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

Abstract

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 DNAencoded 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. (Journal of Biomolecular Screening 2010:937-948)

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“…Isolated mitochondrial fractions were used to obtain toxicological parameters such as IC 50 in order to establish a ranking of compound toxicity, however, one obvious disadvantage is the lack of complexity of the biological system. Nevertheless, isolated mitochondrial fractions can be used as a good indicator to predict drug safety, being in fact in use by several major pharmaceutical companies [2,3].…”

Mitochondrial toxicity assessment in industry – a decade of technology development and insight

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Off-Target Effects of Drugs that Disrupt Human Mitochondrial DNA Maintenance

“…Four human cellular topoisomerases localize to mitochondria: TOP1mt, TOP2α, TOP2β, and a TOP3α long isoform (Zhang H. et al, 2014 ; Pommier et al, 2016 ). Tamoxifen a drug used to prevent breast cancer, tacrine a drug used to treat Alzheimer's disease, and a fluoroquinolone broad-spectrum antibiotic, have all been hypothesized to have off-target effects on mitochondrial topoisomerases (Lawrence et al, 1996 ; Mansouri et al, 2003 ; Larosche et al, 2007 ; Nadanaciva et al, 2010 ; Begriche et al, 2011 ). Mice separately treated for 28 days with tamoxifen and tacrine displayed mtDNA depletion and both of these drugs were demonstrated to inhibit in vitro topoisomerase-mediated plasmid DNA relaxation (Mansouri et al, 2003 ; Larosche et al, 2007 ).…”