AbstractMitochondrial toxicity is a primary source of pre-clinical drug attrition. 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. IC50 determination using a standard multi-well plate reader, shown that the methodology allowed to detect metabolic toxicity induced by azide, antimycin, oligomycin, rotenone and myxothiazol with high sensitivity. Suitability for high-throughput screening applications was evaluated resulting in a Z’-factor > 0.5 and CV% < 20. A pilot screening allowed sensitive detection of commercial drugs that were previously withdrawn from the market due to liver/cardiac toxicity issues, such as camptothecin, ciglitazone, troglitazone, rosiglitazone, and terfenadine, in ten minutes.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.
MitoToxy Assay: a novel cell-based method for the assessment of metabolic toxicity in a multiwell plate format using a lactate FRET nanosensor, Laconic
