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Assay Development for High-Throughput Screening of Chemicals of Toxicological Concern


Adverse human health outcomes – a.k.a., “toxicity” – caused by pharmaceutical or environmental compounds are a major cause of drug development failure and public health concern. Methods to evaluate the potential of chemical compounds to induce toxicity are based largely on animal testing, are low-throughput and expensive while giving little insight into mechanisms of toxicity, and have not changed appreciably in the last 50 years despite enormous advances in science. Multiple efforts, including Tox21 in the U.S., REACH in the E.U., and multiple industrial collaborations, are attempting to develop in vitro methods to assess chemical toxicity. These programs must assess toxicity potential in every organ system and identify pathways and/or targets affected. Given the protean nature of these effects, it is likely that hundreds of in vitro assays will need to be developed and tested for their ability to read out chemical effects on particular cell types and pathways. Progress in the field is currently limited by the relatively small number of pathways and cell types that have been developed into high-throughput screening (HTS)-ready assays, and the artificial nature of many of the assays that have been developed (e.g., immortalized/transformed cell lines, heterologous expression with lack of physiologically accurate regulation). The development of HTS-ready assays which can report on particular pathways and cellular phenotypes across the full spectrum of pathway space and toxicological outcomes is needed. Such assays would need to meet strict performance criteria of robustness, reproducibility, and physiological relevance. The assays developed would need to be capable of being run in 384-well or (ideally) 1536-well format and must allow the testing of >100,000 samples per week. Main requirements The outcome of this contract is expected to be one or more novel assays for targets, pathways, and cellular phenotypes related to any type of xenobiotic toxicity. These assays would utilize human cells, including immortalized cell lines, primary cells, and stem cell derived cells, and must be functional in multiwell format with characteristics suitable for automated high-throughput screening. Such assays should be novel, reflecting new pathways or cellular endpoints than are currently available, and be clearly connected to some type of human toxicological response. Such assays could find utility as in chemical assessment and risk management after validation. Deliverables Phase 1 An assay that meets the requirements listed above and also meets the following: • Develop a working assay in 96-well or denser (384, 1536) microwell format • Characterize the sensitivity, specificity, variability, reproducibility, signal: background, dynamic range, and accuracy of the assay, utilizing standard positive and negative controls, Z’ values >0.5 • Demonstrate the utility of the assay by characterizing its ability to detect the effects of compounds known to affect the pathway/cellular phenotype, with a throughput of at least 10,000 samples/day with workstation automation • Are not duplicative of assays already available commercially • Deliver the assay/SOP to NCATS for evaluation Deliverables Phase 2 • Demonstrate miniaturization of assay to work in at least 384-well (preferably 1536-well) format with same technical specifications as listed above • Demonstrate amenability for HTS by successful testing of >100,000 samples/day in fully automated robotic format with maintenance of assay performance • Deliver final assay/SOP to NCATS for evaluation.
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