SBIR Phase II: New Fluorescent Biosensors for Drug Discovery in Living Cells

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，它将产生新的活细胞分析方法，用于发现新药。目前销售的药物中，大约有一半是针对体内G蛋白偶联受体（gpcr）的，因此，找到针对这些受体、副作用更小的更好的药物，是一个重要的社会目标。在我们身体的许多不同器官中，许多gpcr通过环AMP （cAMP）的变化发出信号。事实上，cAMP信号在大脑中被用来形成记忆，它控制我们心脏的兴奋性，它在糖尿病中起着重要作用。我们的目标是开发一种基因编码的荧光生物传感器，可以在活的人类细胞中使用，以报告药物何时激活GPCR并引起cAMP的变化。这些新的生物传感器将使药物发现团队能够在他们想要影响的活生生的人类细胞的背景下寻找新药。能够在最相关的生物学背景下筛选药物，将有可能更快地找到副作用更少的更好的药物。拟议的项目将为活细胞中的cAMP信号产生遗传编码荧光传感器。传统上，cAMP信号是用单一的、破坏性的终点分析来测量的。这些分析忽略了cAMP信号在细胞内的时间和空间上受到严格控制的事实：简单地测量长时间内cAMP的总积累可能会错过重要的信号事件。更糟糕的是，有许多不同的信号通路可以改变cAMP的水平，单终点检测无法区分它们。在第一阶段，我们创建了一系列绿色或红色荧光原型cAMP生物传感器，证明了创建用于自动筛选平台的强大cAMP传感器是可行的。本提案的目标是：1)通过筛选约2,500个变量来优化绿色荧光传感器产生的亮度和信号，以获得最佳性能；2)基于我们从绿色传感器中学到的知识，创建类似的红色荧光传感器；3)将这些红色和绿色传感器与颜色互补的二酰基甘油传感器结合起来，创建用于区分信号通路的多重传感器；4)将基因编码的传感器包装起来，用于在各种人类细胞系的自动化药物筛选设施中进行病毒传递和表达。

The broader impact/commercial potential of this Small Business Innovation Research (SBIR) Phase II project is that it will generate new live cell assays for the discovery of new drugs. Roughly half of the drugs sold today target G protein coupled receptors (GPCRs) in the body, so finding better drugs that target these receptors, with fewer side effects, is an important societal goal. Many of the GPCRs, in many different organs of our body, signal through changes in cyclic AMP (cAMP). Indeed cAMP signaling is used in the brain to form memories, it controls the excitability of our hearts, and it plays an important role in diabetes. Our goal is to develop a genetically encoded, fluorescent biosensor that can be used in living human cells to report when a drug is activating a GPCR and causing changes in cAMP. These new biosensors will enable drug discovery teams to search for new drugs in the context of the very living, human cells that they want influence. Being able to screen for drugs in the most relevant biological context will make it possible to find better drugs with fewer side effects faster.The proposed project will generate genetically encoded fluorescent sensors for cAMP signaling in living cells. Traditionally, cAMP signaling has been measured with single, destructive end-point assays. These assays ignore the fact that cAMP signaling is tightly controlled in time and space within a cell: simply measuring total cAMP accumulation over an extended time period can miss important signaling events. Worse, there are many different signaling pathways that can change the levels of cAMP, and single end-point assays cannot distinguish among them. In Phase I, we created a series of green or red fluorescent prototype cAMP biosensors that demonstrated it is feasible to create robust cAMP sensors for use in automated screening platforms. The goal of this proposal is to 1) optimize the brightness and signal produced by our green fluorescent sensor by screening ~2,500 variants for optimal properties, 2) create analogous red fluorescent sensors based on what we have learned from the green sensors, 3) combine these red and green sensors with color complementary diacylglycerol sensors to create multiplex sensors for distinguishing signaling pathways and 4) package the genetically encoded sensors for viral delivery and expression in automated drug screening facilities in a variety of human cell lines.