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.

项目描述（由申请人提供）：该项目旨在通过高通量筛选（HTS）程序鉴定细胞表面减少或消除朊病毒蛋白（PrP）表达的小分子化合物。朊病毒疾病是由PrP错误折叠引起的传染性神经退行性疾病。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对朊病毒复制至关重要，但对宿主来说是必不可少的，因此是治疗干预的理想靶点。从细胞表面消耗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。初步分析目前采用384孔格式，通过统计参数和成功的美国药物收集初步筛选评估，符合HTS准备标准。我们现在建议将该分析转移到分子文库生产中心网络（MLPCN），小型化和优化1536孔格式的分析，并筛选分子文库小分子库（MLSMR）。然后，我们将实施二次分析，以消除假阳性命中（毒性试验），确认不同神经元细胞表面PrP的抑制（正交试验），并确定命中的优先级