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

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.