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


Autoři: Yasna Contreras-Baeza aff001;  Sebastián Ceballo aff001;  Robinson Arce-Molina aff001;  Pamela Y. Sandoval aff001;  Karin Alegría aff001;  Luis Felipe Barros aff001;  Alejandro San Martín aff001
Působiště autorů: Centro de Estudios Científicos (CECs), Valdivia, Chile aff001;  Universidad Austral de Chile (UACh), Valdivia, Chile aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0224527

Souhrn

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 A pilot screening allowed sensitive detection of commercial drugs that were previously withdrawn from the market due to liver/cardiac toxicity issues, such as camptothecin, ciglitazone, troglitazone, rosiglitazone, and terfenadine, in ten minutes.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.

Klíčová slova:

Astrocytes – Cell metabolism – Drug metabolism – Drug research and development – Fluorescence resonance energy transfer – Mitochondria – Toxicity – Azides


Zdroje

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