Screening for enhancers of secreted clusterin (sCLU) and evaluation in AD models

分泌型凝聚素 (sCLU) 增强子的筛选和 AD 模型的评估

• 批准号: 10195566
• 负责人: Varghese John
• 金额: $ 42.34万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10195566
• 关键词: APP-PS1; Address; Adverse effects; Affect; Alzheimer' s Disease; Alzheimer' s disease model; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer’s disease biomarker; Amyloid beta-Protein; Apoptotic; Binding Proteins; Biological Assay; Biological Availability; Biological Markers; Biological Process; Brain; Cardiovascular Diseases; Cell Culture Techniques; Cell Death; Cell Line; Cell Survival; Cells; Cellular Stress Response; Central Nervous System Diseases; Consensus; Cytoprotection; Development; Devices; Disease; Disease Progression; Drug Kinetics; Effectiveness; Enhancers; Evaluation; Failure; Functional disorder; Future; Glioblastoma; Goals; Half-Life; Hippocampus (Brain); Homeostasis; Human; Immunoassay; Impaired cognition; In Vitro; Induced pluripotent stem cell derived neurons; Inflammation; Late Onset Alzheimer Disease; Lead; Libraries; Liquid substance; Literature; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Metabolic; Mitochondria; Molecular; Molecular Chaperones; Molecular Target; Nature; Nerve Degeneration; Neurons; Oral; Oxidative Regulation; Oxidative Stress; Pathology; Permeability; Pharmaceutical Preparations; Pharmacology; Phosphotransferases; Photoaffinity Labels; Preclinical Testing; Predisposition; Property; Protein Isoforms; Proteins; Public Health; Research; Role; Senile Plaques; Signal Pathway; Signal Transduction; Slice; Solubility; Stress; Testing; Testis; Therapeutic; Toxic effect; Treatment Efficacy; abeta deposition; base; drug candidate; effectiveness evaluation; efficacy testing; experimental study; extracellular; genetic risk factor; genetic variant; glycogen synthase kinase 3 beta; high throughput screening; hyperphosphorylated tau; immunoregulation; in vivo; induced pluripotent stem cell; innovation; instrument; lipid transport; mouse model; nerve stem cell; neuroblastoma cell; novel; novel therapeutic intervention; programs; proteotoxicity; screening; small molecule; sulfated glycoprotein 2; tau aggregation; tau phosphorylation; tau-1; treatment strategy

PROJECT SUMMARY/ABSTRACT This proposal provides a unique opportunity to identify isoform-specific modulators of clusterin (CLU) and evaluate its role in Alzheimer's disease (AD). Clusterin, also known as apolipoprotein J (Apo J) is a protein originally identified in 1979. Several CLU gene variants are associated with AD and the SNP rs11136000C is the third strongest genetic risk factor for Late Onset Alzheimer's disease (LOAD). There is evidence that enhancing brain levels of sCLU will slow or reverse various molecular mechanisms underlying onset of the AD pathophysiology. This is based on studies that show sCLU promoting the clearance of disease-causing amyloid beta (Aβ) plaques, while reducing cellular stress responses such as oxidative stress and inflammation known to lead to increased kinase activity such as GSK-3β and increased hyperphosphorylated tau involved in progression of AD. Interestingly, the CLU gene variants that confer increased susceptibility to LOAD also reduce sCLU levels and result in increased Aβ deposition and tau neurofibrillary tangles and faster cognitive decline relative to non-carriers. In this proposal a comprehensive research program will be undertaken to identify potent, brain permeable, small molecules that increase levels of sCLU and modulate biomarkers in AD models. This will be accomplished using high throughput screening (HTS) to identify `hits' with potent sCLU enhancing ability, drug-like properties, brain permeability in pharmacokinetics (PK) analyses to guide compound selection for further testing and mechanism-of-action (MOA) studies in iPSC derived neurons and ex-vivo models of AD. In Aim1 we will screen for compounds that increase extracellular concentrations of sCLU. UCLA's large compound library will be screened by using fully automated liquid handling devices and analytical instruments to identify molecules that increase sCLU levels. For these experiments, sensitive and readily formattable AlphaLISA based immunoassays will be optimized to detect different CLU isoforms. In Aim2, we would conduct in-vitro ADME/T testing and in-vivo pharmacokinetics (PK) analyses to guide hit selection including determination of sCLU enhancing potency, characterizing the physiochemical properties including solubility, brain permeability and metabolic stability of candidate sCLU enhancing compounds. In Aim3, we would conduct testing in induced pluripotent stem cells (iPSCs) derived neurons and ex-vivo brain slices. The goal is to evaluate the effectiveness of sCLU enhancers with sufficient oral brain bioavailability and half-life in AD patient-derived neurons as well as in ex-vivo organotypic brain slice cultures from the hippocampi of an AD mouse model. In Aim4, we would identify molecular targets and cell signaling pathways affected by prioritized hits. MOA studies involving target identification by photoaffinity-labeling/purification as well as global and phosphoproteome analyses will be accomplished using state-of-the-art mass spectrometry. Using these approaches, we will identify molecular targets of our sCLU enhancing compounds to identify novel mechanisms involved in regulating brain sCLU levels in AD.

项目摘要/摘要 该建议提供了一个独特的机会，可以识别簇蛋白（CLU）和 评估其在阿尔茨海默氏病（AD）中的作用。簇蛋白，也称为载脂蛋白J（Apo J）是一种蛋白 最初在1979年确定。几种CLU基因变体与AD相关，而SNP RS11136000C为 晚期发病的第三强遗传危险因素（负载）。有证据表明 提高SCLU的大脑水平将减慢或逆转AD发作的各种分子机制 病理生理学。这是基于表明SCLU促进疾病清除率的研究 淀粉样β（Aβ）斑块，同时减少细胞应激反应，例如氧化应激和炎症 已知会导致激酶活性增加，例如GSK-3β，并增加了参与的高磷酸化tau AD的进展。有趣的是，CLU基因变体，会议增加了负载的敏感性 降低SCLU水平，并导致Aβ沉积和TAU神经原纤维缠结和更快的认知 相对于非携带者而下降。在此提案中，将进行全面的研究计划 确定潜在的，大脑可渗透的小分子，以增加SCLU水平并调节AD中的生物标志物 型号。这将使用高吞吐量筛选（HTS）来实现，以识别有效的SCLU 增强能力，类似药物的特性，药代动力学（PK）分析的大脑渗透性 用于进一步测试和行动机理（MOA）研究的复合选择 AD的前体模型。在AIM1中，我们将筛选出增加细胞外浓度的化合物 Sclu。 UCLA的大型复合库将通过使用全自动液体处理设备和 分析工具以识别增加SCLU水平的分子。对于这些实验，敏感和 易于格式的基于α的免疫测定将被优化以检测不同的CLU同工型。在 AIM2，我们将进行体外ADME/T测试和体内药代动力学（PK）分析以指导命中率 选择包括确定SCLU增强效力，表征生理学特性 包括溶解度，脑渗透性和候选SCLU增强化合物的代谢稳定性。在 AIM3，我们将在诱导的多能干细胞（IPSC）衍生的神经元和前体脑中进行测试 切片。目的是通过足够的口服脑生物利用度和 AD患者衍生的神经元以及前体内有机脑切片培养的半衰期 AD鼠标模型的海马。在AIM4中，我们将确定分子靶标和细胞信号通路 受优先命中的影响。 MOA研究涉及通过光性标记/纯化目标识别目标识别的研究 以及将使用最先进的质谱法完成全局和磷光蛋白质组分析。 使用这些方法，我们将识别SCLU增强化合物的分子靶标，以识别新颖 调节AD中脑SCLU水平的机制。