High Throughput Screening for Small-molecules Facilitating Prion Study

传染性海绵状脑病（tse），也被称为朊病毒疾病，是一组不寻常的感染性哺乳动物神经退行性疾病。tse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并发症，tse仍然无法治愈，目前没有治疗方法可以阻止其快速进展。有趣的是，出芽的酵母，酿酒酵母，含有一些非孟德尔遗传元素，这些元素以改变的蛋白质构象的形式传播，被称为酵母朊病毒。酵母的简单性和遗传可追溯性使其成为朊病毒研究的有力模式生物。在本提案中，我们计划以酵母朊病毒[SWI+]系统为平台，通过高通量筛选方法鉴定可以抑制朊病毒繁殖的小分子。[SWI+]是在我们实验室发现的，其蛋白决定因素是Swi1，它是进化上保守的染色质重塑复合体SWI/SNF的一个亚基。我们发现，[SWI+]严重抑制了fl1的表达，fl1是SWI/SNF靶基因，编码酵母丝状生长所需的细胞壁蛋白。通过用ura3编码区替换FLO1-ORF，我们创建了一个忠实的基于flo1启动子- ura3的染色体报告基因。虽然[SWI+]不能在缺乏尿嘧啶的培养基中生长，但等基因非朊病毒细胞可以。因此，在缺乏尿嘧啶的培养基中，可以通过一种简单、安全的生长试验积极选择能够消除[SWI+]的化合物。我们证明，这种具有成本效益的分析方法适用于384孔格式的高通量筛选。我们的试验性筛选已经产生了一些效果（可以有效消除[SWI+]的化合物）。我们预计，从本研究中获得的一些抗朊病毒化合物可能会成为朊病毒研究的有价值的分子探针，进一步研究它们的朊病毒治疗机制将有助于鉴定出新的细胞成分，这对朊病毒化和开发有效的抗朊病毒治疗药物至关重要，这是治疗TSE迫切需要的。由于[SWI+]朊病毒的淀粉样性质，我们还期望一些已鉴定的抗朊病毒化合物也能有效抑制由蛋白质错误折叠引起的非朊病毒淀粉样变性疾病，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).