High Throughput Screening for compounds reducing cell surface prion protein

高通量筛选减少细胞表面朊病毒蛋白的化合物

• 批准号: 8404112
• 负责人: Corinne Ida Lasmezas
• 金额: $ 4.95万
• 依托单位: SCRIPPS FLORIDA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8404112
• 关键词: Alzheimer' s Disease; Amyloid; Biological Assay; Biology; Cell Culture Techniques; Cell Surface Proteins; Cell membrane; Cell surface; Cells; Characteristics; Collaborations; Collection; Detection; Development; Dimethyl Sulfoxide; Disease; Florida; Fluorescence Resonance Energy Transfer; Follow-Up Studies; Infection; Laboratories; Letters; Mediating; Messenger RNA; Miniaturization; Modeling; Molecular Bank; Molecular Probes; Molecular Target; Mus; NIH Program Announcements; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Outcome; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Prion Diseases; Prions; Production; Protein Biosynthesis; PubChem; Readiness; Role; Screening Result; Screening procedure; Series; Specificity; Testing; Therapeutic; Therapeutic Intervention; Time; Toxic effect; Transcription Process; Translations; Validation; analog; base; follow-up; high throughput screening; meetings; miniaturize; neglect; neurotoxicity; novel; pre-clinical; prevent; programs; protein expression; protein metabolism; repository; small molecule; tool; trafficking

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.

描述（由申请人提供）：该项目旨在通过高通量筛选（HTS）程序鉴定减少或消除细胞表面朊病毒蛋白（PrP）表达的小分子化合物。朊病毒病是由PrP错误折叠引起的可传播的神经退行性疾病。这些罕见的致命疾病，从治疗的角度来看，是被忽视的疾病，目前尚无治愈方法。PrP是朊病毒复制所必需的，但对宿主是不可缺少的，因此构成了治疗干预的理想靶点。从细胞表面去除PrP足以消除朊病毒复制。因此，与Weissmann博士合作，我们开发了一种检测方法来筛选能够减少或消除细胞表面PrP表达的化合物。主要检测试剂盒目前采用384孔格式，并通过统计参数和美国药物保藏中心的成功初步筛选评估符合HTS准备标准。我们现在建议将该检测转移到分子库生产中心网络（MLPCN），对1536孔格式的检测进行优化和优化，并筛选分子库小分子库（MLSMR）。然后，我们将实施二次测定以去除假阳性命中（毒性测定），确认不同神经元细胞上细胞表面PrP的抑制（正交测定），并对命中进行优先排序 根据它们在细胞培养物中预防和治愈朊病毒感染的能力。三级试验将包括确定化合物对PrP相对于其他细胞表面蛋白的特异性，并确定其作用模式（PrP合成、降解或运输至质膜）。药物化学将由William Wendy博士进行，以提高所选化合物的效力和特异性。这些研究与NIH计划公告PAR-09-129（“分子库探针生产中心网络（MLPCN）中高重复筛选（HTS）测定的征集”）直接相关。将在由Peter Hodder领导的Scripps佛罗里达的MLPCN实验室进行试验小型化和筛选。该项目的成果将不仅是用于治疗开发的化合物，而且还将收集分子探针来研究PrP生物合成和细胞运输途径。后续研究（超出提案范围）将确定选定化合物的主要分子靶点，研究这些靶点在PrP代谢中的作用，继续SAR并在小鼠朊病毒感染模型中测试最佳化合物，以产生临床前开发的候选化合物。鉴于PrP至少部分介导A？寡聚体诱导的神经毒性，我们的方法不仅可能影响朊病毒疾病，还可能影响阿尔茨海默病。