Dataset Information

MitoToxy assay: A novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic.

ABSTRACT: Mitochondrial toxicity is a primary source of pre-clinical drug attrition, black box warning and post-market drug withdrawal. Methods that detect mitochondrial toxicity as early as possible during the drug development process are required. Here we introduce a new method for detecting mitochondrial toxicity based on MDA-MB-231 cells stably expressing the genetically encoded FRET lactate indicator, Laconic. The method takes advantage of the high cytosolic lactate accumulation observed during mitochondrial stress, regardless of the specific toxicity mechanism, explained by compensatory glycolytic activation. Using a standard multi-well plate reader, dose-response curve experiments allowed the sensitivity of the methodology to detect metabolic toxicity induced by classical mitochondrial toxicants. Suitability for high-throughput screening applications was evaluated resulting in a Z'-factor > 0.5 and CV% < 20 inter-assay variability. A pilot screening allowed sensitive detection of commercial drugs that were previously withdrawn from the market due to liver/cardiac toxicity issues, such as camptothecin, ciglitazone, troglitazone, rosiglitazone, and terfenadine, in ten minutes. We envisage that the availability of this technology, based on a fluorescent genetically encoded indicator, will allow direct assessment of mitochondrial metabolism, and will make the early detection of mitochondrial toxicity in the drug development process possible, saving time and resources.

SUBMITTER: Contreras-Baeza Y

PROVIDER: S-EPMC6822764 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Publications

MitoToxy assay: A novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic.

Contreras-Baeza Yasna Y Ceballo Sebastián S Arce-Molina Robinson R Sandoval Pamela Y PY Alegría Karin K Barros Luis Felipe LF San Martín Alejandro A

PloS one 20191031 10

Mitochondrial toxicity is a primary source of pre-clinical drug attrition, black box warning and post-market drug withdrawal. Methods that detect mitochondrial toxicity as early as possible during the drug development process are required. Here we introduce a new method for detecting mitochondrial toxicity based on MDA-MB-231 cells stably expressing the genetically encoded FRET lactate indicator, Laconic. The method takes advantage of the high cytosolic lactate accumulation observed during mitoc ...[more]

PMID: 31671132

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Project description:In order to establish phage therapy as a standard clinical treatment for bacterial infections, testing of every phage to ensure the suitability and safety of the biological compound is required. While some issues have been addressed over recent years, standard and easy-to-use animal models to test phages are still rare. Testing of phages in highly suitable mammalian models such as mice is subjected to strict ethical regulations, while insect larvae such as the Galleria mellonella model suffer from batch-to-batch variations and require manual operator skills to inject bacteria, resulting in unreliable experimental outcomes. A much simpler model is the nematode Caenorhabditis elegans, which feeds on bacteria, a fast growing and easy to handle organism that can be used in high-throughput screening. In this study, two clinical bacterial strains of Escherichia coli, one Klebsiella pneumoniae, and one Enterobacter cloacae strain were tested on the model system together with lytic bacteriophages that we isolated previously. We developed a liquid-based assay, in which the efficiency of phage treatment was evaluated using a scoring system based on microscopy and counting of the nematodes, allowing increasing statistical significance compared to other assays such as larvae or mice. Our work demonstrates the potential to use Caenorhabditis elegans to test the virulence of strains of Klebsiella pneumoniae, Enterobacter cloacae, and EHEC/EPEC as well as the efficacy of bacteriophages to treat or prevent infections, allowing a more reliable evaluation for the clinical therapeutic potential of lytic phages. IMPORTANCE Validating the efficacy and safety of phages prior to clinical application is crucial to see phage therapy in practice. Current animal models include mice and insect larvae, which pose ethical or technical challenges. This study examined the use of the nematode model organism C. elegans as a quick, reliable, and simple alternative for testing phages. The data show that all the four tested bacteriophages can eliminate bacterial pathogens and protect the nematode from infections. Survival rates of the nematodes increased from <20% in the infection group to >90% in the phage treatment group. Even the nematodes with poly-microbial infections recovered during phage cocktail treatment. The use of C. elegans as a simple whole-animal infection model is a rapid and robust way to study the efficacy of phages before testing them on more complex model animals such as mice.

Dataset Information

MitoToxy assay: A novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic.

Publications

MitoToxy assay: A novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic.

Similar Datasets

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