Lateral-flow immunoassay for detecting drug-induced inhibition of mitochondrial DNA replication and mtDNA-encoded protein synthesis

Nadanaciva, Sashi; Willis, John H.; Barker, Melissa L.; Gharaibeh, Dima N.; Capaldi, Roderick A.; Marusich, Michael F.; Will, Yvonne

doi:10.1016/j.jim.2008.12.002

Cited by 19 publications

(11 citation statements)

References 32 publications

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“…Such toxicities have led to failed clinical trials, “Black Box” warnings and withdrawal of drugs from the market, and have driven the pharmaceutical industry to develop better methods for detection of drug-induced mitochondrial dysfunction (Begriche et al ., 2011; Dykens et al ., 2007; Marroquin et al ., 2007; Nadanaciva et al ., 2009; Pereira et al ., 2009). …”

Section: Drug-induced Mitochondrial and Mtdna Toxicitymentioning

confidence: 99%

Mitochondria as a Target of Environmental Toxicants

Meyer

Leung

Rooney

et al. 2013

Toxicological Sciences

445

329

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Enormous strides have recently been made in our understanding of the biology and pathobiology of mitochondria. Many diseases have been identified as caused by mitochondrial dysfunction, and many pharmaceuticals have been identified as previously unrecognized mitochondrial toxicants. A much smaller but growing literature indicates that mitochondria are also targeted by environmental pollutants. We briefly review the importance of mitochondrial function and maintenance for health based on the genetics of mitochondrial diseases and the toxicities resulting from pharmaceutical exposure. We then discuss how the principles of mitochondrial vulnerability illustrated by those fields might apply to environmental contaminants, with particular attention to factors that may modulate vulnerability including genetic differences, epigenetic interactions, tissue characteristics, and developmental stage. Finally, we review the literature related to environmental mitochondrial toxicants, with a particular focus on those toxicants that target mitochondrial DNA. We conclude that the fields of environmental toxicology and environmental health should focus more strongly on mitochondria.

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Section: Drug-induced Mitochondrial and Mtdna Toxicitymentioning

confidence: 99%

Mitochondria as a Target of Environmental Toxicants

Meyer

Leung

Rooney

et al. 2013

Toxicological Sciences

445

329

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“…Both the antiComplex IV-subunit 1 antibody and the anti-Complex V-α subunit antibody have been shown to identify proteins localized in the mitochondria. 10,12,13 Optimal concentrations of each antibody for the assay were determined by titrating each of the primary antibodies separately with a varying amount of the appropriate isotype-specific secondary antibody. The fluorescence signal due to Complex IV-subunit 1 was measured as the Mean CircSpot Total Intensity in channel 2, and the fluorescence signal due to Complex V-α subunit was measured as the Mean RingSpot Total Intensity in channel 3 (see Materials and Methods).…”

Section: Hcs Assay Performancementioning

confidence: 99%

“…9 Moreover, the proteins involved in oxidative phosphorylation have to fall below a critical threshold in order for mitochondrial ATP synthesis to be impaired, the threshold varying for each cell type. 3 Hence, compounds that alter mtDNA-encoded protein levels have usually been detected by Western blotting 6 or lateral-flow dipstick immunoassays 10 of cells grown in the compound of interest for several days, but neither of these techniques is amenable to high-throughput screening (HTS). To address this problem, we have developed a high-content screening (HCS) assay that identifies compounds that affect mtDNA-encoded protein levels in adherent eukaryotic cells.…”

Section: Introductionmentioning

confidence: 99%

High-Content Screening for Compounds That Affect mtDNA-Encoded Protein Levels in Eukaryotic Cells

et al. 2010

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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-α subunit, a nuclear DNAencoded 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. (Journal of Biomolecular Screening 2010:937-948)

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“…Recent data suggest that CMX157, like TFV, has a low potential to induce mitochondrial toxicity, showing no effect on biogenesis of a mitochondrially encoded and expressed protein or a nuclear-encoded, cytoplasmically translated mitochondrial protein at concentrations more than 10-fold higher than the clinical target. In these studies CMX157 and TFV were identical in their toxicity to mitochondria (29,49). Studies designed to evaluate the preclinical safety of CMX157 were encouraging.…”

Section: Discussionmentioning

confidence: 86%

Development of Hexadecyloxypropyl Tenofovir (CMX157) for Treatment of Infection Caused by Wild-Type and Nucleoside/Nucleotide-Resistant HIV

Lanier

Ptak

Lampert

et al. 2010

Antimicrob Agents Chemother

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CMX157 is a lipid (1-0-hexadecyloxypropyl) conjugate of the acyclic nucleotide analog tenofovir (TFV) with activity against both wild-type and antiretroviral drug-resistant HIV strains, including multidrug nucleoside/ nucleotide analog-resistant viruses. CMX157 was consistently >300-fold more active than tenofovir against multiple viruses in several different cell systems. CMX157 was active against all major subtypes of HIV-1 and HIV-2 in fresh human peripheral blood mononuclear cells (PBMCs) and against all HIV-1 strains evaluated in monocyte-derived macrophages, with 50% effective concentrations (EC 50 s) ranging between 0.20 and 7.2 nM. The lower CMX157 EC 50 s can be attributed to better cellular uptake of CMX157, resulting in higher intracellular levels of the active antiviral anabolite, TFV-diphosphate (TFV-PP), inside target cells. CMX157 produced >30-fold higher levels of TFV-PP in human PBMCs exposed to physiologically relevant concentrations of the compounds than did TFV. Unlike conventional prodrugs, including TFV disoproxil fumarate (Viread), CMX157 remains intact in plasma, facilitating uptake by target cells and decreasing relative systemic exposure to TFV. There was no detectable antagonism with CMX157 in combination with any marketed antiretroviral drug, and it possessed an excellent in vitro cytotoxicity profile. CMX157 is a promising clinical candidate to treat wild-type and antiretroviral drug-resistant HIV, including strains that fail to respond to all currently available nucleoside/nucleotide reverse transcriptase inhibitors.

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Lateral-flow immunoassay for detecting drug-induced inhibition of mitochondrial DNA replication and mtDNA-encoded protein synthesis

Cited by 19 publications

References 32 publications

Mitochondria as a Target of Environmental Toxicants

Mitochondria as a Target of Environmental Toxicants

High-Content Screening for Compounds That Affect mtDNA-Encoded Protein Levels in Eukaryotic Cells

Development of Hexadecyloxypropyl Tenofovir (CMX157) for Treatment of Infection Caused by Wild-Type and Nucleoside/Nucleotide-Resistant HIV

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