title: Lateral-flow immunoassay for detecting drug-induced inhibition of mitochondrial DNA replication and mtDNA-encoded protein synthesis.
abstract: Drug-induced mitochondrial toxicity can occur as a result of inhibition of mitochondrial DNA (mtDNA) replication as with certain nucleoside reverse transcriptase inhibitors or inhibition of mtDNA-encoded protein synthesis as with certain antibacterials. Both types of dysfunction have the overall effect of reducing the level of proteins encoded by mtDNA. A lateral-flow immunoassay which measures the levels of both a mtDNA-encoded protein and a nuclear DNA-encoded protein allows simple and rapid determination of the ratio of these 2 proteins and, hence, identifies changes in mtDNA-encoded protein levels. Here, we describe an assay that compares the level of Complex IV (cytochrome c oxidase), a mitochondrial protein which has 3 subunits encoded by mtDNA and made by mitochondrial ribosomes, with that of frataxin, a protein encoded by nuclear DNA and made by cytosolic ribosomes. We tested a selection of antibacterials and antiretrovirals in cells and show that the ratio of Complex IV: frataxin decreases when a drug inhibits either mtDNA replication or mtDNA-encoded protein synthesis. The results obtained with the assay were confirmed by Western blotting and immunocytochemical analysis. The assay has high reproducibility, requires small amounts of sample, is quantitative, and is able to identify drugs which ultimately lead to a decrease in mtDNA-encoded proteins.
authors.affiliation: MitoSciences Inc., Eugene, OR 97403, USA. sashi.nadanaciva@pfizer.com
authors.forename: Sashi
authors.initials: S
authors.lastname: Nadanaciva
authors.forename: John H
authors.initials: JH
authors.lastname: Willis
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Barker
authors.forename: Dima
authors.initials: D
authors.lastname: Gharaibeh
authors.forename: Roderick A
authors.initials: RA
authors.lastname: Capaldi
authors.forename: Michael F
authors.initials: MF
authors.lastname: Marusich
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Will
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D044966:Anti-Retroviral Agents / Q000494:pharmacology; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects*; D004272:DNA, Mitochondrial / Q000187:drug effects*; D003576:Electron Transport Complex IV / Q000096:biosynthesis; D006801:Humans; D007118:Immunoassay; D033862:Iron-Binding Proteins / Q000096:biosynthesis; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000096:biosynthesis; D014176:Protein Biosynthesis / Q000187:drug effects*; D015203:Reproducibility of Results; D000096923:Frataxin
chemical_list: D000900:Anti-Bacterial Agents; D044966:Anti-Retroviral Agents; D004272:DNA, Mitochondrial; D003576:Electron Transport Complex IV; D033862:Iron-Binding Proteins; D024101:Mitochondrial Proteins; D000096923:Frataxin