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（由适用提供）：该项目旨在通过高通量筛选（HTS）程序来识别细胞表面上prion蛋白（PRP）的表达的小分子化合物。 prion疾病是由PRP折叠引起的透明神经退行性疾病。从治疗的角度来看，无法治愈这些罕见，致命的，并且是被忽视的疾病。 PRP对于prion复制至关重要，但对于宿主来说是必不可少的，因此构成了治疗干预的理想目标。从TH表面耗尽的PRP足以消除prion复制。因此，与Weissmann博士合作，我们开发了一项评估，以筛选能够在细胞表面降低或废除PRP表达的化合物。主要测定法当前是384孔格式，并符合通过统计参数和成功的美国药物收集的成功初步筛查评估的HTS准备标准。现在，我们建议将测定法转移到分子文库生产中心网络（MLPCN），微型化并优化1536井格式的测定法，并筛选分子库小分子存储库（MLSMR）。然后，我们将实施次要测定以去除假阳性命中（毒性测定），确认在不同的神经元细胞（正交分析）上抑制细胞表面PRP并优先级。 根据它们预防和治愈细胞培养中事先感染的能力。第三纪将包括确定PRP与其他细胞表面蛋白的化合物的特异性，并确定其作用方式（PRP合成，降解或运输到质膜）。威廉·鲁什（William Roush）博士将进行药物化学，以提高所选化合物的效力和特异性。这些研究与NIH计划公告直接相关（“分子库中高吞吐量筛选（HTS）的测定探测中心网络（MLPCN）的测定法。分析和筛选将在Petipps Florida Levers of Peter Project will Will Will Project demult demult demult demult demult demult demult demult demult demund demult n drow demind。研究PRP生物合成的分子问题和细胞运输途径，其后续研究是超出提案范围的，将确定所选化合物的主要分子靶标，这些靶标在PRP代谢中的作用，继续在PRP代谢中，继续SAR并在最佳的MED semation pre the Press prepatit Prot Prepation中，低聚物诱导的神经毒性，我们的方法不仅可能影响王室疾病，还会影响阿尔茨海默氏病。