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.; Bawaskar, Prachi; Yuen, Samantha L.; Li, Ji; Muretta, Joseph M.; Gillispie, Gregory D.; Thomas, David D.

doi:10.1177/1087057116680151

Cited by 58 publications

(110 citation statements)

References 33 publications

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“…The fluorescence lifetime measurement is prone to interference from fluorescent compounds, so a stringent fluorescent compound filter was used to flag 26 compounds as potential false-positives due to interference from compound fluorescence. 18 This filter did not change significantly the mean or standard deviation of the histogram (Figures 3A and 3B). FRET efficiency from all compounds that passed the fluorescent compound filter were averaged from four independent NCC screens.…”

Section: Resultsmentioning

confidence: 86%

“…Fluorescent compounds were flagged as potential false positives due to interference from compound fluorescence by the spectral recording method based on the assessment of the similarity index of each well from the pilot NCC screens. 18 T-test was performed on the data and the left and right 5% tails were classified as outliers and removed. After removal of fluorescent compounds, histogram of the average FRET distribution from all compounds in the screens was plotted and fitted to a Gaussian curve to obtain a mean and standard deviation (SD).…”

Section: Methodsmentioning

confidence: 99%

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An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors

et al. 2017

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Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor-receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

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Section: Resultsmentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors

et al. 2017

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“…This instrument enables high-throughput fluorescence lifetime detection at high precision by utilizing a unique direct waveform-recording technology (40). The performance of this FLTPR has been previously demonstrated with FRET-based HTS that targets sarcoplasmic reticulum Ca-ATPase (SERCA) and the ryanodine receptor (14,15).…”

Section: Fluorescence Data Acquisitionmentioning

confidence: 99%

“…Fluorescence lifetime measurements were carried out by a high-precision FLTPR (Fluorescence Innovations, Inc., Minneapolis, MN) (13)(14)(15). FM-labeled donor actin was excited with a 473-nm microchip laser (Bright Solutions, Cura Carpignano, Italy), and emission was filtered with 488-nm long pass and 517/20-nm band pass filters (Semrock, Rochester, NY) (13,18).…”

Section: Fluorescence Data Acquisitionmentioning

confidence: 99%

“…We hypothesize that this TR-FRET approach can be used as a tool to screen for compounds that rescue defects in actomyosin structure and function. Small-molecule modulators of actomyosin structural dynamics represent potential leads for future drug development, and this search is greatly facilitated by recent developments in high-throughput FRET-based screening methods, which measure the effects of compounds on the distance between donor and acceptor probes on interacting proteins (1,(12)(13)(14)(15).…”

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High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

Guhathakurta

Próchniewicz

Grant

et al. 2018

Journal of Biological Chemistry

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We have used a novel time-resolved FRET (TR-FRET) assay to detect small-molecule modulators of actin-myosin structure and function. Actin-myosin interactions play crucial roles in the generation of cellular force and movement. Numerous mutations and post-translational modifications of actin or myosin disrupt muscle function and cause life-threatening syndromes. Here, we used a FRET biosensor to identify modulators that bind to the actin-myosin interface and alter the structural dynamics of this complex. We attached a fluorescent donor to actin at Cys-374 and a nonfluorescent acceptor to a peptide containing the 12 N-terminal amino acids of the long isoform of skeletal muscle myosin's essential light chain. The binding site on actin of this acceptor-labeled peptide (ANT) overlaps with that of myosin, as indicated by () a similar distance observed in the actin-ANT complex as in the actin-myosin complex and () a significant decrease in actin-ANT FRET upon binding myosin. A high-throughput FRET screen of a small-molecule library (NCC, 727 compounds), using a unique fluorescence lifetime readout with unprecedented speed and precision, showed that FRET is significantly affected by 10 compounds in the micromolar range. Most of these "hits" alter actin-activated myosin ATPase and affect the microsecond dynamics of actin detected by transient phosphorescence anisotropy. We conclude that the actin-ANT TR-FRET assay enables detection of pharmacologically active compounds that affect actin structural dynamics and actomyosin function. This assay establishes feasibility for the discovery of allosteric modulators of the actin-myosin interaction, with the ultimate goal of developing therapies for muscle disorders.

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Recent advances in cellular biosensor technology to investigate tau oligomerization

2021

Bioengineering & Transla Med

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Tau is a microtubule binding protein which plays an important role in physiological functions but it is also involved in the pathogenesis of Alzheimer's disease (AD) and related tauopathies. While insoluble and β-sheet containing tau neurofibrillary tangles have been the histopathological hallmark of these diseases, recent studies suggest that soluble tau oligomers, which are formed prior to fibrils, are the primary toxic species. Substantial efforts have been made to generate tau oligomers using purified recombinant protein strategies to study oligomer conformations as well as their toxicity. However, no specific toxic tau species has been identified to date, potentially due to the lack of cellular environment. Hence, there is a need for cell-based models for direct monitoring of tau oligomerization and aggregation. This review will summarize the recent advances in the cellular biosensor technology, with a focus on fluorescence resonance energy transfer (FRET), bimolecular fluorescence complementation (BiFC) and split luciferase complementation (SLC) approaches, to monitor formation of tau oligomers and aggregates in living cells. We will discuss the applications of the cellular biosensors in examining the heterogeneous tau conformational ensembles and factors affecting tau self-assembly, as well as detecting cell-to-cell propagation of tau pathology. We will also compare the advantages and limitations of each type of tau biosensors, and highlight their translational applications in biomarker development and therapeutic discovery.

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

Cited by 58 publications

References 33 publications

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors

An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors

High-throughput screen, using time-resolved FRET, yields actin-binding compounds that modulate actin–myosin structure and function

Recent advances in cellular biosensor technology to investigate tau oligomerization

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