Development of Pathway Selective Cells and Assays for Sensitive Detection of Environmental Toxicants via High Throughput Label-free Cell Based Screening

Environmental pollutants that impact critical biological pathways involved in human development and physiology can have unpredictable effect on health. Among these critical pathways, various receptor tyrosine kinases, sonic hedgehog, transforming growth factor beta, Notch and WNT are known or suspected mediators of the effect of numerous environmental toxicants produced during manufacturing, present in the air and water supply. The interconnected nature of these pathways, and finely-tuned spatial and temporal regulation, make them simultaneously broad targets for impactful environmental pollutants and challenging to efficiently model in the laboratory.§Previously, direct modeling of the effect of environmental toxicants on critical developmental and physiological pathways in a comprehensive or holistic manner involved the use of animal models which are expensive, labor-intensive, time consuming, low throughput, and may not always be highly predictive of human responses. Many of these key developmental pathways overlap with targets of modern oncology drug discovery effort, due to their misregulation in many cancers. A multitude of sophisticated, high-throughput assays and technologies focused on the identification of modulators of these pathways have been developed and marketed for drug discovery applications of the last decade. Among these, label-free cell-based assays provide an opportunity to assess the impact of toxicants on biological pathways in a relatively holistic and biologically relevant context compared to traditional HTS assays. While label-free, cell-based approaches to assessment of pathway modulators lack the ability to detect tissue and organism-level effects, they have proven to be predictive of toxicity and efficacy of small molecule drugs in the clinic, in part due to the choice of biologically relevant human cell lines matching the biological context of the pathways being assayed. §This project will help us to research and develop for eventual commercialization a panel of validated cell lines, protocols, and data analysis methods to assess toxic effects of molecules on the critical developmental pathways outlined above on existing, commercially available high throughput label-free screening systems. The first phase consists of initial feasibility studies using receptor tyrosine kinase pathways to broadly assess which pathways can potentially be interrogated, and for each, what toxicantclasses may be detected. The optimization of product design and production techniques for stable scale-up will be conducted, followed by assay optimization for each pathway-specific product. Finally, assay miniaturization, testing under HTS automation workflows, development and validation of dataanalysis parameters, and preliminary validation of assay selectivity will be conducted to generate a product suitable of commercialization.