2 | ||
1 | ||
1 |
2 |
2 | ||
2 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 |
2 | ||
1 | ||
1 |
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 | ||
1 |
1 | ||
1 |
Macroautophagy is a major cellular degradation pathway for long-lived proteins and cellular organelles to maintain cellular homeostasis. Reduced autophagy has been implicated in neurodegenerative diseases, metabolic syndrome, and tumorigenesis. In contrast, increased autophagy has been shown to protect against tissue injury and aging. Here we employed a cell-based quantitative high-throughput image screening (qHTS) for autophagy modulators using mouse embryonic fibroblasts (MEFs) that are stably expressing GFP-LC3. The library of pharmacologically active compounds (LOPAC) was used to screen for the autophagy modulators in compounds alone or in combination with the lysosome inhibitor chloroquine (CQ). The GFP-LC3 puncta were then quantified to measure autophagic flux. The primary screening revealed 173 compounds with efficacy more than 40%. These compounds were cherry-picked and re-tested at multiple different concentrations using the same assay. A number of novel autophagy inducers, inhibitors, and modulators with dual-effects on autophagy were identified from the cherry-pick screening. Interestingly, we found a group of compounds that induce autophagy are related to dopamine receptors and are commonly used as clinical psychiatric drugs. Among them, indatraline hydrochloride (IND), a dopamine inhibitor, and chlorpromazine hydrochloride (CPZ) and fluphenazine dihydrochloride (FPZ), two dopamine receptor antagonists, were further evaluated. We found that FPZ-induced autophagy through mTOR inhibition but IND and CPZ induced autophagy in an mTOR-independent manner. Our data suggest that image-based autophagic flux qHTS can efficiently identify autophagy inducers and inhibitors.
|
High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress.Thorne N, Malik N, Shah S, Zhao J, Class B, Aguisanda F, Southall N, Xia M, McKew JC, Rao M, Zheng WStem Cells Transl Med , (5), 613-27, 2016. Article Pubmed UNLABELLED: Astrocytes are the predominant cell type in the nervous system and play a significant role in maintaining neuronal health and homeostasis. Recently, astrocyte dysfunction has been implicated in the pathogenesis of many neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Astrocytes are thus an attractive new target for drug discovery for neurological disorders. Using astrocytes differentiated from human embryonic stem cells, we have developed an assay to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. This phenotypic oxidative stress assay has been optimized for high-throughput screening in a 1,536-well plate format. From a screen of approximately 4,100 bioactive tool compounds and approved drugs, we identified a set of 22 that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress. Nine of these compounds were also found to be protective of induced pluripotent stem cell-differentiated astrocytes in a related assay. These compounds are thought to confer protection through hormesis, activating stress-response pathways and preconditioning astrocytes to handle subsequent exposure to hydrogen peroxide. In fact, four of these compounds were found to activate the antioxidant response element/nuclear factor-E2-related factor 2 pathway, a protective pathway induced by toxic insults. Our results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development.
SIGNIFICANCE: Astrocytes play a key role in neurological diseases. Drug discovery efforts that target astrocytes can identify novel therapeutics. Human astrocytes are difficult to obtain and thus are challenging to use for high-throughput screening, which requires large numbers of cells. Using human embryonic stem cell-derived astrocytes and an optimized astrocyte differentiation protocol, it was possible to screen approximately 4,100 compounds in titration to identify 22 that are cytoprotective of astrocytes. This study is the largest-scale high-throughput screen conducted using human astrocytes, with a total of 17,536 data points collected in the primary screen. The results demonstrate the relevancy and utility of using astrocytes differentiated from human stem cells as a disease model for drug discovery and development.
|