SBIR Phase I: New Fluorescent Biosensors for Signaling in Living Cells

这个小企业创新研究（SBIR）第一阶段项目将确定开发基因编码生物传感器的可行性，以监测环磷酸腺苷（cAMP）的水平，cAMP是药物信号传导途径的重要第二信使成分。与现有的基于FRET的生物传感器依赖于两个荧光分子之间的能量转移不同，该传感器将采用单个循环排列的荧光绿色蛋白。这种绿色传感器可以与其他第二信使的不同颜色的生物传感器耦合，以产生信号通路的多个组分的同时读数，当被药物激活时，该信号通路发生变化。该项目更广泛的影响/商业潜力是，报告多个细胞信号传导事件的多重遗传编码测定将通过改善关于时间的信息，生理相关组织中的位置和通路串扰。这些测定是均相的，不需要多个步骤或细胞裂解。制药行业的一个日益增长的趋势是在原代细胞培养中进行筛选，而遗传编码检测非常适合于此。该提案中开发的技术代表了荧光活细胞测定的新创新，具有降低成本和提高药物发现可靠性的强大潜力，以找到安全有效的药物，提供更好的治疗结果和改善人类健康。

This Small Business Innovation Research (SBIR) Phase I project will establish the feasibility of developing a genetically-encoded biosensor to monitor levels of cyclic adenosine monophosphate (cAMP), an important second messenger component of drug signaling pathways. Unlike existing FRET-based biosensors that depend upon energy transfer between two fluorescent molecules, this sensor will employ a single, circularly permuted fluorescent green protein. This green sensor can be coupled with differently colored biosensors for other second messengers to produce simultaneous readouts for multiple components of a signaling pathway that change when activated by a drug.The broader impact/commercial potential of this project is that multiplex, genetically-encoded assays reporting multiple cell signaling events would expand the depth of knowledge about signal transduction by improving information on the timing, location and pathway cross-talk in physiologically relevant tissues. These assays are homogenous, do not require multiple steps, or cell lysis. A growing trend in the pharmaceutical industry is screening in primary cell cultures, and genetically encoded assays are ideally suited for this. The technology to be developed in this proposal represents a new innovation in fluorescent live-cell assay and has strong potential to reduce the cost and improve the reliability of drug discovery to find safe and effective drugs that provide better treatment outcomes and improved human health.