SBIR Phase II: A novel multicolor cell line engineering platform that enables high-throughput microscopy-based screening of living cells for drug discovery

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase II project is the development of new tools to understand the dynamic behavior of cellular machinery that is disrupted in disease. Unraveling the dynamic aspects of cellular physiology that may be targeted therapeutically requires new technologies capable of profiling the response of entire signaling pathways to pharmacological intervention targeted at single pathway nodes. The availability of physiologically relevant live-cell models that are compatible with visualizing and quantifying the spatiotemporal regulation of disease-relevant signal transduction pathways and cellular machinery will be key to enabling this approach. The ability to monitor multiple facets of key cancer signaling pathways in this way represents a valuable opportunity to identify potent and selective therapeutic inhibitors of "undruggable" targets, such as the Ras protein, which is a crucial driver of more than 30% of cancers. By enabling development of a robust and scalable high-throughput live-cell assay platform, this technology may reduce the time and cost to pinpoint the mechanism of action and off-target effects of pharmaceutical chemicals, thus delivering new capabilities to rapidly and cost-effectively identify safe and effective therapeutics. This SBIR Phase II project will develop a robust and flexible platform for rapid generation of precision-engineered, multicolor fluorescent cell lines and associated high-throughput microscopy-based assays. This platform contrasts with industry standard methods for developing such cell lines and assays, which are lengthy and inflexible. The project comprises optimization and execution of four components: 1) Generation of a panel of cell lines compatible with rapid, reliable stable reporter integration; 2) Delivery of a library of approximately 25 multicolor reporters of the Ras/MAPK pathway; 3) Rapid generation and validation of a library of approximately 100 validated stable reporter cell lines expressing all therapeutically relevant mutations and isoforms of the Ras/MAPK pathway; and 4) 384-well plate assay development and screening of these Ras/MAPK reporter cells using tool compounds. The project aims to demonstrate the capability of the platform to rapidly pinpoint compound mechanism of action and potential off-target effects by monitoring multiple facets of previously inaccessible biology associated with a critical, high-value oncology target in live cells. The standardized platform established in the course of this project will allow rapid expansion to additional clinically relevant signaling pathways.