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

在早期的研究中，建立了一套工作流程，用于使用细胞活力测试来筛选人类候选的衰老(衰老细胞杀伤)化合物。利用我们的筛选工作流程，我们测试了天然抗衰老化合物的面板杀死衰老细胞的能力。我们已经在肺成纤维细胞、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步的感光筛选。黄酮类化合物的感光活性具有细胞类型特异性。总体而言，姜黄酮甲是最有效的杀伤细胞，在所有被测试的细胞类型中都有显著的活性。Fisetin在所有被测试的细胞类型中也有显著意义，尽管程度不同，单核细胞中的活性最强。其他化合物中的每一种都表现出细胞类型特异性的感官分解活性。此外，对于在最大剂量下可感光的化合物，我们在衰老和非衰老细胞中进行全剂量反应，以确定每种化合物最特异和最敏感的感光剂量。为了测量每种化合物的细胞内浓度，我们发展了多种反应监测质谱学方法来检测化合物，并发现某些衰老细胞对感受性化合物的摄取更高，这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感光剂量下摄取菲斯汀、槲皮素和芹菜素的速率高于健康肾上皮细胞，因此可能比健康细胞对药物更敏感。 我们现在已经从几种细胞类型(单核细胞、肾皮质和近端小管上皮细胞)收集了300多个样本，这些细胞用最佳剂量的黄烷素和各自的控制条件处理。接下来，我们将进行质谱分析，并通过鉴定药物在衰老细胞中参与的蛋白质和生物学途径，确定这些药物分解衰老或优先杀死敏感细胞的机制。然后，我们将使用基因干预研究来验证这些发现。

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.