Towards Therapeutics for Neurodegenerative Diseases

没有一种FDA批准的药物可以阻止甚至减缓中枢神经系统的神经退化。尽管 花费了数十亿美元，无论是产业界还是学术界都未能开发出一种延缓速度的药物 阿尔茨海默病(AD)、帕金森氏病(PD)和克雅氏病(CJD)的进展 肌萎缩侧索硬化症(ALS)和额颞性痴呆(FTD)。尽管存在这些障碍，但仍有 在了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。稳步积累的 实验数据证明，每种神经退行性疾病都是由一种不同的蛋白质引起的，这些蛋白质 获得变得自繁殖的替代结构，即Pron(Meyer-Luehmann等人，2006年； Clavaguera等人，2009；Frost and Diamond，2009；Olanow and Prusiner，2009；Brundin等人，2010；Cushman 等人，2010年；Colby和Prusiner出版)。这些发现是最令人满意的，因为它们提供了一个扩大 支持现在被认为是有先见之明的推测的大量证据(Prusiner，1984,2001)。 了解到所有或大部分神经退行性疾病都是由普恩引起的，可能会给出几个 有效疗法发展的新视角。首先，治愈受感染的培养细胞的药物 在实验动物或人类身上，带有普鲁恩的基因可能并不能预示成功。这就是我们对 治愈培养细胞但不能延长小鼠或人类寿命的抗疟疾药物奎纳克林 (Colinger等人，2009年；Ghaemmaghami等人，2009年)。即使奎纳克林的水平几乎增加了 在P-gp转运蛋白(Mdr1)被敲除的小鼠脑中，P-gp转运蛋白(Mdr1)被敲除的小鼠大脑中的P-gp转运蛋白(Mdr1)增加了100倍，超过了一半的有效 浓度(EC50)，但不能延长孵育时间(Ghaemmaghami等人， ^2009)。我们收集了令人信服的证据，表明对此最有可能的解释是 治疗失败是PrP^*^的构象改变，导致了抗药性的Prion菌株。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 这些药物能够延长小鼠的孵化时间(Ghaemmaghami等人，2010年；Gallardo-Godoy等人，2011年)。 发现可用于治疗神经退行性变的药物面临着巨大的挑战。话虽如此， 我们迫切需要更多的化验方法来衡量“命中”和“领先”的有效性，以便继续进行 药物发现的过程。在TG研究之前对培养细胞中铅有效性的更具预测性的分析 啮齿动物对于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 时间和资源的数量。 为了改进我们对化学库和先导化合物组的体外评估，我们计划使用 用于高通量筛选(HTS)的新获得的OPERA共聚焦显微镜系统。这是一种高级 系统将允许我们测量亚细胞隔间中的PrP^和PrP^‘^。尽管成功了 采用ELISA来测量HTS中的这些PrP异构体，我们预计 OPERA系统将使我们能够从基于细胞的分析中更准确地预测哪些化合物将进步 通过动物模型成功。 生物发光法对实验性瘙痒病和阿尔茨海默病在体读数的适应性 老鼠(Tamgtiney等人，2009a；Watts等人，2011年)极大地促进了我们的药物疗效研究。计划 在研究计划中，将改进这些小鼠模型，并将工作扩展到大鼠身上。 大基因小鼠-在mdrl基因敲除背景上，荧光素酶报告基因在控制下表达 正在培育用于药物疗效研究的GFAP启动子。类似的表达荧光素酶的小鼠正在研究中 其中小鼠PrP基因已被敲除，人(Hu)/小鼠(Mo)嵌合体转基因 也表达了。这些小鼠在接种CJD(MM1)病毒80天后发病(Giles等人，2010年)。 在另一组研究中，我们使用RNAi文库来识别参与 PrP蛋白的形成和复制。这些研究为新药的鉴定提供了一条途径 抗病毒药物的靶标。计划将更多的重点放在这些研究上，因为似乎有可能 具有不同作用模式的药物将是最有可能成功治疗神经退行性变的途径 疾病。

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.