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MINNEAPOLIS, MN, United States

Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: STTR | Phase: Phase II | Award Amount: 860.49K | Year: 2015

DESCRIPTION provided by applicant We seek to develop and commercialize a powerful technology platform to increase dramatically the effectiveness of drug discovery using high throughput screening HTS based on fluorescence lifetime FLT readouts of FRET in live cells This breakthrough is enabled by a combination of two complementary and synergistic technologies fluorescent biosensor engineering University of Minnesota UMN and fluorescence instrumentation engineering Fluorescence Innovations FI In Phase I we achieved our aims producing the first truly high throughput and high precision applications of FLT in living cells applied to a specific biosensor SERCA We also surpassed our aims by developing new instrumentation for high throughput spectral recording carrying out a SERCA screen on a compund library and developing FRET biosensors based on several new targets In addition several pharmaceutical companies expressed interest in this technology platform so UMN and FI started a new drug discovery company Photonic Pharma for the ultimate commercialization of these combined technologies In Phase II we will further develop this technology platform and apply it to a diverse array of targets thus clearly demonstrating the high potential for commercialization In In Aim we will seek improvements in hardware and software that will enhance future commercialization potential including a new instrument that combines FLT and spectral recording increased digitizer speed higher density plates connectivity with robotics and software improvements In Aim in order to demonstrate that our technology platform is widely applicable we will expand our list of biosensors focusing on targets of high commercial potential for which world leading experts are in close proximity at UMN These disease targets include heart failure drug abuse and addiction inflammation and cancer and muscle dystonia Biosensors will be engineered and expressed in live cells and screened against standard test and validation libraries to optimize the screening assay We will then carry out several large scale compound screens using optimized HTS assays Hit compounds will be retested as a function of compound concentration dose response and then subjected to functional assays performed by the above world class experts at UMN Promising hits may become leads for future medicinal chemistry development but the primary goal is to validate the discovery platform We are confident that the research conducted under these aims will set us up nicely for the commercialization phase by validating our business plan to combine UMN expertise in biosensor engineering and cell culture with FI expertise in instrumentation led by Photonic Pharma an emerging early phase drug discovery start up company Thus our business plan is not to sell instruments but to leverage our unique combination of biosensor and instrumentation expertise to develop and apply a technology platform that accelerates the early phase of drug discovery PUBLIC HEALTH RELEVANCE Fluorescence Innovations Inc in collaboration with the University of Minnesota proposes to greatly improve the technology for drug discovery during the crucial early stages of the process This technology has great commercial potential because it will make the process of drug discovery much more effective and efficient and is applicable to a wide range of health issues including heart failure diabetes cancer and drug addiction

Lloyd W.R.,University of Michigan | Wilson R.H.,University of Michigan | Chang C.-W.,University of Michigan | Gillispie G.D.,Fluorescence Innovations, Inc. | Mycek M.-A.,University of Michigan
Biomedical Optics Express | Year: 2010

A fiber-optic system was developed to rapidly acquire tissue fluorescence wavelength-time matrices (WTMs) with high signal-to-noise ratio (SNR). The essential system components (473 nm microchip laser operating at 3 kHz repetition frequency, fiber-probe assemblies, emission monochromator, photomultiplier tube, and digitizer) were assembled into a compact and clinically-compatible unit. Data were acquired from fluorescence standards and tissue-simulating phantoms to test system performance. Fluorescence decay waveforms with SNR > 100 at the decay curve peak were obtained in less than 30 ms. With optimized data transfer and monochromator stepping functions, it should be feasible to acquire a full WTM at 5 nm emission wavelength intervals over a 200 nm range in under 2 seconds. © 2010 Optical Society of America. Source

Muretta J.M.,University of Minnesota | Kyrychenko A.,University of Kansas Medical Center | Ladokhin A.S.,University of Kansas Medical Center | Kast D.J.,University of Minnesota | And 2 more authors.
Review of Scientific Instruments | Year: 2010

We describe a high-performance time-resolved fluorescence (HPTRF) spectrometer that dramatically increases the rate at which precise and accurate subnanosecond-resolved fluorescence emission waveforms can be acquired in response to pulsed excitation. The key features of this instrument are an intense (1 μJ/pulse), high-repetition rate (10 kHz), and short (1 ns full width at half maximum) laser excitation source and a transient digitizer (0.125 ns per time point) that records a complete and accurate fluorescence decay curve for every laser pulse. For a typical fluorescent sample containing a few nanomoles of dye, a waveform with a signal/noise of about 100 can be acquired in response to a single laser pulse every 0.1 ms, at least 105 times faster than the conventional method of time-correlated single photon counting, with equal accuracy and precision in lifetime determination for lifetimes as short as 100 ps. Using standard single-lifetime samples, the detected signals are extremely reproducible, with waveform precision and linearity to within 1% error for single-pulse experiments. Waveforms acquired in 0.1 s (1000 pulses) with the HPTRF instrument were of sufficient precision to analyze two samples having different lifetimes, resolving minor components with high accuracy with respect to both lifetime and mole fraction. The instrument makes possible a new class of high-throughput time-resolved fluorescence experiments that should be especially powerful for biological applications, including transient kinetics, multidimensional fluorescence, and microplate formats. © 2010 American Institute of Physics. Source

Gauer J.W.,University of Minnesota | Sisk R.,University of Minnesota | Murphy J.R.,University of Minnesota | Jacobson H.,University of Minnesota | And 3 more authors.
Biophysical Journal | Year: 2012

The C2A domain is one of two calcium ion (Ca2+)- and membrane-binding domains within synaptotagmin I (Syt I), the identified Ca 2+ sensor for regulated exocytosis of neurotransmitter. We propose that the mechanistic basis for C2A's response to Ca2+ and cellular function stems from marginal stability and ligand-induced redistributions of protein conformers. To test this hypothesis, we used a combination of calorimetric and fluorescence techniques. We measured free energies of stability by globally fitting differential scanning calorimetry and fluorescence lifetime spectroscopy denaturation data, and found that C2A is weakly stable. Additionally, using partition functions in a fluorescence resonance energy transfer approach, we found that the Ca2+- and membrane-binding sites of C2A exhibit weak cooperative linkage. Lastly, a dye-release assay revealed that the Ca2+- and membrane-bound conformer subset of C2A promote membrane disruption. We discuss how these phenomena may lead to both cooperative and functional responses of Syt I. © 2012 by the Biophysical Society. Source

Peng Y.,Montana State University | Veneziano S.E.,Montana State University | Gillispie G.D.,Fluorescence Innovations, Inc. | Broderick J.B.,Montana State University
Journal of Biological Chemistry | Year: 2010

Pyruvate formate-lyase-activating enzyme (PFL-AE) activates pyruvate formate-lyase (PFL) by generating a catalytically essential radical on Gly-734 of PFL. Crystal structures of unactivated PFL reveal that Gly-734 is buried 8 Å from the surface of the protein in what we refer to here as the closed conformation of PFL. We provide here the first experimental evidence for an alternate open conformation of PFL in which: (i) the glycyl radical is significantly less stable; (ii) the activated enzyme exhibits lower catalytic activity; (iii) the glycyl radical undergoes less H/D exchange with solvent; and (iv) the Tm of the protein is decreased. The evidence suggests that in the open conformation of PFL, the Gly-734 residue is located not in its buried position in the enzyme active site but rather in a more solvent-exposed location. Further, we find that the presence of the PFL-AE increases the proportion of PFL in the open conformation; this observation supports the idea that PFL-AE accesses Gly-734 for direct hydrogen atom abstraction by binding to the Gly-734 loop in the open conformation, thereby shifting the closed ↔ open equilibrium of PFL to the right. Together, our results lead to a model in which PFL can exist in either a closed conformation, with Gly-734 buried in the active site of PFL and harboring a stable glycyl radical, or an open conformation, with Gly-734 more solvent-exposed and accessible to the PFL-AE active site. The equilibrium between these two conformations of PFL is modulated by the interaction with PFL-AE. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Source

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