Elsevier

SLAS Discovery

Volume 15, Issue 8, September 2010, Pages 937-948
SLAS Discovery

Original Articles
High-Content Screening for Compounds That Affect mtDNA-Encoded Protein Levels in Eukaryotic Cells

https://doi.org/10.1177/1087057110373547Get rights and content
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Compounds that interfere with the synthesis of either mitochondrial DNA or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial adenosine triphosphate (ATP) production. Toxicity caused by these compounds is seldom identified in 24- to 72-h cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. To address this problem, the authors developed a 96-well format, high-content screening (HCS) assay that measures, in eukaryotic cells, the level of Complex IV–subunit 1, an mtDNA-encoded protein synthesized on mitochondrial ribosomes, and the level of Complex V–α subunit, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The effect of several antibiotics and antiretrovirals on these 2 proteins was assessed, in transformed human liver epithelial cells, 6 days after compound treatment. The results confirmed effects of drugs known to reduce mtDNA-encoded protein levels and also revealed novel information showing that several fluoroquinolones and a macrolide, josamycin, impaired expression of mtDNA-encoded proteins. The HCS assay was robust with an average Z′ factor of 0.62. The assay enables large-scale screening of compounds to identify those that potentially affect mtDNA-encoded protein levels and can be implemented within a screening paradigm to minimize compound attrition.

Key words

high-content screening
mitochondrial
ribosome
antibiotic
antiretroviral

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