High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

Schaaf, Tory; Peterson, Kurt C.; Grant, Benjamin D.; Yuen, Samantha L.; Bawaskar, Prachi; Li, Ji; Muretta, Joesph M.; Gillispie, Gregory D.; Thomas, David D.

doi:10.1016/j.bpj.2016.11.1791

Cited by 17 publications

(52 citation statements)

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“…Our approach to developing this drug discovery pipeline has been consistent to previous efforts with other biosensor systems (e.g. tau and TNFR1, amongst others) 84,[90][91][92][103][104][105] . At the core of any fluorescent assay is the potential of artifacts being introduced into the model system through the labeling of protein with synthetic fluorophores or fusion to large fluorescent XFPs.…”

Section: Discussionsupporting

confidence: 58%

“…Another concern with HTS campaigns is the physiological relevance of the cellular platform being deployed. The choice of HEK293 cells for our FRET HTS primary assay implementation is strongly rooted in the thorough vetting of this cell lines performance in multiple HTS campaigns that interrogated a wide range of protein targets 84,[90][91][92][103][104][105] . We acknowledge that not using neuronal cell lines in a primary HTS platform targeting neurodegeneration imparts a disconnect between the physiological and epigenetic link between cell type and disease environment.…”

Section: Discussionmentioning

confidence: 99%

“…The core of our cellular bio-sensor system builds upon our established, state-of-the-art, HEK293/fluorescent lifetime plate reader (FLT-PR) platform designed to monitor specific protein interactions and structural changes in living cells. [83][84][85] Fluorescence lifetime (FLT) detection increases the precision of FRET-based screening by a factor of 30 compared with conventional fluorescence intensity detection 81 , and provides exquisite sensitivity to resolve minute structural changes within protein ensembles. The improved signal-to-noise of FLT detection is due to FLT's being an inherent property of the XFP and not sensitive to fluctuations in steady state intensity.…”

Section: Biosensor Engineeringmentioning

confidence: 99%

“…A major challenge to any fluorescent based HTS platform stems from potential fluorescent interference due to fluorescent compounds in the library. Fluorescent compounds (FC) were flagged using a spectral similarity index filter which evaluates the ratio of intensity from two bandwidths that span the donor emission spectrum (see 84 for more detail) and are excluded as potential false-positives 84,[89][90][91][92] . FLT for all compounds that passed the FC filter are presented in Fig.…”

Section: Pilot Hts With the Lopac Library For The Oligomer And Monomementioning

confidence: 99%

“…After resuspension and dilution in PBS, the biosensor cells were constantly and gently stirred using a magnetic stir bar at room temperature, keeping the cells in suspension and evenly distributed to avoid clumping. During screening, cells (50 µl/well for 384-well; 10 µl/well for 1536-well) were dispensed by a Multidrop Combi liquid dispenser from Thermo (Pittsburg, PA, USA) into the assay plates containing the compounds and allowed to incubate at room temperature for 90 minutes before readings were taken by the fluorescence lifetime plate reader and spectral unmixing plate reader (Fluorescence Innovations, Inc) as described previously 108,109 . Reproducible FRET hits for retesting were purchased from Tocris (Minneapolis, MN, USA), Sigma (St. Louis, MO, USA), or Invitrogen (Carlsbad, CA, USA), depending on availability.…”

Section: Lopac Library and Liquid Handlingmentioning

confidence: 99%

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Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor

Braun

Liao

Horvath

et al. 2020

Preprint

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Preventing or reversing the pathological misfolding and self-association of alpha-synuclein (aSyn) can rescue a broad spectrum of pathological cellular insults that manifest in Parkinson's Disease (PD), Dementia with Lewy bodies (DLB), and other alpha-synucleinopathies. We have developed a high-throughput, FRET-based drug discovery platform that combines high-resolution protein structural detection in living cells with an array of functional and biophysical assays to identify novel lead compounds that protect SH-SY5Y cells from aSyn induced cytotoxicity as well as inhibiting seeded aSyn aggregation, even at nanomolar concentrations.Our combination of cellular and cell-free assays allow us to distinguish between direct aSyn binding or indirect mechanisms of action (MOA). We focus on targeting oligomers with the requisite sensitivity to detect subtle protein structural changes that may lead to effective therapeutic discoveries for PD, DLB, and other alphasynucleinopathies. Pilot high-throughput screens (HTS) using our aSyn cellular FRET biosensors has led to the discovery of the first nanomolar-affinity small molecules that disrupt toxic aSyn oligomers in cells and inhibit cell death. Primary neuron assays of aSyn pathology (e.g. phosphorylation of mouse aSyn PFF) show rescue of pathology for two of our tested compounds. Subsequent seeded thioflavin-t (ThioT) aSyn aggregation assays demonstrate these compounds deter or block aSyn fibril assembly. Other hit compounds identified in our HTS are known to modulate oxidative stress, autophagy, and ER stress, providing validation that our biosensor is sensitive to indirect MOA as well. Author contributionsA.R.B. designed and conducted the experiments. E.E.L provided assistance with cell-based assays and western blot experiments. M.C.Y produce and purified recombinant protein. M.H. and K.L. performed primary neuron PFF assays. D.D.T. provided expertise on FRET and HTS, and provided comments and edits to the manuscript. M.E. and R.B. performed statistical model development and analysis on the PFF pathology model. E.E.L. provided comments and edits to the manuscript. A.R.B. and J.N.S. wrote the manuscript. Competing interestsDavid D. Thomas holds equity in and serves as executive officer for Photonic Pharma LLC, a company that owns intellectual property related to technology used in part of this project. These relationships have been reviewed and managed by the University of Minnesota in accordance with its conflict-of-interest polices.

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Section: Discussionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Section: Biosensor Engineeringmentioning

confidence: 99%

Section: Pilot Hts With the Lopac Library For The Oligomer And Monomementioning

confidence: 99%

Section: Lopac Library and Liquid Handlingmentioning

confidence: 99%

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Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor

Braun

Liao

Horvath

et al. 2020

Preprint

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Conformational states of TNFR1 as a molecular switch for receptor function

Huber

Sachs

2020

Protein Science

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Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that plays a key role in the regulation of the inflammatory pathway. While inhibition of TNFR1 has been the focus of many studies for the treatment of autoimmune diseases such as rheumatoid arthritis, activation of the receptor is important for the treatment of immunodeficiency diseases such as HIV and neurodegenerative diseases such as Alzheimer's disease where a boost in immune signaling is required. In addition, activation of other TNF receptors such as death receptor 5 or FAS receptor is important for cancer therapy. Here, we used a previously established TNFR1 fluorescence resonance energy transfer (FRET) biosensor together with a fluorescence lifetime technology as a high-throughput screening platform to identify a novel small molecule that activates TNFR1 by increasing inter-monomeric spacing in a ligandindependent manner. This shows that the conformational rearrangement of pre-ligand assembled receptor dimers can determine the activity of the receptor. By probing the interaction between the receptor and its downstream signaling molecule (TRADD) our findings support a new model of TNFR1 activation in which varying conformational states of the receptor act as a molecular switch in determining receptor function. K E Y W O R D Sconformational states, FRET, high-throughput screening, small molecule activator, structural dynamics, TNFR1 signaling | INTRODUCTIONThe receptors and ligands in the tumor necrosis factor (TNF) superfamily have unique structural attributes that couple them directly to signaling pathways that are responsible for a wide range of cellular activities such as cell proliferation, differentiation, or death. 1,2 Within this superfamily, tumor necrosis factor receptor 1 (TNFR1) is a characteristic member and a central mediator in the signal transduction of the inflammatory pathway. 3 Stimulation by the native ligands of TNFR1, tumor necrosis factor-alpha (TNFα) and lymphotoxin-alpha (LTα), leads to the recruitment of TNFR1 associated death domain (TRADD) followed by IκBα degradation and NF-κB activation. 4 While over-activation of TNFR1 results in excessive NF-κB activation, which has been associated with several autoimmune diseases such as rheumatoid arthritis, 3-5 lack of NF-κB activation has been implicated in diseases related to immune deficiency and cell death such as HIV, neurodegeneration, and tissue degeneration. [6][7][8][9] Hence, there is a need for increased activation of TNFR1 beyond its native activation to promote NF-κB activation for treatment of these diseases.

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Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies

Lim

Ding

et al. 2019

Alzheimer's & Dementia

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Objective: Understanding the heterogeneous pathology in Alzheimer's disease and related tauopathies is one of the most urgent and fundamental challenges facing the discovery of novel disease-modifying therapies. Through monitoring ensembles of toxic and nontoxic tau oligomers spontaneously formed in cells, our biosensor technology can identify tool compounds that modulate tau oligomer structure and toxicity, providing much needed insight into the nature and properties of toxic tau oligomers. Background: Tauopathies are a group of neurodegenerative disorders characterized by pathologic aggregation of the microtubule binding protein tau. Recent studies suggest that tau oligomers are the primary toxic species in tauopathies. New/Updated Hypothesis: We hypothesize that tau biosensors capable of monitoring tau oligomer conformation are able to identify tool compounds that modulate the structure and conformation of these tau assemblies, providing key insight into the unique structural fingerprints of toxic tau oligomers. These fingerprints will provide gravely needed biomarker profiles to improve staging of early tauopathy pathology and generate lead compounds for potential new therapeutics. Our time-resolved fluorescence resonance energy transfer biosensors provide us an exquisitely sensitive technique to monitor minute structural changes in monomer and oligomer conformation. In this proof-ofconcept study, we identified a novel tool compound, MK-886, which directly binds tau, perturbs the conformation of toxic tau oligomers, and rescues tau-induced cytotoxicity. Furthermore, we show that MK-886 alters the conformation of tau monomer at the proline-rich and microtubule binding regions, stabilizing an on-pathway oligomer. Major Challenges for the Hypothesis: Our approach monitors changes in the ensemble of assemblies that are spontaneously formed in cells but does not specifically isolate or enrich unique toxic tau species. However, time-resolved fluorescence resonance energy transfer does not provide highresolution, atomic scale information, requiring additional experimental techniques to resolve the structural features stabilized by different tool compounds. David D. Thomas holds equity in and serves as an executive officer for Photonic Pharma LLC, a company that owns intellectual property related to technology used in part of this project. These relationships have been reviewed and managed by the University of Minnesota in accordance with its conflict-of-interest polices.

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High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA

Cited by 17 publications

References 0 publications

Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor

Potent inhibitors of toxic alpha-synuclein oligomers identified via cellular time-resolved FRET biosensor

Conformational states of TNFR1 as a molecular switch for receptor function

Targeting the ensemble of heterogeneous tau oligomers in cells: A novel small molecule screening platform for tauopathies

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