High Throughput Screening for Small-molecules Facilitating Prion Study

描述：传染性海绵状脑病(TSEs)，又称Pron病，是一组罕见的感染性哺乳动物神经退行性疾病。TSEs的病原体被认为是正常功能宿主细胞蛋白(PrPC)的错误折叠形式(PrPSc)。部分由于基于蛋白质的病理的复杂性，TSE仍然是无法治愈的，目前还没有治疗方法可以阻止它们的快速发展。有趣的是，萌芽酵母，酿酒酵母，包含几个非孟德尔遗传元素，这些基因元素以改变的蛋白质构象传递，被称为酵母普里恩。它的简单性和遗传可追踪性使酵母成为研究普恩病毒的强大模式生物。在这项提案中，我们计划使用酵母普里恩[SWI+]系统作为一个平台，通过高通量筛选的方法来识别能够抑制普恩病毒传播的小分子。[Swi+]是在我们实验室发现的，其蛋白质决定簇是Swi1，它是进化上保守的染色质重塑复合体SWI/SNF的一个亚单位。我们发现，[SWI+]严重抑制了FLO1的表达，FLO1是一种编码酵母丝状生长所需的细胞壁蛋白的SWI/SNF靶基因。通过用URA3编码区替换FLO1-ORF，我们创建了一个忠实的FLO1启动子-基于URA3的染色体报告基因。虽然[SWI+]不能在缺乏尿嘧啶的培养液中生长，但同基因的非Prion细胞可以生长。因此，能够消除[SWI+]的化合物可以在缺乏尿嘧啶的培养基中进行简单、安全的生长试验进行阳性选择。我们证明，这种经济有效的分析方法适用于384孔格式的高通量筛查。我们的试点屏幕已经产生了一些点击(化合物，可以有效地消除[SWI+])。我们预计，从本研究中获得的一些抗Prion化合物将有可能成为Pron研究的有价值的分子探针，进一步研究它们的Prion固化机制将有助于鉴定新的细胞成分，这些新的细胞成分对于Pri化和开发治疗TSE迫切需要的有效的抗Prion治疗药物是非常重要的。由于[SWI+]Prion的淀粉样蛋白的性质，我们还预计一些已识别的反Prion化合物也可以有效地抑制由蛋白质错误折叠引起的非Prion淀粉样变性疾病，如阿尔茨海默病(AD)、帕金森病(PD)和肌萎缩侧索硬化症(ALS)。

DESCRIPTION: Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of unusual infectious mammalian neurodegenerative disorders. The pathogen of TSEs is believed to be a misfolded form (PrPSc) of a normal functional host cellular protein (PrPC). Partly due to the complication of protein-based pathology, TSEs remain incurable and currently there is no treatment that can halt their rapid progression. Intriguingly, the budding yeast, Saccharomyces cerevisiae, contains several non-Mendelian genetic elements that are transmitted as altered protein conformations and are termed as yeast prions. Its simplicity and genetic trackability has made yeast a powerful model organism for prion research. In this proposal, we plan to use the yeast prion [SWI+] system as a platform to identify small molecules that can inhibit prion propagation through a high-throughput screen approach. [SWI+] was discovered in our laboratory, whose protein determinant is Swi1, a subunit of an evolutionarily conserved chromatin-remodeling complex - SWI/SNF. We found that the expression of FLO1, a SWI/SNF target gene encoding a cell wall protein required for yeast filamentous growth is severely suppressed by [SWI+]. By replacing the FLO1-ORF with the URA3-coding region, we created a faithful FLO1promoter-URA3-based chromosomal reporter. While [SWI+] cannot grow in media lacking uracil, the isogenic non-prion cells can. Thus, chemical compounds that can eliminate [SWI+] can be positively selected in a simple, safe growth assay in media lacking uracil. We demonstrate that this cost-effective assay is suitable for high-throughput screens in a 384-well format. Our pilot screens have already yielded a number of hits (chemical compounds that can effectively eliminate [SWI+]). We anticipate that some anti-prion compounds obtained from this study will likely become valuable molecular probes for prion research and further investigation of their prion- curing mechanism will lead to identification of novel cellular components important for prionization and development of effective anti-prion therapeutic drugs that are urgently needed for TSE treatment. Due to the amyloid nature of the [SWI+] prion, we also expect that some identified anti-prion compounds are also effective in suppressing non-prion amyloidogenic diseases resulted from protein misfolding, such as Alzheimer's disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS).