High Throughput Screening for Small-molecules Facilitating Prion Study

高通量筛选小分子促进朊病毒研究

• 批准号: 9094598
• 负责人: LIMING LI
• 金额: $ 28.86万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9094598
• 关键词: Acids; Affect; Alzheimer' s Disease; Amyloid; Amyotrophic Lateral Sclerosis; Animal Model; Architecture; Biological Assay; Biology; Caenorhabditis elegans; Cell Wall; Cells; Chemicals; Cherry - dietary; Chromatin Remodeling Factor; Code; Collection; Complication; Development; Disease; Dose; Effectiveness; Event; Gene Targeting; Genes; Genetic; Goals; Growth; Hippocampus (Brain); Homeostasis; In Vitro; Investigation; Laboratories; Lead; Libraries; Modeling; Molecular; Molecular Conformation; Molecular Probes; Mus; Nature; Neurodegenerative Disorders; Neurons; Open Reading Frames; Parkinson Disease; Pathology; Pharmaceutical Preparations; Pilot Projects; PrPC Proteins; PrPSc Proteins; Prion Diseases; Prions; Protein Conformation; Proteins; Reporter; Reporting; Research; Saccharomyces; Saccharomyces cerevisiae; Saccharomycetales; Specificity; Structure-Activity Relationship; System; Testing; Therapeutic; Toxic effect; Universities; Uracil; Yeasts; alpha synuclein; amyloidogenesis; base; cellular targeting; cost effective; counterscreen; genetic element; high throughput analysis; high throughput screening; non-prion; novel; pathogen; protein aggregate; protein folding; protein misfolding; public health relevance; response; small molecule; small molecule inhibitor; therapeutic development; transmission process; yeast prion

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).

描述：传染性海绵状脑病 (TSE)，也称为朊病毒病，是一组不寻常的传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性宿主细胞蛋白 (PrPC) 的错误折叠形式 (PrPSc)。部分由于基于蛋白质的病理学并发症，TSE 仍然无法治愈，目前没有任何治疗方法可以阻止其快速进展。有趣的是，芽殖酵母（酿酒酵母）含有几种非孟德尔遗传元件，这些元件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踪性使其成为朊病毒研究的强大模型生物。在本提案中，我们计划使用酵母朊病毒[SWI+]系统作为平台，通过高通量筛选方法识别可以抑制朊病毒繁殖的小分子。 [SWI+]是我们实验室发现的，其蛋白质决定簇是Swi1，是进化上保守的染色质重塑复合物SWI/SNF的一个亚基。我们发现 FLO1（一种编码酵母丝状生长所需细胞壁蛋白的 SWI/SNF 靶基因）的表达被 [SWI+] 严重抑制。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，但同基因非朊病毒细胞可以。因此，可以在缺乏尿嘧啶的培养基中通过简单、安全的生长测定来肯定地选择可以消除[SWI+]的化合物。我们证明这种经济高效的检测方法适用于 384 孔格式的高通量筛选。我们的试点筛选已经产生了许多成果（可以有效消除 [SWI+] 的化合物）。我们预计，从这项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中有价值的分子探针，对其朊病毒治愈机制的进一步研究将导致鉴定出对于朊病毒化和开发有效的抗朊病毒治疗药物至关重要的新型细胞成分。 TSE 治疗急需的药物。由于[SWI+]朊病毒的淀粉样蛋白性质，我们还预计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，例如阿尔茨海默病（AD）、帕金森病（PD）和肌萎缩侧索硬化症（ALS）。