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

描述(由申请人提供)：在HIV-1相关神经认知障碍(HAND)患者中，突触树突状变性与认知能力下降相关。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，该方法基于对融合到绿色荧光蛋白(PSD95-GFP)的支架蛋白(PSD95-GFP)簇的检测，并建立了荧光点代表功能性突触的方法。在暴露于HIV-1感染细胞释放的包括HIV-1蛋白Tat在内的因子后，PSD95-GFP斑点丢失。在接触TAT的过程中，控制突触数量的信号通路发生了变化；一条通路调节突触丢失，另一条通路调节突触恢复。逆转兴奋性突触丢失的药物被认为是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)分析的发展，并将其用于筛选类药物分子库，长期目标是发现逆转手部认知能力下降的药物。这一无偏见的筛选将识别通过目前未知的信号通路作用的化合物，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分析将适用于HCA平台。进展将是三个里程碑：1)以足够的规模和密度可靠地生产表达PSD95-GFP的皮质神经元，用于高通量筛选；2)使用HCA平台对PSD95-GFP进行高分辨率成像和点状计数的定量分析；以及3)使用HCA平台通过阳性相对于阴性对照药物产生可重复性和统计学上显著的突触恢复。在达到这些里程碑后，HCA检测将在R33阶段扩大规模并实现自动化。自动化化验将通过筛选药理活性化合物(LOPAC)文库进行验证。LOPAC结果将用于确定分析窗口、方差和重复性。然后，将使用HCA来筛选从具有药理活性和结构多样性的目标化合物库中选择的10,000种化合物的集合。化学信息学将被用来挑选化合物进行确认和验证。已确认的化合物将对非预期活性进行分类，如非特异性突触效应。在筛选完成后，该分析将为高通量规模实施和/或主导化合物优化做好准备。该项目将为指导改善手部患者功能的药物的开发提供基础，并将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变过程中都很常见，因此在这项测试中检测呈阳性的化合物可能被证明是有用的药物，可以诱导多种原因导致的神经损伤患者的康复。

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.