Towards Therapeutics for Neurodegenerative Diseases

神经退行性疾病的治疗方法

• 批准号: 8411612
• 负责人: STANLEY B PRUSINER
• 金额: $ 59.25万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8411612
• 关键词: Academia; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Animal Model; Animals; Antimalarials; Biological Assay; Bioluminescence; Brain; Breeding; Cells; Creutzfeldt-Jakob Syndrome; Cultured Cells; Data; Development; Diamond; Disease; Drug Targeting; Drug resistance; FDA approved; Failure; Frontotemporal Dementia; Human; In Vitro; Industry; Knock-out; Lead; Libraries; Luciferases; Measures; Microscope; Mus; Nerve Degeneration; Neurodegenerative Disorders; Parkinson Disease; Pathogenesis; Pharmaceutical Preparations; PrP; PrP gene; Prions; Process; Protein Isoforms; Proteins; Quinacrine; RNA Interference; Rattus; Reporter; Research; Resolution; Resources; Rodent; Route; Scrapie; Structure; System; Therapeutic; Time; Transgenes; Work; advanced system; base; drug discovery; drug efficacy; experience; high throughput screening; improved; in vivo; mouse model; programs; promoter; small molecule libraries; success

There is not a single FDA-approved drug that halts or even slows neurodegeneration in the CNS. Despite spending billions of dollars, neither industry nor academia has been able to develop a single drug that slows the progression of Alzheimer's (AD), Parkinson's (PD) and Creutzfeldt-Jakob (CJD) diseases as well as amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD). Despite this roadblock, there have been impressive advances in understanding the pathogenesis of all these disorders. A steady accumulation of experimental data argues that a different protein causes each neurodegenerative disease and these proteins acquire alternative structures that become self-propagating i.e., prions (Meyer-Luehmann et al., 2006; Clavaguera et al., 2009; Frost and Diamond, 2009; Olanow and Prusiner, 2009; Brundin et al., 2010; Cushman et al., 2010; Colby and Prusiner, In press). These findings are most gratifying since they provide an enlarging body of evidence in support of what now are regarded as prescient speculations (Prusiner, 1984, 2001). The understanding that all or most ofthe neurodegenerative diseases are caused by prions may give several new perspectives on the development of effective therapeutics. First, drugs that cure cultured cells infected with prions may not predict success in experimental animals or humans. Such is our experience with the antimalarial drug quinacrine that cured cultured cells but failed to extend the lives of either mice or humans (Collinge et al., 2009; Ghaemmaghami et al., 2009). Even when the level of quinacrine was increased almost 100-fold, in the brains of mice in which the P-gp transporter (Mdr1) has been knocked out, above the halfeffective concentration (EC50) in cultured cells, it failed to extend the incubation time (Ghaemmaghami et al., ^ 2009). We were able to gather convincing evidence showing that the most likely explanation for this therapeutic failure was a conformational change in PrP^*^ resulting in a drug-resistant prion strain. These results prompted us to develop a broad drug discovery program that has begun to identify lead compounds that are able to extend incubation times in mice (Ghaemmaghami et al., 2010; Gallardo-Godoy et al., 2011). Discovering drugs that can be used to treat neurodegeneration presents substantial challenges. With that said, we desperately need additional assays for measuring the efficacy of "hits" and "leads" to proceed through the process of drug discovery. More predictive assays of lead efficacy in cultured cells prior to studies in Tg rodents are critical to the development of effective therapeutics. Such cell assays could save an immense amount of time and resources. To improve our in vitro assessments of chemical libraries as well as groups of lead compounds, we plan to use a newly acquired Opera confocal microscope system for high throughput screening (HTS). This advanced system will allow us to measure both PrP^ and PrP^'^ in subcellular compartments. Despite successfully adapting ELISAs for measuring these PrP isoforms in HTS, we anticipate that the robust resolution of the Opera system will allow us to predict more accurately, from cell-based assays, which compounds will advance successfully through animal models. The adaption of bioluminescence to the in vivo readout of experimental scrapie and Alzheimer's disease in Tg mice (Tamgtiney et al., 2009a; Watts et al., 2011) has greatly facilitated our drug efficacy studies. Plans for improving these mouse models and extending the work into rats are described below in the research plan. Bigenic mice-on the Mdrl knockout background and with a luciferase reporter expressed under control of the Gfap promoter-are being bred for use in drug efficacy studies. Similar mice expressing luciferase are being bred, in which the mouse PrP gene has been knocked out and a chimeric human (Hu)/mouse (Mo) transgene is also expressed. Such mice become ill <80 days after inoculation with CJD(MM1) prions (Giles et al., 2010). In another set of studies, we have used RNAi libraries to identify auxiliary proteins that participate in the formation and replication of PrP¿*^ prions. These studies provide a route into the identification of new drug targets for antiprion drugs. More emphasis on these studies is planned since it seems likely that cocktails of drugs with different modes of action will be the most likely routes to successful treatment of neurodegenerative illnesses.

没有一种FDA批准的药物可以阻止甚至减缓CNS中的神经变性。尽管 花费数十亿美元，无论是工业界还是学术界都无法开发出一种减缓 阿尔茨海默病（AD）、帕金森病（PD）和克雅氏病（CJD）的进展以及 肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）。尽管有这些障碍， 在理解所有这些疾病的发病机制方面取得了令人印象深刻的进展。一个稳定的积累 实验数据表明，不同的蛋白质导致每种神经退行性疾病， 获得变为自传播的替代结构即，朊病毒（Meyer-Luehmann等，二○ ○六年; Cladenera等人，2009; Frost和Diamond，2009; Olanow和Prusiner，2009; Brundin等人，2010年;库什曼 例如，2010;科尔比和Prusiner，出版中）。这些发现是最令人满意的，因为它们提供了一个扩大的 一系列证据支持现在被视为有先见之明的推测（Prusiner，1984，2001）。 所有或大部分神经退行性疾病都是由朊病毒引起的，这一认识可能会给我们带来一些 开发有效疗法的新观点。首先，治疗培养细胞感染的药物 在实验动物或人类中的成功可能并不预示着。这就是我们的经验， 抗疟疾药物奎纳克林治愈了培养的细胞，但未能延长小鼠或人类的生命 （Collinge等人，2009; Ghaemmaghami等人，2009年）。即使当奎纳克林的浓度增加到 100倍，在P-gp转运蛋白（Mdr 1）被敲除的小鼠大脑中， 虽然在培养细胞中的浓度（EC 50），但它未能延长孵育时间（Ghaemmaghami等人， ^ 2009）。我们能够收集到令人信服的证据表明，最有可能的解释是， 治疗失败的原因是PrP的构象变化导致产生抗药性朊病毒株。这些 这些结果促使我们开发了一个广泛的药物发现计划，该计划已经开始识别先导化合物， 其能够延长小鼠中的孵育时间（Ghaemmaghami等人，2010; Gallardo-Godoy等人，2011年）。 发现可用于治疗神经退行性变的药物存在重大挑战。话虽如此， 我们迫切需要额外的测定来测量“命中”和“引导”的功效， 药物发现的过程。在Tg研究之前，在培养细胞中进行更预测性的铅疗效测定 啮齿类动物对开发有效的治疗方法至关重要。这样的细胞分析可以节省大量的 大量的时间和资源。 为了改进我们对化学库以及先导化合物组的体外评估，我们计划使用 一个新收购的Opera共聚焦显微镜系统的高通量筛选（HTS）。这种先进 系统将允许我们测量亚细胞区室中的PrP^和PrP ^“。尽管成功 调整ELISA用于测量HTS中的这些PrP亚型，我们预计， Opera系统将使我们能够更准确地预测，从基于细胞的分析，哪些化合物将取得进展 成功地通过动物模型。 生物发光对实验性瘙痒症和阿尔茨海默病体内读数的适应性 小鼠（Tamgtiney等人，2009 a; Watts等人，2011）极大地促进了我们的药物疗效研究。计划 改进这些小鼠模型并将工作扩展到大鼠的研究计划如下所述。 Bigenic小鼠-在Mdrl敲除背景下，并具有在Mdrl基因的控制下表达的荧光素酶报告基因。 Gfap启动子-正在培育用于药物功效研究。类似的表达荧光素酶的小鼠 培育，其中小鼠PrP基因已被敲除，并且嵌合人（Hu）/小鼠（Mo）转基因 也表达了。这些小鼠在接种CJD（MM 1）朊病毒后<80天发病（Giles等人，2010年）。 在另一组研究中，我们使用RNAi文库来鉴定参与转录的辅助蛋白。 朊病毒的形成和复制。这些研究为新药的鉴定提供了途径 抗朊病毒药物的靶点计划更多地强调这些研究，因为似乎很可能是鸡尾酒， 具有不同作用模式的药物将是成功治疗神经退行性疾病的最有可能的途径。 疾病。