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

描述（由申请方提供）：突触树突变性与HIV-1相关神经认知障碍（HAND）患者的认知下降相关。开发了基于共聚焦成像的测定以基于检测融合至绿色荧光蛋白（PSD 95-GFP）的支架蛋白PSD 95的簇来检测完整的突触后密度（PSD），并且确定荧光斑点代表功能性突触。PSD 95-GFP斑点在暴露于由HIV-1感染的细胞释放的因子（包括HIV-1蛋白达特）后丢失。控制突触数量的信号通路在暴露于达特期间发生变化;一条通路调节突触丢失，另一条调节突触恢复。逆转兴奋性突触丧失的药物被认为具有诱导认知恢复的最大潜力。本申请描述了基于突触成像的高含量分析（HCA）测定的开发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中认知下降的药物。这种无偏见的筛选将确定化合物通过目前未识别的信号通路，控制神经毒性挑战过程中的突触恢复。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PSD 95-GFP的皮质神经元以用于高通量筛选，2）使用HCA平台对PSD 95-GFP进行高分辨率成像和对斑点计数进行定量分析，以及3）使用HCA平台由阳性相对于阴性对照药物产生的可再现且统计学显著的突触恢复。在达到这些里程碑后，HCA检测试剂盒将在R33阶段扩大规模并自动化。将通过筛选生物活性化合物库（LOPAC）验证自动化试验。LOPAC结果将用于确定试验窗口、方差和重现性。然后，HCA将用于筛选10，000种化合物的集合，这些化合物选自具有生物活性和结构多样性的化合物的靶向文库。化学信息学将用于挑选化合物进行确认和验证。确认的化合物将被分类用于非期望的活性，例如非特异性突触发生效应。在筛选完成时，测定将准备好用于高通量规模实施和/或先导化合物优化。该项目将为指导开发改善HAND患者功能的药物提供基础，并为研究突触功能提供有效的平台。由于突触丧失对于许多神经退行性过程是常见的，因此在该测定中测试为阳性的化合物可以证明可用作诱导具有由多种病因引起的神经损伤的患者恢复的药物。

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.