Research paper Lateral-flow immunoassay for detecting drug-induced inhibition of mitochondrial DNA replication and mtDNA-encoded protein synthesis Sashi Nadanaciva a, ⁎, John H. Willis a , Melissa L. Barker a , Dima Gharaibeh a , Roderick A. Capaldi a , Michael F. Marusich a , Yvonne Will b a MitoSciences Inc., 1850 Millrace Drive, Eugene, OR 97403, United States b Exploratory Safety Differentiation, Pfizer Inc., Eastern Point Road, Groton, CT 06340, United States article info abstract Article history: Received 7 September 2008 Received in revised form 14 December 2008 Accepted 15 December 2008 Available online 17 January 2009 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. © 2008 Elsevier B.V. All rights reserved. Keywords: Lateral-flow immunoassay Mitochondria Antibacterials Antiretrovirals Nucleoside reverse transcriptase inhibitors Toxicity 1. Introduction Mitochondria are essential for a cell's survival. These organelles make ~95% of the cell's ATP, and are also involved in fatty acid oxidation, steroid synthesis, heme synthesis and apoptosis. Mitochondria contain several copies of double- stranded circular DNA molecules, a DNA replicating enzyme known as DNA polymerase γ, and their own ribosomes. Each mitochondrial DNA (mtDNA) molecule encodes 13 proteins, all of which are subunits of the oxidative phosphorylation complexes, as well as 22 types of tRNA and 2 types of rRNA (Wallace and Starkov, 2000). Given the complexity of mitochondria, it is not surprising that they can by impaired by drugs in a variety of ways (Dykens et al., 2007; Chan et al., 2005; Dykens and Will, 2007; Wallace, 2008). For instance, drug-induced mitochondrial toxicity can occur as a result of impairment of mtDNA replication. Nucleoside reverse transcriptase inhibitors (NRTIs) which inhibit the HIV reverse transcriptase have been reported to inhibit the off-target, DNA polymerase γ, leading to impairment of mtDNA replication and, hence, to depletion of the 13 proteins encoded by mtDNA (Brinkman Journal of Immunological Methods 343 (2009) 1–12 Abbreviations: mtDNA, mitochondrial DNA; NRTI, nucleoside reverse transcriptase inhibitor; PBS, phosphate buffered saline; HEPES, 4-(2-Hydro- xyethyl)piperazine-1-ethanesulfonic acid; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; Ab, antibody; mAb, monoclonal anti- body; PD, population doubling; SD, standard deviation; mABS, milli absorbance; ddC, 2′,3′-dideoxycytidine; ddI, 2′,3′-dideoxyinosine; d4T, 2′,3′-didehydro-2′,3′- deoxythymidine. ⁎ Corresponding author. Current address: Exploratory Safety Differentia- tion, Pfizer Inc., Eastern Point Road, Groton, CT 06340, United States. Tel.: +1 860 686 1031; fax: +1 860 441 9637. E-mail address: sashi.nadanaciva@pfizer.com (S. Nadanaciva). 0022-1759/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jim.2008.12.002 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim