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: 8507710
• 负责人: Corinne Ida Lasmezas
• 金额: $ 4.8万
• 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/8507710
• 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; 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; screening; 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 错误折叠引起的传染性神经退行性疾病。这些罕见、致命且从治疗角度来看被忽视的疾病无法治愈。 PrP 对于朊病毒复制至关重要，但对于宿主来说却是可有可无的，因此构成了治疗干预的理想靶点。从细胞表面耗尽 PrP 足以消除朊病毒复制。因此，我们与 Weissmann 博士合作，开发了一种方法来筛选能够减少或消除细胞表面 PrP 表达的化合物。主要检测目前采用 384 孔格式，根据统计参数和美国药物库的成功初步筛选评估，满足 HTS 准备标准。我们现在建议将测定转移到分子库生产中心网络 (MLPCN)，小型化和优化 1536 孔格式的测定，并筛选分子库小分子存储库 (MLSMR)。然后，我们将进行二次测定，以消除假阳性命中（毒性测定），确认不同神经元细胞表面 PrP 的抑制（正交测定）并优先考虑命中 根据它们在细胞培养物中预防和治疗朊病毒感染的能力。第三次测定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、降解或运输到质膜）。 William Roush 博士将进行药物化学研究，以增强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（“在分子库探针生产中心网络 (MLPCN) 中征集高通量筛选 (HTS) 测定法”）直接相关。测定小型化和筛选将在 Peter Hodder 领导的佛罗里达州斯克里普斯 MLPCN 实验室进行。该项目的成果不仅是用于治疗开发的化合物，而且 用于研究 PrP 生物合成和细胞运输途径的分子探针集合。后续研究超出了提案的范围，将确定选定化合物的主要分子靶标，研究这些靶标在 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，以便产生临床前开发的候选化合物。鉴于 PrP 至少部分介导， 由于寡聚物引起的神经毒性，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氏病。