title: Validation of a HTS-amenable assay to detect drug-induced mitochondrial toxicity in the absence and presence of cell death.
abstract: Drug-induced mitochondrial dysfunction is known to contribute to late stage compound attrition. Recently, assays that identify mitochondrial dysfunction have been developed but many require expensive reagents, specialized equipment, or specialized expertise such as isolation of mitochondria. Here, we validate a new 384-well format cell-based dual parameter assay that uses commonly available detection methods to measure both mitochondrial toxicity and cytotoxicity. In our initial evaluation, antimycin A, CCCP, nefazodone, flutamide, and digitonin were tested in K562 cells in both glucose- and galactose-supplemented media with a 2h incubation. The assay was able to correctly differentiate these compounds into mitochondrial toxicants and non-mitochondrial toxicants, and had excellent reproducibility. We next tested 74 compounds in K562 cells in both types of media and show that the assay was able to correctly identify some of the compounds as mitochondrial toxicants. Moreover, the assay could be simplified, without loss of information, by using K562 cells in galactose-containing medium alone. This simple, robust assay can be positioned as a rapid, early readout of mitochondrial and cellular toxicity. However, since the assay fails to identify some mitochondrial toxicants, further assays may be required to detect mitochondrial toxicity once lead compounds have been selected.
authors.affiliation: Compound Safety Prediction, Pfizer Inc., Groton, CT, USA.
authors.forename: Rachel
authors.initials: R
authors.lastname: Swiss
authors.forename: Andrew
authors.initials: A
authors.lastname: Niles
authors.forename: James J
authors.initials: JJ
authors.lastname: Cali
authors.forename: Sashi
authors.initials: S
authors.lastname: Nadanaciva
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Will
mesh_terms: D001681:Biological Assay; D016923:Cell Death; D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D064420:Drug-Related Side Effects and Adverse Reactions; D022781:Hepatocytes; D057166:High-Throughput Screening Assays; D006801:Humans; D020014:K562 Cells; D008928:Mitochondria / Q000187:drug effects*; D032383:Myocytes, Cardiac; D015203:Reproducibility of Results; D013234:Stem Cells / Q000166:cytology
keywords: Glucose/galactose; High throughput; Mitochondria; Toxicity