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BOZEMAN, MT, United States

Grant
Agency: National Science Foundation | Branch: | Program: STTR | Phase: Phase I | Award Amount: 150.00K | Year: 2007

This Small Business Technology Transfer (STTR) Phase I research project aims to develop new probes for targeting specific cellular compartments. This will be achieved by producing a library of green fluorescent-tagged proteins using a high throughput insertion strategy. Availability of these reagents would be of great value to cell biologists interested in cellular trafficking or other areas that would require the use of compartment-specific markers.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 568.65K | Year: 2015

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.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: SMALL BUSINESS PHASE II | Award Amount: 692.38K | Year: 2014

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.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 348.18K | Year: 2011

DESCRIPTION (provided by applicant): Project Summary/Abstract Drug discovery depends crucially upon reliable assays for biological activity. Live cell assays provide a rich environment for measuring biological activity. Coupled with genetically encodedfluorescent biosensors, live cell assays have the potential to provide read-outs with unprecedented specificity for particular signaling pathways. Although widely used for basic research applications in living cells, genetically encoded fluorescent biosensors have had little impact on drug discovery because of difficulties in measuring and interpreting fluorescence intensity read-outs, including poor signal to noise ratios, variability in cell expression, and interference from fluorescence emitted by compounds. This Phase 1 project will demonstrate the feasibility of a new strategy that combines highly specific biosensors with extremely fast fluorescence lifetime measurements to produce the speed, sensitivity and specificity needed for high throughput screening applications. This approach employs an alternative fluorescence measurement based on fluorescence lifetime that is much faster than time-correlated single photon counting (TCSPC), yet also more precise. It operates in non-imaging mode which makes forsimple data interpretation and minimizes background fluorescence. It goes far beyond the expected incremental improvements to image-based technologies. Our preliminary data demonstrates the tremendous potential for robust live cell assays when lifetime methodology is applied to measuring genetically encoded fluorescent sensors. Our specific aims will accomplish the vital proof of principle steps and set the direction for our long term objectives of producing a robust live cell drug discovery platform within5 years. PUBLIC HEALTH RELEVANCE: New assays for biological activity are urgently needed to develop safe and effective drugs that provide better treatment outcomes and improved human health. This proposal addresses the technical challenges associated with using fluorescent live-cell assays and has strong potential to reduce the cost and improve the reliability of drug discovery processes.


Grant
Agency: NSF | Branch: Standard Grant | Program: | Phase: SMALL BUSINESS PHASE I | Award Amount: 164.42K | Year: 2013

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.

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