title: High-throughput assays for assessing mitochondrial dysfunction caused by compounds that impair mtDNA-encoded protein levels in eukaryotic cells.
abstract: Compounds that impair the synthesis of either mitochondrial DNA (mtNDA) or mtDNA-encoded proteins reduce the levels of 13 proteins essential for oxidative phosphorylation, leading to a decrease in mitochondrial ATP production. Toxicity caused by these compounds is seldom identified in 24 to 72 hr cytotoxicity assays due to the low turnover rates of both mtDNA and mtDNA-encoded proteins. Here, we describe three high-throughput screening assays that detect compounds that affect mtDNA-encoded protein levels. All three assays measure the levels of two proteins, one a mtDNA-encoded protein synthesized on mitochondrial ribosomes and the other, a nuclear DNA-encoded protein synthesized on cytosolic ribosomes. The first assay measures the levels of these two proteins by quantitative image analysis and requires a high-content imaging system. The second assay is an in-cell immunoassay that utilizes infrared dyes for detection of the two proteins and, thus, requires a LI-COR Odyssey system. The third assay is an in-cell immunoassay that utilizes colorimetric detection of the two proteins and requires an absorbance microplate reader.
authors.affiliation: Compound Safety Prediction, Worldwide Medicinal Chemistry, Pfizer Inc., Groton, Connecticut, USA.
authors.forename: Sashi
authors.initials: S
authors.lastname: Nadanaciva
authors.forename: James
authors.initials: J
authors.lastname: Murray
authors.forename: Casey
authors.initials: C
authors.lastname: Wilson
authors.forename: David F
authors.initials: DF
authors.lastname: Gebhard
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Will
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003124:Colorimetry; D004272:DNA, Mitochondrial / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects; D057166:High-Throughput Screening Assays; D006801:Humans; D007090:Image Interpretation, Computer-Assisted; D007118:Immunoassay; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D004272:DNA, Mitochondrial; D024101:Mitochondrial Proteins