Evaluating the Cell-Type Specificity and Cellular Targets of Senotherapuetic Compounds with Unknown Mechanisms

评估具有未知机制的治疗化合物的细胞类型特异性和细胞靶点

• 批准号: 10913050
• 负责人: Nathan Basisty
• 金额: $ 5.7万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10913050
• 关键词: Adipocytes; Apigenin; Biological; Biological Assay; Cell Aging; Cell Culture Techniques; Cell Survival; Cells; Dietary Flavonoid; Dose; Epithelial Cells; Fibroblasts; Flavonoids; Human; Intervention; Intervention Studies; Lung; Mass Spectrum Analysis; Measures; Modeling; Natural Compound; Pathway interactions; Pharmaceutical Preparations; Predisposition; Proteins; Proximal Kidney Tubules; Quercetin; Sampling; Smooth Muscle Myocytes; Specificity; Testing; Vascular Smooth Muscle; cell killing; cell type; cellular targeting; detection method; efficacy evaluation; fisetin; flavanoid; gene therapy; genetic manipulation; kidney cortex; monocyte; multiple reaction monitoring; novel; renal epithelium; response; screening; senescence; uptake

In early studies, established a workflow for the screening of candidate senolytic (senescence cell killing) compounds in human using a cell viability assay. Using our screening workflow, we test panels of natural senolytic compounds for the ability to kill senescent cells. We have carried out initial senolytic screens in lung fibroblasts, monocytes, preadipocytes, renal proximal tubule epithelial cells, renal cortical epithelial cells and vascular smooth muscle cells. Flavonoid compounds showed cell-type specificity in their senolytic activities. Gingeronone A was the most effective overall in killing cells, with significant activity in all cell types tested. Fisetin was also significant in all cell types tested, albeit to different degrees, with the strongest activity in monocytes. Each of the other compounds showed cell-type specificity in senolytic activity. Further, for compounds that are senolytic at the maximal dose, we are performed a full dose responses in both senescent and non-senescent cells to establish the most specific and sensitive senolytic doses of each compound. Toward our AIM of measuring the intracellular concentrations of each compound, we developed multiple reaction monitoring mass spectrometry methods for the detection of compounds, and have found that uptake of the senolytic compounds are higher in some senescent cell types, possibly explaining their susceptibility to the compounds. For example, senescent renal epithelial cells uptake fisetin, quercetin, and apigenin at higher rates than healthy renal epithelial cells at the maximal senolytic doses, thus may be more sensitive to the drugs than the healthy cells. We have now collected over 300 samples from several cell types (monocytes, renal cortical and proximal tubule epithelial cells) treated with optimal senolytic doses of flavanoids, and respective control conditions. Next we will perform mass spectrometry analysis and identify the mechanisms by which these drugs are senolytic, or preferentially killing seenscent cells, by identifying the protein and biological pathways engaged by the drugs in senescent cells. We will then validate these findings using genetic intervention studies.

在早期研究中，使用细胞活力测定法建立了用于筛查人类候选鼻溶细胞（衰老细胞杀死）化合物的工作流程。使用我们的筛选工作流程，我们测试了天然鼻溶解化合物的面板，以杀死衰老细胞。我们在肺成纤维细胞，单核细胞，前脂肪细胞，肾近端小管上皮细胞，肾皮质上皮细胞和血管平滑肌细胞中进行了初始的鼻溶液筛查。类黄酮化合物在其鼻溶性活性中表现出细胞类型的特异性。姜酮A是杀死细胞中最有效的总体，在所有细胞类型中都具有显着的活性。 Fisetin在所有测试的细胞类型中也很重要，尽管在不同程度上，单核细胞的活性最强。其他每种化合物在鼻溶性活性中均显示出细胞类型的特异性。此外，对于以最大剂量为鼻溶剂的化合物，我们在衰老和非阳性细胞中都进行了全剂量反应，以建立每种化合物的最特异性和敏感的鼻溶剂剂量。为了测量每种化合物的细胞内浓度，我们开发了多种反应监测质谱法以检测化合物，并发现在某些衰老细胞类型中摄取鼻溶性化合物的摄取较高，可能解释了它们对化合物的敏感性。例如，衰老的肾上皮细胞在最大鼻溶剂剂量下以比健康的肾上皮细胞更高的速率摄取fisetin，槲皮素和丙酸蛋白，因此可能比健康细胞更敏感。 现在，我们从几种细胞类型（单核细胞，肾皮质和近端小管上皮细胞）中收集了300多种样品，这些细胞用最佳的类黄酮类药物和各自的对照条件处理。接下来，我们将进行质谱分析，并确定这些药物是鼻溶剂或优先杀死月经细胞的机制，通过识别衰老细胞中药物参与的蛋白质和生物学途径。然后，我们将使用遗传干预研究验证这些发现。