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Ding Y.,University of Alberta | Li J.,Peking University | Enterina J.R.,University of Alberta | Shen Y.,University of Alberta | And 12 more authors.
Nature Methods | Year: 2015

We have developed a versatile new class of genetically encoded fluorescent biosensor based on reversible exchange of the heterodimeric partners of green and red dimerization-dependent fluorescent proteins. We demonstrate the use of this strategy to construct both intermolecular and intramolecular ratiometric biosensors for qualitative imaging of caspase activity, Ca 2+ concentration dynamics and other second-messenger signaling activities. © 2015 Nature America, Inc.


Tewson P.,Montana Molecular, Llc | Westenberg M.,Montana Molecular, Llc | Zhao Y.,University of Alberta | Campbell R.E.,University of Alberta | And 3 more authors.
PLoS ONE | Year: 2012

Phospholipase C produces two second messengers - diacylglycerol (DAG), which remains in the membrane, and inositol triphosphate (IP 3), which triggers the release of calcium ions (Ca 2+) from intracellular stores. Genetically encoded sensors based on a single circularly permuted fluorescent protein (FP) are robust tools for studying intracellular Ca 2+ dynamics. We have developed a robust sensor for DAG based on a circularly permuted green FP that can be co-imaged with the red fluorescent Ca 2+ sensor R-GECO for simultaneous measurement of both second messengers. © 2012 Tewson et al.


Tewson P.H.,Montana Molecular, Llc | Quinn A.M.,Montana Molecular, Llc | Hughes T.E.,Montana Molecular, Llc | Hughes T.E.,Montana State University
Journal of Biomolecular Screening | Year: 2013

There is a growing need in drug discovery and basic research to measure multiple second-messenger components of cell signaling pathways in real time and in relevant tissues and cell types. Many G-protein-coupled receptors activate the heterotrimeric protein, Gq, which in turn activates phospholipase C (PLC). PLC cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce two second messengers: diacylglycerol (DAG), which remains in the plasma membrane, and inositol triphosphate (IP3), which diffuses through the cytosol to release stores of intracellular calcium ions (Ca2+). Our goal was to create a series of multiplex sensors that would make it possible to simultaneously measure two different components of the Gq pathway in living cells. Here we describe new fluorescent sensors for DAG and PIP2 that produce robust changes in green or red fluorescence and can be combined with one another, or with existing Ca2+ sensors, in a live-cell assay. These assays can detect multiple components of Gq signaling, simultaneously in real time, on standard fluorescent plate readers or live-cell imaging systems. © 2013 Society for Laboratory Automation and Screening.


Grant
Agency: National Science Foundation | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 149.42K | 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.


Described herein are novel fluorescent sensors for cyclic adenosine monophosphate (cAMP) that are based on single fluorescent proteins. These sensors use less visible spectrum than FRET-based sensors, produce robust changes in fluorescence, and can be combined with one another, or with other sensors, in a multiplex assay on standard fluorescent plate readers or live cell imaging systems.


Described herein are novel fluorescent sensors for Diacyl Glycerol (DAG) and hosphatidylinositol 4,5-bisphosphate (PIP2) that are based on circularly permuted fluorescent proteins. These sensors use less visible spectrum than FRET-based sensors, produce robust changes in fluorescence, and can be combined with one another, or with other sensors, in amultiplex assay on standard fluorescent plate readers or live cell imaging systems.


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: 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: 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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