title: 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-alpha 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.
authors.affiliation: Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc., Groton, CT 06340, USA. sashi.nadanaciva@pfizer.com
authors.forename: Sashi
authors.initials: S
authors.lastname: Nadanaciva
authors.forename: Keith
authors.initials: K
authors.lastname: Dillman
authors.forename: David F
authors.initials: DF
authors.lastname: Gebhard
authors.forename: Alka
authors.initials: A
authors.lastname: Shrikhande
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Will
mesh_terms: D000900:Anti-Bacterial Agents / Q000302:isolation & purification / Q000494:pharmacology; D044966:Anti-Retroviral Agents / Q000302:isolation & purification / Q000494:pharmacology; D002461:Cell Line, Transformed; D004272:DNA, Mitochondrial / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D004526:Efficiency; D003576:Electron Transport Complex IV / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005057:Eukaryotic Cells / Q000187:drug effects* / Q000378:metabolism; D024841:Fluoroquinolones / Q000302:isolation & purification / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D015570:Josamycin / Q000302:isolation & purification / Q000494:pharmacology; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D025261:Mitochondrial Proton-Translocating ATPases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D010085:Oxidative Phosphorylation / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology*
chemical_list: D000900:Anti-Bacterial Agents; D044966:Anti-Retroviral Agents; D004272:DNA, Mitochondrial; D024841:Fluoroquinolones; D024101:Mitochondrial Proteins; D011500:Protein Synthesis Inhibitors; C021983:complex V (mitochondrial oxidative phosphorylation system); D003576:Electron Transport Complex IV; D025261:Mitochondrial Proton-Translocating ATPases; D015570:Josamycin