scholarly journals A fluorescence polarization-based screening assay for nucleic acid polymerase elongation activity

2007 ◽  
Vol 365 (2) ◽  
pp. 194-200 ◽  
Author(s):  
Santano P. Mestas ◽  
Aaron J. Sholders ◽  
Olve B. Peersen
Keyword(s):  
Nucleic Acid ◽  
Fluorescence Polarization ◽  
Screening Assay ◽  
Nucleic Acid Polymerase ◽  
Elongation Activity

scholarly journals Application of QuantiGene™ Nucleic Acid Quantification Technology for High Throughput Screening

2000 ◽  
Vol 5 (5) ◽  
pp. 343-351 ◽  
Author(s):  
Usha Warrior ◽  
Yihong Fan ◽  
Caroline A. David ◽  
Julie A. Wilkins ◽  
Evelyn M. McKeegan ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
High Throughput ◽  
High Throughput Screening ◽  
Molecular Probes ◽  
Epithelial Cell Line ◽  
Reporter System ◽  
Screening Assay ◽  
Human Lung Epithelial Cell ◽  
High Throughput Screening Assay ◽  
Time Required

To identify inhibitors of interleukin-8 (IL-8) production, a high throughput assay was developed using the Quanti-Gene™ nucleic acid quantification kit that employs branched-chain DNA (bDNA) technology to measure the mRNA directly from cells. Unlike polymerase chain reaction and other technologies that employ target amplification, the QuantiGene system uses signal amplification. To perform the assay, various molecular probes capable of hybridizing with IL-8 mRNA were designed and synthesized. A human lung epithelial cell line was treated with interleukin-la (IL-la) to stimulate the IL-8 gene expression and the mRNA was measured using the QuantiGene system. The QuantiGene assay was sensitive, flexible, and reproducible and achieved equivalent or better sensitivity than promoter-reporter assays, and eliminated the time required for constructing a promoter-reporter system. Our data show that bDNA technology has the potential to be used as a high throughput screening assay.

scholarly journals Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Gangan Yan ◽  
Dongsheng Li ◽  
Yuan Lin ◽  
Zhenghao Fu ◽  
Haiyan Qi ◽  
...  
Keyword(s):  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Antiviral Agents ◽  
Competitive Inhibitor ◽  
Rapid Screening ◽  
Screening Assay ◽  
Hydrogen Bond Interactions ◽  
Main Protease ◽  
Fluorescence Polarization Assay

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. Results In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. Conclusions This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

scholarly journals Optimization of Fluorescently Labeled Nrf2 Peptide Probes and the Development of a Fluorescence Polarization Assay for the Discovery of Inhibitors of Keap1-Nrf2 Interaction

2011 ◽  
Vol 17 (4) ◽  
pp. 435-447 ◽  
Author(s):  
Daigo Inoyama ◽  
Yu Chen ◽  
Xinyi Huang ◽  
Lesa J. Beamer ◽  
Ah-Ng Tony Kong ◽  
...  
Keyword(s):  
Small Molecule ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Dynamic Range ◽  
Antioxidant Response ◽  
Binding Affinities ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Peptide Amide

Activation of the antioxidant response element (ARE) upregulates enzymes involved in detoxification of electrophiles and reactive oxygen species. The induction of ARE genes is regulated by the interaction between redox sensor protein Keap1 and the transcription factor Nrf2. Fluorescently labeled Nrf2 peptides containing the ETGE motif were synthesized and optimized as tracers in the development of a fluorescence polarization (FP) assay to identify small-molecule inhibitors of the Keap1-Nrf2 interaction. The tracers were optimized to increase the dynamic range of the assay and their binding affinities to the Keap1 Kelch domain. The binding affinities of Nrf2 peptide inhibitors obtained in our FP assay using FITC-9mer Nrf2 peptide amide as the probe were in good agreement with those obtained previously by a surface plasmon resonance assay. The FP assay exhibits considerable tolerance toward DMSO and produced a Z′ factor greater than 0.6 in a 384-well format. Further optimization of the probe led to cyanine-labeled 9mer Nrf2 peptide amide, which can be used along with the FITC-9mer Nrf2 peptide amide in a high-throughput screening assay to discover small-molecule inhibitors of Keap1-Nrf2 interaction.

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