Incorporating Phase I/II drug metabolism in HTS via micro scale co-culture

PROJECT SUMMARY High throughput screensHTSare routinely performed in both drug discovery and toxicity testingwhere libraries of compounds are screened for their effects on a given biological endpointi edruggable targetcell toxicityetcA challenge with this approach is that drugs chemicals act by targeting specific biomoleculesbut are often metabolized to different compounds before they reach their target in the bodySince most HTS reporter cell lines exhibit limited drug metabolismthey often fail to report drugs or chemicals that are metabolized to a more active formresulting in a false negativeor are rapidly detoxified clearedresulting in a false positiveEnhancing existing reporter assays with metabolic competence to increase the in vivo predictability of these assays while retaining their compatibility with existing HTS infrastructure would be invaluable in identifying novel drug candidates and identifying potentially hazardous chemicalsWe have developed a microtiter plate based device for high throughput co culture dubbed the Metabolism Integrated Co culture RepOrterMicroTiter plate or MICRO MTFigThe MICRO MT incorporates physiological levels of chemical metabolism into HTS assays by enabling the co culture of metabolically competent liver cells with existing reporter cell assays in a technically simple manner that seamlessly integrates with existing robotic infrastructuree gno new equipment requiredWhile co culture of a reporter cell assay with hepatocytes has been pursued as a viable solution to this problemtraditional Transwellco culture assays are not well suited for robotic HTS due to increased robotic handling and analytical incompatibilityThe MICRO MT is a simple HTS retrofit that fits seamlessly into existing infrastructurePreliminary data with the fungal natural productaflatoxin BAFBdemonstrated that Hekcells in monoculture were insensitive to AFBbut that HepaRG co culture with Hekcells in the MICRO MT conferred assay sensitivity of Hekcells to AFBComprehensive characterization of the MICRO MT will move the assay toward commercial use in the HTStox marketdrug developmentmetabolite discovery and beyond PROJECT NARRATIVE This proposal will validate the Metabolism Integrated Co culture RepOrterMICRO MTfor high throughput coculture to improve the predictability of non animal drug and toxicity testingSpecificallythe MICRO MT adds drug metabolism to existing high throughput screening assays to improve physiological relevance and increase accuracy of existing assaysMore accurate drug screening and toxicity assays will lead to the discovery of novel drug candidatesreduce reliance on animal testing and facilitate identification of potentially hazardous chemicals