS）检测。这些体外筛选系统将与缺乏多药耐药（mdr）基因la和b的新型Tg小鼠相补充，因此可以克服传统上与在中枢神经系统中获得大量测试化合物相关的问题。将mdrla和1b基因的敲除与控制PrPc表达的诱导转基因系统结合起来，将使我们能够测量PrPSc的清除率，作为CNS中高浓度推定治疗药物的功能。除了促进新化合物的功效测试，这些新的Tg小鼠模型也将有助于验证我们的体外筛选靶点。为了扩大在抗朊病毒药物发现中有用的相关HTS检测的数量，我们还建议进行shRNA筛选，以鉴定参与朊病毒表达、复制和清除的非prp基因。这项研究可能会揭示朊病毒疾病的生物学，并且重要的是，导致新的HTS检测的发展，以确定非prp靶点的选择性配体，从而减少或抑制朊病毒疾病的进展。大量高温热通量测定方法的发展可能最终导致一系列治疗性化合物，这些化合物在协同使用时可以阻止朊病毒的传播，从而阻止疾病的进展。

DESCRIPTION (provided by applicant): The neurodegenerative diseases include Alzheimer's, Parkinson's and prion disease, all of which are disorders of protein processing. Toward developing effective therapies for the protein processing diseases, we have focused on prion disease as a model system. The study of prion disease is advantageous since robust cell and transgenic (Tg) mouse models of disease are available. These cell and mouse systems facilitate measurements of disease progression and therapeutic intervention. Due to the commonality amongst the protein processing diseases, it is thought that lessons learnt in developing therapies for prion disease may have important implications for disorders like Alzheimer's and Parkinson's diseases that affect vastly greater numbers of people. Prion diseases are characterized by the misfolding of the cellular isoform of the prion protein, designated PrPc, to the disease causing isoform, denoted PrPSc. We have identified critical points in the replication of PrPSc that may be suitable for therapeutic intervention with small molecules. These include, expression and localization of the substrate, PrPc; subsequent conversion to PrPSc; and cellular clearance of PrPSc. In this application we propose to generate new screening procedures for compounds that might serve as therapeutics for prion diseases. Our goal is to develop robust, high throughput system (HTS) assays for PrPc, PrPSc accumulation, and PrPSc clearance. These in vitro screening systems will be complemented with new Tg mice that lack the multi-drug resistance (mdr) genes la and b and hence, can overcome problems traditionally associated with achieving substantial concentrations of test compounds in the CNS. Coupling the knockout of the mdrla and 1b genes with an inducible transgene system that governs PrPc expression, will allow us to measure the clearance of PrPSc as a function of high concentrations of putative therapeutics in the CNS. Besides facilitating testing of the efficacy of novel compounds, these new Tg mouse models will also aid in validating our in vitro screening targets. So as to expand the number of relevant HTS assays useful in anti-prion drug discovery, we also propose to undertake a shRNA screen to identify non-PrP genes that are involved with prion expression, replication and clearance. This study may shed light on the biology of prion disease, and importantly, lead to the development of novel HTS assays to identify selective ligands for non-PrP targets that reduce or inhibit the progression of prion disease. The development of a substantial array of HTS assays may eventually lead to an armamentarium of therapeutic compounds, which when used in concert halt the propagation of prions and hence, the progression of disease.