Identification of small molecules for neurological complications of HIV and substance abuse comorbidity

摘要 艾滋病毒相关性神经认知障碍(手)和药物滥用共病仍然很普遍 尽管联合抗逆转录病毒治疗(CART)。这个项目背后的创新假设是，逆转 以突触树突状细胞和线粒体损伤为特征的手营养缺陷 有效预防或改善HIV和强迫性吸毒的认知衰退。为了检验这一假设， 我们将进行吞吐量筛选活动(HTS)来确定一个新发现的基因的调节子 调节机制，广泛调节营养支持和线粒体生物发生在关键的皮质和 我们发现海马神经细胞群在神经艾滋病和依赖中都下调了。 我们预计，能够上调这一机制的小分子将使神经元对 氧化应激和神经变性，导致神经元损伤和额叶下部减少，这是一种 艾滋病毒患者认知功能障碍的主要原因以及依赖者强制服药和饮酒。我们 将采用1536孔板格式的基于细胞的发光报告分析与分层的 筛选640,000个化合物Scripps药物发现文库并确认其有效性的方法 ~500个类药物分子。将执行HIT-验证以识别小分子调节剂并消除 采用平行正交法的非特异性效应器和使用细胞毒性的非靶点评估 使用与疾病相关的细胞类型的反筛选。为了确定热门脚手架系列的优先顺序，我们将选择 确认来自现有复合库和其他商业来源的命中。热门的脚手架将是 经过分类以去除难处理的分子。我们将从最有希望的热门作品中挑选3-5个分子系列， 将被用来验证其选择性、效力和无细胞毒性。使用来自 申请实验室(加利福尼亚州斯克里普斯和佛罗里达州斯克里普斯)，2-4系列的铅将被制定并重新测试 为了效力/选择性，目的是推进能够在体外引起适当反应的先导 上述检测，并可评估对细胞表达水平的适当表型反应。 随后将进行体外和体内药代动力学(PK)研究，以确定1-2个顶级支架，以便进一步 调查。最后，最有希望的2-3化合物具有良好的药物代谢和药代动力学。 (DMPK)的特性将被选择用于体内测试，包括高口服生物利用度，并将扩大和 在啮齿动物药效模型中进行了体内测试。基因表达程序表达调控的有效性 正在进行的研究将通过突触、树突和线粒体的定量聚合酶链式反应来验证 基因和RNA序列与基因集浓缩分析(GSEA)相结合。然后我们将测试一下 预防或改善可归因于前额叶的认知障碍的优化探针化合物 联合用药前后HIV-1转基因大鼠的海马区退行性改变 或者是酒精自我管理。

Summary HIV-associated neurocognitive disorders (HANDs) and substance abuse comorbidity remain prevalent despite combination antiretroviral therapy (cART). The innovative hypothesis behind this project is that reversing trophic deficits that characterize HANDs and manifest as synaptodendritic and mitochondrial injury can effectively prevent or ameliorate cognitive decline in HIV and compulsive drug taking. To test this hypothesis, we will perform a throughput screening campaign (HTS) to identify modulators of a newly identified gene regulatory mechanism that broadly regulates trophic support and mitochondrial biogenesis in key cortical and hippocampal neuronal populations and that we found are downregulated in both neuroAIDS and dependence. We expect that small molecules that are able to upregulate this mechanism will make neurons more resilient to oxidative stress and neurodegeneration, resulting in a reduction of neuronal injury and hypofrontality, which is a key cause of cognitive dysfunction in HIV and compulsive drug and alcohol taking in dependent individuals. We will employ cell-based luminescence reporter assays in a 1536-well plate format in conjunction with a tiered approach to screen the >640,000-compound Scripps Drug Discovery Library (SDDL) and confirm the potency of ~500 drug-like molecules. Hit-validation will be performed to identify small-molecule regulators and eliminate nonspecific effectors using parallel orthogonal assays and off-target assessment using cytotoxicity counterscreens employing disease-relevant cell types. To prioritize hit scaffold series, we will select analogs of confirmed hits from both the existing compound libraries and other commercial sources. Hit scaffolds will be triaged to remove intractable molecules. We will select 3-5 molecular series from the most promising hits which, will be profiled to verify the selectivity, potency, and lack of cytotoxicity. Using an orchestrated effort from the applicant laboratories (Scripps California and Scripps Florida), leads in 2-4 series will be formulated and retested for potency/selectivity with the aim of advancing leads that can elicit the appropriate in vitro response in the aforementioned assays and can be evaluated for appropriate phenotypic responses to cell-expression levels. This will be followed by in vitro and in vivo pharmacokinetics (PK) studies to identify 1-2 top scaffolds for further investigation. Finally, the most promising 2-3 compounds with favorable drug metabolism and pharmacokinetics (DMPK) properties will be selected for in vivo testing, including high oral bioavailability, and will be scaled up and tested in vivo in rodent efficacy models. Efficacy in regulating the expression of the gene expression program under study will be validated by quantitative polymerase chain reaction of synaptic, dendritic, and mitochondrial genes and RNA-Seq in conjunction with Gene Set Enrichment Analyses (GSEA). We will then test the ability of the optimized probe compounds to prevent or ameliorate cognitive impairment that is attributable to prefrontal and hippocampal degenerative changes in HIV-1 transgenic rats with and without concomitant compulsive drug or alcohol self-administration.