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

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。通过检测与绿色荧光蛋白（PSD95- gfp）融合的支架蛋白PSD95簇，建立了一种基于共聚焦成像的检测完整突触后密度（psd）的方法，并确定荧光点代表功能突触。暴露于HIV-1感染细胞释放的因子（包括HIV-1蛋白Tat）后，PSD95-GFP点丢失。控制突触数量的信号通路在暴露于Tat时发生改变；一种途径调控突触丧失，另一种途径调控突触恢复。据推测，逆转兴奋性突触丧失的药物具有诱导认知恢复的最大潜力。本申请描述了一种基于突触成像的高含量分析（HCA）测定方法的发展及其用于筛选药物样分子库的用途，其长期目标是发现逆转HAND认知能力下降的药物。这种无偏见的筛选将识别通过目前尚未确定的信号通路起作用的化合物，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，基于共聚焦显微镜的突触恢复试验将适用于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产生表达PSD95-GFP的皮质神经元，以进行高通量筛选；2)使用HCA平台对PSD95-GFP进行高分辨率成像和对点计数进行定量分析；3)使用HCA平台对阳性对照药物相对于阴性对照药物产生可重复且具有统计学意义的突触恢复。在达到这些里程碑后，HCA检测将在R33期扩大规模并实现自动化。自动分析将通过筛选药理学活性化合物库（LOPAC）进行验证。LOPAC结果将用于确定分析窗口、方差和可重复性。然后，HCA将用于筛选从具有药理活性和结构多样的化合物的目标文库中选择的10,000种化合物。化学信息学将用于筛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，例如非特异性突触形成效应。在筛选完成后，该分析将准备进行高通量规模实施和/或先导化合物优化。本项目将为指导开发改善HAND患者功能的药物提供基础，并为突触功能的研究提供一个有效的平台。由于突触丢失在许多神经退行性过程中是常见的，因此在该试验中检测阳性的化合物可能被证明是有用的药物，可诱导由多种病因引起的神经损伤患者恢复。

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.