High-content screening for compounds that affect mtDNA-encoded protein levels in eukaryotic cells

J Biomol Screen. 2010 Sep;15(8):937-48. doi: 10.1177/1087057110373547. Epub 2010 Jul 12.

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.

Publication types

  • Evaluation Study
  • Validation Study

MeSH terms

  • Anti-Bacterial Agents / isolation & purification
  • Anti-Bacterial Agents / pharmacology
  • Anti-Retroviral Agents / isolation & purification
  • Anti-Retroviral Agents / pharmacology
  • Cell Line, Transformed
  • DNA, Mitochondrial / drug effects
  • DNA, Mitochondrial / genetics*
  • DNA, Mitochondrial / metabolism
  • Efficiency
  • Electron Transport Complex IV / drug effects
  • Electron Transport Complex IV / genetics
  • Electron Transport Complex IV / metabolism
  • Epithelial Cells / drug effects
  • Epithelial Cells / metabolism
  • Eukaryotic Cells / drug effects*
  • Eukaryotic Cells / metabolism
  • Fluoroquinolones / isolation & purification
  • Fluoroquinolones / pharmacology
  • High-Throughput Screening Assays / methods*
  • Humans
  • Josamycin / isolation & purification
  • Josamycin / pharmacology
  • Liver / drug effects
  • Liver / metabolism
  • Mitochondrial Proteins / drug effects
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism*
  • Mitochondrial Proton-Translocating ATPases / drug effects
  • Mitochondrial Proton-Translocating ATPases / genetics
  • Mitochondrial Proton-Translocating ATPases / metabolism
  • Oxidative Phosphorylation / drug effects
  • Protein Synthesis Inhibitors / analysis
  • Protein Synthesis Inhibitors / isolation & purification*
  • Protein Synthesis Inhibitors / pharmacology*

Substances

  • Anti-Bacterial Agents
  • Anti-Retroviral Agents
  • DNA, Mitochondrial
  • Fluoroquinolones
  • Mitochondrial Proteins
  • Protein Synthesis Inhibitors
  • complex V (mitochondrial oxidative phosphorylation system)
  • Electron Transport Complex IV
  • Mitochondrial Proton-Translocating ATPases
  • Josamycin