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

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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.