SBIR-STTR Award

High throughput screens (HTS) are routinely performed in both drug discovery and toxicity testing, where libraries of compounds are screened for their effects on a given biological endpoint (i.e. druggable target, cell toxicity, etc.). A challenge with this approach is that drugs/chemicals act by targeting specific biomolecules, but are often metabolized to different compounds before they reach their target in the body. Since most HTS reporter cell lines exhibit limited drug metabolism, they often fail to report drugs or chemicals that are metabolized to a more active form (resulting in a false negative) or are rapidly detoxified/cleared (resulting in a false positive). Enhancing 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 chemicals. We have developed a microtiter plate based device for high-throughput co-culture dubbed the Metabolism Integrated Co-culture RepOrter - MicroTiter plate or MICRO-MT (Fig. 1). The 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 infrastructure (e.g. no new equipment required). While co-culture of a reporter cell assay with hepatocytes has been pursued as a viable solution to this problem, traditional Transwell® co-culture assays are not well suited for robotic HTS due to increased robotic handling and analytical incompatibility. The MICRO-MT is a simple HTS retrofit that fits seamlessly into existing infrastructure. Preliminary data with the fungal natural product, aflatoxin B1 (AFB1) demonstrated that Hek293 cells in monoculture were insensitive to AFB1, but that HepaRG co-culture with Hek293 cells in the MICRO-MT conferred assay sensitivity of Hek293 cells to AFB1. Comprehensive characterization of the MICRO-MT will move the assay toward commercial use in the HTStox market, drug development, metabolite discovery and beyond.

Project Terms:
Aflatoxin B1; Aflatoxins; Animal Model; Animal Testing; Aromatase; base; Benzo(a)pyrene; Biological; Biological Assay; Biology; Cell Line; cell type; Cells; Cellular Assay; Chemicals; Coculture Techniques; commercialization; Competence; cost; Custom; Cyclophosphamide; Cytochrome P450; cytotoxicity; Data; density; Detection; Devices; Dimensions; drug candidate; drug development; drug discovery; drug metabolism; Drug or Chemical; Drug Screening; drug testing; Drug toxicity; druggable target; Enzymes; Equipment; Estrogen Receptors; Exhibits; experimental study; Feedback; Foundations; Glucuronides; Glutathione; Goals; Hazardous Chemicals; Hepatocyte; high throughput screening; improved; in vivo; Injections; Laboratories; Lead; Libraries; Metabolic; Metabolism; Molds; Natural Products; novel therapeutics; operation; Performance; Pharmaceutical Preparations; Pharmacology; Phase; Physiological; predictive test; Production; Protocols documentation; prototype; Publishing; Reporter; Reporting; Research Infrastructure; Robotics; routine screening; screening; Screening Result; Small Business Innovation Research Grant; small molecule; stem; Sulfides; Testing; Time; Toxic effect; Toxicity Tests; Transactivation; Universities; Wisconsin; Work;

In place of animal testing, high throughput screening (HTS) is used to discover potential drugs and identify chemicals that are toxic to humans. Federal efforts to reduce toxicity testing in animal models that use HTS have found that traditional HTS tests lack critical biology, including drug/chemical metabolism and signaling between different cell types that would result in a more accurate prediction of human toxicities. Researchers in drug discovery and chemical testing lack a simple, affordable and high-throughput way to co-culture different cell-types together to improve the human relevance of their tests. Onexio Biosystems is developing an HTS testing platform that supports co-culture and multi-culture to improve human relevance in drug discovery and chemical toxicity testing applications. Known as the microDUO, this versatile platform technology supports the intercellular signaling necessary to improve human relevance, while maintaining full compatibility with standard HTS instrumentation. The microDUOs innovation lies in the integration of micro-scale diffusion channels between adjacent pairs or groups of wells (test tubes) in and HTS compatible format. In this grant proposal, we will continue and expand our Phase 1 SBIR R&D efforts to develop high-priority predictive toxicity assays in the MicroDUO. We will 1)confer metabolic competence (drug metabolism) to new cell types including GI, lung and ovary cells by coculturing these cells with liver hepatocyes 2)coculture different cell types from the breast to rebuild the molecular and cellular events that occur during breast cancer progression and test how chemicals in the environment might drive cancer progression. 3) test MicroDUO performance across multiple HTS laboratories and 4)produce a microscope compatible MicroDUO to enable improved understanding of cellular interactions and toxicities in co-culture.

Public Health Relevance Statement:
PROJECT NARRATIVE This proposal will expand the development of high-priority predictive toxicity assays in the microDUO to reduce the use of animal models and better predict risks of chemical exposures to human populations. The microDUO conferred metabolic competence assays developed in Phase I will be expanded to include multiple tissue types and a new assay to understand chemical effects on breast cancer risk will be developed. Improved, imaging- based assays endpoints will be developed in the proposed new clear bottom MicroDUO. More accurate toxicity assays will lead to the discovery of novel drug candidates and accurate identification of potentially hazardous chemicals.

Project Terms:
Anabolism; Animal Model; Animal Testing; Applications Grants; Area; base; Biological Assay; Biology; Breast; breast cancer progression; Breast Cancer Risk Factor; Carcinoma; Cardiac; Cell Communication; cell type; Cells; Chemical Exposure; Chemicals; Coculture Techniques; Competence; Complex; cytotoxicity; Development; Devices; Diffusion; Disease; Dose; drug candidate; drug development; drug discovery; drug metabolism; Drug Targeting; End Point Assay; Endocrine; Environment; Equipment; Estrogens; Event; experimental study; Exposure to; Gastrointestinal tract structure; Goals; Hazardous Chemicals; Hepatocyte; high standard; high throughput screening; Human; Image; improved; in vivo; Infrastructure; innovation; instrumentation; Interview; Kidney; Lab-On-A-Chips; Laboratories; Lead; Letters; Liver; liver metabolism; Lung; malignant breast neoplasm; Measures; Metabolic; Metabolism; Microscope; Modeling; Molecular; National Institute of Environmental Health Sciences; nephrotoxicity; novel therapeutics; Ovary; paracrine; Performance; Pharmaceutical Preparations; Phase; Poison; Population; programs; Reader; Reporter; Reproducibility; Research; research and development; Research Personnel; response; Risk; screening; Signal Transduction; Small Business Innovation Research Grant; steroid metabolism; Technology; Testing; Tissues; Toxic effect; toxicant; Toxicity Tests; Toxicology; Tube; tumor microenvironment; tumor progression; Xenobiotic Metabolism