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. 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 geneticallyencoded 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 A pilot screening allowed sensitive detection of commercial drugs that were previously withdrawn from the market due to liver/cardiac toxicity issues, such as camptothecin, ciglitazone, troglitazone, rosiglitazone, and terfenadine, in ten minutes.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. IntroductionHigh-throughput screening (HTS) is a fundamental step in the hierarchical and long drug development process. Discovering and developing a drug starts with libraries consisting of thousands of chemicals, which are screened for a target activity using in vitro assays. Hits are selected for subsequent stages, then tested for activity and toxicity in cells, tissues, animal models, followed by clinical trials. Most candidates fail in clinical trials, as they are found to be ineffective in a physiological context or produce unwanted side effects.Rate of failure of lead compounds during the drug development process is called attrition, which can be quantified giving a wide view about the whole efficacy of drug development chain.Pre-clinical and clinical safety, pharmacokinetics or bioavailability issues, and rationalization of the company portfolio are the main sources of drug attrition (Waring et al. 2015). However, toxicity-related side-effects are by far the most important source of attrition during pre-clinical stages, being responsible for 59% of failures (Waring et al. 2015). For instance, a comprehensive study combined data on drug attrition from AstraZeneca, Eli Lilly, GlaxoSmithKline, and Pfizer, which revealed that during 2000 and 2010 a total of 211 from 356 lead compounds were rejected due to safety concerns in the pre-clinical stage (Waring et al. 2015). Attrition at later stages of the development chain produces a higher economic impact and is the most important determinant of overall efficiency. These data suggest that although minimizing safety-related attrition has been a significant area...

Signaling Microdomains in the Spotlight: Visualizing Compartmentalized Signaling Using Genetically Encoded Fluorescent Biosensors

“…A recent screening effort by Yasna Contreras-Baeza and colleagues also suggests that FP-based biosensors can be used for pre-clinical toxicity testing. Using a FRET-based lactate biosensor, Laconic (143), to monitor lactate accumulation induced by mitochondrial toxicants, the authors performed a pilot screen in Laconic-expressing MDA-MB-231 cells using commercial drugs that were previously withdrawn from the market due to liver/cardiac toxicity issues (144). This approach consistently reported the known cellular toxicities identified for these molecules, highlighting a potential strategy for direct assessment and early detection of mitochondrial toxicity in the drug development pipeline, thus helping save time, money, and lives.…”

Highly responsive single-fluorophore indicator to explore lactate dynamics in high calcium environments

“…To monitor lactate dynamics with improved spatial/temporal resolution in living systems, we developed a Förster Resonance Energy Transfer (FRET)based indicator called Laconic 9 . This tool has been instrumental in the discovery of mechanisms involved in neurometabolic coupling [10][11][12][13][14] , identification of new monocarboxylate transporters (MCT) 15 and channels 16 , improved monocarboxylate transporter characterization 17 , development of high-throughput methodologies to detect mitochondrial toxicity 18 , and a complete set of methodologies to evaluate lactate dynamics in intact systems 19 .…”