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

这个小企业创新研究（SBIR）二期项目的更广泛的影响/商业潜力是开发新的工具来理解在疾病中被破坏的细胞机制的动态行为。揭示细胞生理学的动态方面，可能是靶向治疗需要新的技术能够分析整个信号通路的反应，针对单个通路节点的药物干预。与疾病相关的信号转导通路和细胞机制的时空调节的可视化和量化相兼容的生理相关活细胞模型的可用性将是实现这一方法的关键。以这种方式监测关键癌症信号通路的多个方面的能力代表了一个宝贵的机会，可以识别“不可药物”靶点的有效和选择性治疗抑制剂，例如Ras蛋白，它是超过30%的癌症的关键驱动因素。通过开发强大且可扩展的高通量活细胞检测平台，该技术可以减少查明药物化学物质的作用机制和脱靶效应的时间和成本，从而提供快速且经济有效地识别安全有效疗法的新能力。SBIR二期项目将开发一个强大而灵活的平台，用于快速生成精密工程、多色荧光细胞系和相关的高通量显微镜检测。该平台与开发此类细胞系和检测的行业标准方法形成对比，后者冗长且不灵活。该项目包括四个部分的优化和执行：1)生成与快速、可靠、稳定的报告集成兼容的细胞系面板；2)提供大约25个Ras/MAPK通路的多色报告文库；3)快速生成和验证大约100个经过验证的稳定报告细胞系的文库，表达所有治疗相关的突变和Ras/MAPK途径的同种异构体；4) 384孔板法利用工具化合物开发和筛选这些Ras/MAPK报告细胞。该项目旨在展示该平台的能力，通过监测与活细胞中关键的高价值肿瘤靶标相关的先前无法获得的生物学的多个方面，快速确定复合作用机制和潜在的脱靶效应。在该项目过程中建立的标准化平台将允许快速扩展到其他临床相关的信号通路。

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.