Scalable assay for drugs to reverse synapse loss during HIV-1 neurotoxicity

逆转 HIV-1 神经毒性期间突触损失的药物的可扩展测定

• 批准号: 8678350
• 负责人: Stanley A Thayer
• 金额: $ 26.88万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8678350
• 关键词: AIDS neuropathy; Affect; Algorithms; Biological Assay; Biological Neural Networks; Biological Preservation; Cell Culture Techniques; Cell Death; Cells; Cherry - dietary; Collection; Computer software; Custom; DLG4 gene; Detection; Development; Disease; Drug usage; Ensure; Etiology; Excitatory Synapse; Exposure to; Foundations; Green Fluorescent Proteins; HIV Infections; HIV-1; Hour; Image; Image Analysis; Impaired cognition; Impairment; Lasers; Lead; Libraries; Liquid substance; Microscope; Morphology; N-Methyl-D-Aspartate Receptors; NMDA receptor antagonist; Nerve Degeneration; Neurocognitive; Neurodegenerative Disorders; Neurologic; Neurons; Optics; Pathway interactions; Patients; Pharmaceutical Preparations; Phase; Process; Production; Proteins; Protocols documentation; Receptor Activation; Recovery; Relative (related person); Reproducibility; Resolution; Robotics; Scaffolding Protein; Scanning; Series; Signal Pathway; Structure; Synapses; Testing; Time; Transgenes; Transgenic Mice; Triage; Validation; Virus; base; cheminformatics; cognitive function; cognitive recovery; density; drug development; high throughput screening; image processing; improved; improved functioning; meetings; neurotoxic; neurotoxicity; postsynaptic; prevent; public health relevance; release factor; research study; response; scale up; screening; synaptic function; synaptogenesis

DESCRIPTION (provided by applicant): Synaptodendritic degeneration correlates with cognitive decline in patients with HIV-1-associated neurocognitive disorders (HAND). A confocal imaging-based assay was developed to detect intact postsynaptic densities (PSDs) based on detection of clusters of the scaffolding protein PSD95 fused to green fluorescent protein (PSD95-GFP) and it was established that fluorescent puncta represent functional synapses. PSD95-GFP puncta were lost following exposure to factors released by HIV-1 infected cells including the HIV-1 protein Tat. The signaling pathways that control synapse number changed during exposure to Tat; one pathway regulated synapse loss and the other regulated synapse recovery. Drugs that reverse excitatory synapse loss are hypothesized to have the greatest potential for inducing cognitive recovery. This application describes the development of a high content analysis (HCA) assay based on synaptic imaging and its use to screen a library of drug-like molecules with the long-term objective to discover drugs that reverse cognitive decline in HAND. This unbiased screen will identify compounds acting via currently unidentified signaling pathways that control synapse recovery during a neurotoxic challenge. In the R21 Phase, a confocal microscope-based synapse recovery assay will be adapted to a HCA platform. Progress will be marked by three milestones: 1) reliable production of cortical neurons expressing PSD95-GFP at sufficient scale and density for high-throughput screening, 2) high resolution imaging of PSD95-GFP and quantitative analysis of puncta counts using the HCA platform, and 3) reproducible and statistically significant synapse recovery produced by positive relative to negative control drugs using the HCA platform. Upon meeting these milestones the HCA assay will be scaled up and automated in the R33 Phase. The automated assay will be validated by screening the library of pharmacologically active compounds (LOPAC). LOPAC results will be used to determine assay window, variance and reproducibility. The HCA will then be used to screen a collection of 10,000 compounds selected from targeted libraries of pharmacologically active and structurally diverse compounds. Cheminformatics will be used to cherry-pick compounds for confirmation and validation. Confirmed compounds will be triaged for non-desired activity such as non-specific synaptogenic effects. At the completion of the screen, the assay will be ready for high throughput-scale implementation and/or lead compound optimization. This project will provide a foundation to guide the development of drugs to improve function in HAND patients and will provide an efficient platform for studies of synaptic function. Because synapse loss is common to many neurodegenerative processes, compounds that test positive in this assay may prove useful as drugs to induce recovery in patients with neurological impairments resulting from multiple etiologies.

描述（由申请人提供）：突触树突变性与 HIV-1 相关神经认知障碍 (HAND) 患者的认知能力下降相关。基于对与绿色荧光蛋白融合的支架蛋白 PSD95 (PSD95-GFP) 簇的检测，开发了一种基于共聚焦成像的测定法来检测完整的突触后密度 (PSD)，并确定荧光点代表功能性突触。 PSD95-GFP 斑点在暴露于 HIV-1 感染细胞释放的因子（包括 HIV-1 蛋白 Tat）后丢失。在接触 Tat 期间，控制突触数量的信号通路发生了变化；一种途径调节突触损失，另一种途径调节突触恢复。据推测，逆转兴奋性突触丧失的药物最有可能诱导认知恢复。该申请描述了基于突触成像的高内涵分析 (HCA) 测定的开发，及其用于筛选类药物分子库的用途，其长期目标是发现逆转 HAND 认知衰退的药物。这种公正的筛选将识别通过目前未识别的信号通路发挥作用的化合物，这些信号通路在神经毒性挑战期间控制突触恢复。在 R21 阶段，基于共焦显微镜的突触恢复测定将适用于 HCA 平台。进展将以三个里程碑为标志：1）以足够的规模和密度可靠地生产表达 PSD95-GFP 的皮层神经元，以进行高通量筛选；2）PSD95-GFP 的高分辨率成像和使用 HCA 平台对斑点计数进行定量分析； 3) 使用 HCA 平台，相对于阴性对照药物，阳性对照药物产生可重复且具有统计显着性的突触恢复。达到这些里程碑后，HCA 检测将在 R33 阶段扩大规模并实现自动化。自动检测将通过筛选药理活性化合物库 (LOPAC) 进行验证。 LOPAC 结果将用于确定测定窗口、方差和再现性。然后，HCA 将用于筛选 10,000 种化合物，这些化合物选自具有药理活性和结构多样化的目标化合物库。化学信息学将用于挑选化合物进行确认和验证。已确认的化合物将根据非期望的活性进行分类，例如非特异性突触作用。筛选完成后，该测定将准备好进行高通量规模实施和/或先导化合物优化。该项目将为指导改善 HAND 患者功能的药物开发奠定基础，并将为突触功能研究提供有效的平台。由于突触丢失在许多神经退行性过程中很常见，因此在该测定中测试呈阳性的化合物可能被证明可用作药物，以诱导因多种病因引起的神经损伤患者的康复。