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

2016 ◽  
Vol 22 (3) ◽  
pp. 262-273 ◽  
Author(s):  
Tory M. Schaaf ◽  
Kurt C. Peterson ◽  
Benjamin D. Grant ◽  
Prachi Bawaskar ◽  
Samantha Yuen ◽  
...  
Keyword(s):  
High Precision ◽  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Resonance Energy ◽  
Living Cells ◽  
Spectral Unmixing ◽  
Detection Methods ◽  
Response Curves

A robust high-throughput screening (HTS) strategy has been developed to discover small-molecule effectors targeting the sarco/endoplasmic reticulum calcium ATPase (SERCA), based on a fluorescence microplate reader that records both the nanosecond decay waveform (lifetime mode) and the complete emission spectrum (spectral mode), with high precision and speed. This spectral unmixing plate reader (SUPR) was used to screen libraries of small molecules with a fluorescence resonance energy transfer (FRET) biosensor expressed in living cells. Ligand binding was detected by FRET associated with structural rearrangements of green fluorescent protein (GFP, donor) and red fluorescent protein (RFP, acceptor) fused to the cardiac-specific SERCA2a isoform. The results demonstrate accurate quantitation of FRET along with high precision of hit identification. Fluorescence lifetime analysis resolved SERCA’s distinct structural states, providing a method to classify small-molecule chemotypes on the basis of their structural effect on the target. The spectral analysis was also applied to flag interference by fluorescent compounds. FRET hits were further evaluated for functional effects on SERCA’s ATPase activity via both a coupled-enzyme assay and a FRET-based calcium sensor. Concentration-response curves indicated excellent correlation between FRET and function. These complementary spectral and lifetime FRET detection methods offer an attractive combination of precision, speed, and resolution for HTS.

scholarly journals Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

2016 ◽  
Vol 22 (3) ◽  
pp. 250-261 ◽  
Author(s):  
Tory M. Schaaf ◽  
Kurt C. Peterson ◽  
Benjamin D. Grant ◽  
David D. Thomas ◽  
Gregory D. Gillispie
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Structural Changes ◽  
Fluorescent Protein ◽  
Fluorescence Emission ◽  
Resonance Energy ◽  
Living Cells ◽  
Spectral Unmixing ◽  
Fluorescence Emission Spectrum ◽  
Plate Reader

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

scholarly journals Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening

Biosensors ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 99 ◽  
Author(s):  
Tory Schaaf ◽  
Ang Li ◽  
Benjamin Grant ◽  
Kurt Peterson ◽  
Samantha Yuen ◽  
...  
Keyword(s):  
High Precision ◽  
Small Molecule ◽  
High Throughput ◽  
Fluorescence Lifetime ◽  
High Throughput Screening ◽  
Structural Changes ◽  
Dynamic Range ◽  
Resonance Energy ◽  
Fluorescence Measurements ◽  
Fret Biosensors

We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.

scholarly journals A High-Throughput Screening Strategy to Identify Inhibitors of SSB Protein–Protein Interactions in an Academic Screening Facility

2017 ◽  
Vol 23 (1) ◽  
pp. 94-101 ◽  
Author(s):  
Andrew F. Voter ◽  
Michael P. Killoran ◽  
Gene E. Ananiev ◽  
Scott A. Wildman ◽  
F. Michael Hoffmann ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Small Molecule Inhibitors ◽  
Klebsiella Pneumonia ◽  
Protein Protein Interactions ◽  
Response Curves ◽  
Single Stranded Dna

Antibiotic-resistant bacterial infections are increasingly prevalent worldwide, and there is an urgent need for novel classes of antibiotics capable of overcoming existing resistance mechanisms. One potential antibiotic target is the bacterial single-stranded DNA binding protein (SSB), which serves as a hub for DNA repair, recombination, and replication. Eight highly conserved residues at the C-terminus of SSB use direct protein–protein interactions (PPIs) to recruit more than a dozen important genome maintenance proteins to single-stranded DNA. Mutations that disrupt PPIs with the C-terminal tail of SSB are lethal, suggesting that small-molecule inhibitors of these critical SSB PPIs could be effective antibacterial agents. As a first step toward implementing this strategy, we have developed orthogonal high-throughput screening assays to identify small-molecule inhibitors of the Klebsiella pneumonia SSB-PriA interaction. Hits were identified from an initial screen of 72,474 compounds using an AlphaScreen (AS) primary screen, and their activity was subsequently confirmed in an orthogonal fluorescence polarization (FP) assay. As an additional control, an FP assay targeted against an unrelated eukaryotic PPI was used to confirm specificity for the SSB-PriA interaction. Nine potent and selective inhibitors produced concentration–response curves with IC50 values of <40 μM, and two compounds were observed to directly bind to PriA, demonstrating the success of this screen strategy.

Discovery of Small Molecule Inhibitors of Huntingtin Exon 1 Aggregation by FRET-Based High-Throughput Screening in Living Cells

2020 ◽  
Vol 11 (15) ◽  
pp. 2286-2295 ◽  
Author(s):  
Chih Hung Lo ◽  
Nitin K. Pandey ◽  
Colin Kin-Wye Lim ◽  
Zhipeng Ding ◽  
Meixin Tao ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Living Cells ◽  
Exon 1

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

Time-Resolved Luminescent High-Throughput Screening Platform for Lysosomotropic Compounds in Living Cells

ACS Sensors ◽  
2020 ◽  
Author(s):  
Ke-Jia Wu ◽  
Chun Wu ◽  
Feng Chen ◽  
Sha-Sha Cheng ◽  
Dik-Lung Ma ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Living Cells ◽  
Time Resolved ◽  
Screening Platform

High-Throughput Affinity Selection Mass Spectrometry Using SAMDI-MS to Identify Small-Molecule Binders of the Human Rhinovirus 3C Protease

2021 ◽  
pp. 247255522110232
Author(s):  
Michael D. Scholle ◽  
Doug McLaughlin ◽  
Zachary A. Gurard-Levin
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Human Rhinovirus ◽  
Binding Potential ◽  
Affinity Selection ◽  
Response Curves ◽  
Desorption Ionization ◽  
Self Assembled

Affinity selection mass spectrometry (ASMS) has emerged as a powerful high-throughput screening tool used in drug discovery to identify novel ligands against therapeutic targets. This report describes the first high-throughput screen using a novel self-assembled monolayer desorption ionization (SAMDI)–ASMS methodology to reveal ligands for the human rhinovirus 3C (HRV3C) protease. The approach combines self-assembled monolayers of alkanethiolates on gold with matrix-assisted laser desorption ionization time-of-flight (MALDI TOF) mass spectrometry (MS), a technique termed SAMDI-ASMS. The primary screen of more than 100,000 compounds in pools of 8 compounds per well was completed in less than 8 h, and informs on the binding potential and selectivity of each compound. Initial hits were confirmed in follow-up SAMDI-ASMS experiments in single-concentration and dose–response curves. The ligands identified by SAMDI-ASMS were further validated using differential scanning fluorimetry (DSF) and in functional protease assays against HRV3C and the related SARS-CoV-2 3CLpro enzyme. SAMDI-ASMS offers key benefits for drug discovery over traditional ASMS approaches, including the high-throughput workflow and readout, minimizing compound misbehavior by using smaller compound pools, and up to a 50-fold reduction in reagent consumption. The flexibility of this novel technology opens avenues for high-throughput ASMS assays of any target, thereby accelerating drug discovery for diverse diseases.

scholarly journals A small molecule UPR modulator for diabetes identified by high throughput screening

2021 ◽  
Author(s):  
Valeria Marrocco ◽  
Tuan Tran ◽  
Siying Zhu ◽  
Seung Hyuk Choi ◽  
Ana M. Gamo ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening
Sign in / Sign up

Export Citation Format

Share Document