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High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress

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ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The 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, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.


Flowchart of assays to assess the activity of 66 follow-up compounds found active in the primary high-content oxidative stress screening assay. Abbreviations: ARE-bla, antioxidant response element β-lactamase assay; cpds, compounds; iPSC, induced pluripotent stem cells; LOPAC1280, Library of Pharmacologically Active Compounds; NPC, NIH Chemical Genomics Center Pharmaceutical Collection.
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Figure 5: Flowchart of assays to assess the activity of 66 follow-up compounds found active in the primary high-content oxidative stress screening assay. Abbreviations: ARE-bla, antioxidant response element β-lactamase assay; cpds, compounds; iPSC, induced pluripotent stem cells; LOPAC1280, Library of Pharmacologically Active Compounds; NPC, NIH Chemical Genomics Center Pharmaceutical Collection.

Mentions: Additional secondary assays were performed to prioritize hit compounds and to eliminate false positives due to assay artifacts (Fig. 5). We used the luminescence-based ATP content assay (CTG assay) to eliminate autofluorescent [49], colored, or cytotoxic hit compounds in an assay involving astrocytes not treated with H2O2 (supplemental online data). Many of the 66 hits identified in the primary screen have been reported to cause apoptosis or cytotoxicity in various cell lines (e.g., melphalan and mycophenolic acid [50, 51]). We found that 33 compounds decreased cell viability or were autofluorescent or colored (supplemental online Table 9), leaving 34 hit compounds (including nitrovin) to evaluate in additional assays. An example of the activity of a colored compound that is considered a false positive in the assay is shown in supplemental online Fig. 11A. In addition, data from this CTG assay confirmed what we saw from the primary screening assay—that many compounds were cytotoxic at the highest concentration and were cytoprotective against oxidative stress at lower concentrations (supplemental online Fig. 11B), thus yielding biphasic CRCs for some parameters in the primary and secondary assays (e.g., nitrovin activity in the ARE-bla assay described below).


High-Throughput Phenotypic Screening of Human Astrocytes to Identify Compounds That Protect Against Oxidative Stress
Flowchart of assays to assess the activity of 66 follow-up compounds found active in the primary high-content oxidative stress screening assay. Abbreviations: ARE-bla, antioxidant response element β-lactamase assay; cpds, compounds; iPSC, induced pluripotent stem cells; LOPAC1280, Library of Pharmacologically Active Compounds; NPC, NIH Chemical Genomics Center Pharmaceutical Collection.
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Related In: Results  -  Collection

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Figure 5: Flowchart of assays to assess the activity of 66 follow-up compounds found active in the primary high-content oxidative stress screening assay. Abbreviations: ARE-bla, antioxidant response element β-lactamase assay; cpds, compounds; iPSC, induced pluripotent stem cells; LOPAC1280, Library of Pharmacologically Active Compounds; NPC, NIH Chemical Genomics Center Pharmaceutical Collection.
Mentions: Additional secondary assays were performed to prioritize hit compounds and to eliminate false positives due to assay artifacts (Fig. 5). We used the luminescence-based ATP content assay (CTG assay) to eliminate autofluorescent [49], colored, or cytotoxic hit compounds in an assay involving astrocytes not treated with H2O2 (supplemental online data). Many of the 66 hits identified in the primary screen have been reported to cause apoptosis or cytotoxicity in various cell lines (e.g., melphalan and mycophenolic acid [50, 51]). We found that 33 compounds decreased cell viability or were autofluorescent or colored (supplemental online Table 9), leaving 34 hit compounds (including nitrovin) to evaluate in additional assays. An example of the activity of a colored compound that is considered a false positive in the assay is shown in supplemental online Fig. 11A. In addition, data from this CTG assay confirmed what we saw from the primary screening assay—that many compounds were cytotoxic at the highest concentration and were cytoprotective against oxidative stress at lower concentrations (supplemental online Fig. 11B), thus yielding biphasic CRCs for some parameters in the primary and secondary assays (e.g., nitrovin activity in the ARE-bla assay described below).

View Article: PubMed Central - PubMed

ABSTRACT

Using astrocytes differentiated from human embryonic stem cells, an assay was developed to identify compounds that protect against oxidative stress, a condition associated with many neurodegenerative diseases. The 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, 22 were identified that acutely protect human astrocytes from the consequences of hydrogen peroxide-induced oxidative stress.

No MeSH data available.