A high-throughput dual parameter assay for assessing drug-induced mitochondrial dysfunction provides additional predictivity over two established mitochondrial toxicity assays

Abstract

Mitochondrial toxicity is a major reason for safety-related compound attrition and post-market drug withdrawals, highlighting the necessity for higher-throughput screens that can identify this mechanism of toxicity during the early stages of drug discovery. Here, we present the validation of a 384-well dual parameter plate-based assay capable of measuring oxygen consumption and extracellular acidification in intact cells simultaneously. The assay showed good reproducibility and robustness and is suitable for use with both suspension cells and adherent cells. To determine if the assay provides additional value in detecting mitochondrial toxicity over existing platforms, 200 commercially available drugs were tested in the assay using HL60 suspension cells as well as in two conventional mitochondrial toxicity assays: an oxygen consumption assay that uses isolated mitochondria and a cell-based assay that uses HepG2 cells grown in glucose and galactose media. The combination of the dual parameter assay and the isolated mitochondrial oxygen consumption assay identified more compounds that caused mitochondrial impairment than any other combination of the three assays or each of the three assays on its own. Furthermore, novel information was obtained from the dual parameter assay on drugs not previously reported to cause mitochondrial impairment.