High Throughput Screening for compounds reducing cell surface prion protein

描述(由申请人提供)：该项目旨在通过高通量筛选(HTS)计划识别减少或取消细胞表面PrP表达的小分子化合物。PrP病是由PrP错误折叠引起的遗传性神经退行性疾病。从治疗的角度来看，这些罕见的、致命的、被忽视的疾病是无法治愈的。PrP对于PrP复制是必不可少的，但对宿主来说却是不可缺少的，因此构成了治疗干预的理想靶点。从TH细胞表面去除PrP足以消除PrP复制。因此，我们与魏斯曼博士合作，开发了一种检测方法，以筛选能够减少或消除细胞表面PrP表达的化合物。一次化验目前是384孔的格式，符合HTS的准备标准，通过统计参数和成功的美国药物收集初步筛选进行评估。我们现在建议将检测转移到分子库生产中心网络(MLPCN)，将1536孔格式的检测小型化并进行优化，并筛选分子库小分子存储库(MLSMR)。然后，我们将进行二次检测以消除假阳性命中(毒性分析)，确认细胞表面PrP对不同神经细胞的抑制(正交法)，并对命中进行优先排序 根据它们在细胞培养中预防和治疗普恩病毒感染的能力。第三级分析将包括确定PrP化合物相对于其他细胞表面蛋白的特异性，并确定其作用方式(PrP的合成、降解或转运到质膜)。威廉·罗什博士将进行药物化学研究，以提高选定化合物的效力和特异性。这些研究直接与NIH项目公告PAR-09-129(“分子文库探针生产中心网络(MLPCN)高通量筛选(HTS)分析征集”)相关。化验小型化和筛查将在彼得·霍德领导的佛罗里达州斯克里普斯的MLPCN实验室进行。该项目的结果将不仅是用于治疗开发的化合物，而且还将是一系列分子探针，用于研究PrP的生物合成和细胞运输途径。不在该提案范围内的后续研究将确定选定化合物的主要分子靶点，研究这些靶点在PrP新陈代谢中的作用，继续进行SAR，并在小鼠Prion感染模型中测试最佳化合物(S)，以产生临床前开发的候选化合物。鉴于PrP至少部分地介导了A寡聚体引起的神经毒性，我们的方法可能不仅会影响普里恩疾病，还会影响阿尔茨海默病。

DESCRIPTION (provided by applicant): The project aims at identifying small-molecule compounds reducing or abolishing expression of prion protein (PrP) at the cell surface through a high-throughput screening (HTS) program. Prion diseases are transmisible neurodegenerative diseases caused by misfolding of PrP. There is no cure for these rare, fatal and, from a therapeutic standpoint, neglected diseases. PrP is essential for prion replication but dispensable for the host, thus constituting an ideal target for therapeutic intervention. Depleting PrP from th cell surface is sufficient to abrogate prion replication. Therefore, in collaboration with Dr. Weissmann, we developed an assay to screen for compounds able to reduce or abolish PrP expression at the cell surface. The primary assay is currently in a 384-well format and fulfills HTS readiness criteria as assessed by statistical parameters and successful preliminary screening of the US Drug Collection. We now propose to transfer the assay to the Molecular Libraries Production Centers Network (MLPCN), miniaturize and optimize the assay for the 1536- well format and screen the Molecular Libraries Small Molecule Repository (MLSMR). We will then implement secondary assays to remove false positive hits (toxicity assay), confirm suppression of cell surface PrP on different neuronal cells (orthogonal assays) and prioritize hits according to their capacity to prevent and cure prion infection in cell culture. Tertiary assays wil consist in determining the specificity of the compound for PrP versus other cell surface proteins and determining its mode of action (PrP synthesis, degradation or trafficking to the plasma membrane). Medicinal chemistry will be conducted by Dr. William Roush to enhance potency and specificity of selected compounds. These studies are directly relevant to the NIH program announcement PAR-09-129 ("Solicitation of Assays for High Throughput Screening (HTS) in the Molecular Libraries Probe Production Centers Network (MLPCN). Assay miniaturization and screening will be performed at the MLPCN laboratory of Scripps Florida led by Peter Hodder. The outcome of the project will be not only compounds for therapeutical development but also a collection of molecular probes to study PrP biosynthetic and cellular trafficking pathways. Follow-up studies, which are outside the scope of the proposal, will determine primary molecular targets of selected compounds, study the role of these targets in PrP metabolism, continue SAR and test the best compound(s) in mouse prion infection models in order to generate a candidate for pre-clinical development. Given that PrP mediates, at least in part, A¿ oligomer-induced neurotoxicity, our approach may impact not only prion diseases, but also Alzheimer's disease.