scholarly journals Development of a High-Throughput Fluorescence Polarization DNA Cleavage Assay for the Identification of FEN1 Inhibitors

2013 ◽  
Vol 18 (5) ◽  
pp. 567-575 ◽  
Author(s):  
Claire McWhirter ◽  
Michael Tonge ◽  
Helen Plant ◽  
Ian Hardern ◽  
Willem Nissink ◽  
...  
Keyword(s):  
High Throughput ◽  
Dna Cleavage ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Screening Assay ◽  
Flap Endonuclease 1 ◽  
High Throughput Screening Assay ◽  
Cleavage Assay ◽  
Compound Collection ◽  
Dna Substrate

Flap endonuclease-1 (FEN1) is a highly conserved metallonuclease and is the main human flap endonuclease involved in the recognition and cleavage of single-stranded 5′ overhangs from DNA flap structures. The involvement of FEN1 in multiple DNA metabolism pathways and the identification of FEN1 overexpression in a variety of cancers has led to interest in FEN1 as an oncology target. In this article, we describe the development of a 1536-well high-throughput screening assay based on the change in fluorescence polarization of a FEN1 DNA substrate labeled with Atto495 dye. The assay was subsequently used to screen 850 000 compounds from the AstraZeneca compound collection, with a Z′ factor of 0.66 ± 0.06. Hits were followed up by IC50 determination in both a concentration-response assay and a technology artifact assay.

scholarly journals Development of a High-Throughput Fluorescence Polarization Assay to Detect Inhibitors of the FAK–Paxillin Interaction

2019 ◽  
Vol 25 (1) ◽  
pp. 21-32 ◽  
Author(s):  
Timothy Marlowe ◽  
Carlos Alvarado ◽  
Andrew Rivera ◽  
Felicia Lenzo ◽  
Rohini Nott ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Focal Adhesion ◽  
High Throughput ◽  
Fluorescence Polarization ◽  
High Throughput Screening ◽  
Drug Target ◽  
Scaffolding Protein ◽  
Cancer Drug ◽  
Screening Assay ◽  
High Throughput Screening Assay

Focal adhesion kinase (FAK) is a promising cancer drug target due to its massive overexpression in multiple solid tumors and its critical role in the integration of signals that control proliferation, invasion, apoptosis, and metastasis. Previous FAK drug discovery and high-throughput screening have exclusively focused on the identification of inhibitors that target the kinase domain of FAK. Because FAK is both a kinase and scaffolding protein, the development of novel screening assays that detect inhibitors of FAK protein–protein interactions remains a critical need. In this report, we describe the development of a high-throughput fluorescence polarization (FP) screening assay that measures the interactions between FAK and paxillin, a focal adhesion–associated protein. We designed a tetramethylrhodamine (TAMRA)-tagged paxillin peptide based on the paxillin LD2 motif that binds to the focal adhesion targeting (FAT) domain with significant dynamic range, specificity, variability, stability, and a Z’-factor suitable for high-throughput screening. In addition, we performed a pilot screen of 1593 compounds using this FP assay, showing its feasibility for high-throughput drug screening. Finally, we identified three compounds that show dose-dependent competition of FAT–paxillin binding. This assay represents the first described high-throughput screening assay for FAK scaffold inhibitors and can accelerate drug discovery efforts for this promising drug target.

High Throughput Screening Assay to Identify Modulators of IL-17 Expression

2018 ◽  
Vol 20 (9) ◽  
pp. 804-819 ◽  
Author(s):  
Mohamed Boudjelal ◽  
Ana Maria Ruiz-Avendano ◽  
Gonzalo Colmenarejo ◽  
Sergio A. Senar-Sancho ◽  
Ashley Barnes ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals A high-throughput screening assay based on automated microscopy for monitoring antibiotic susceptibility of Mycobacterium tuberculosis phenotypes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Sadaf Kalsum ◽  
Blanka Andersson ◽  
Jyotirmoy Das ◽  
Thomas Schön ◽  
Maria Lerm
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Imaging System ◽  
Aggregate Size ◽  
Live Cell ◽  
Screening Assay ◽  
High Throughput Screening Assay

Abstract Background Efficient high-throughput drug screening assays are necessary to enable the discovery of new anti-mycobacterial drugs. The purpose of our work was to develop and validate an assay based on live-cell imaging which can monitor the growth of two distinct phenotypes of Mycobacterium tuberculosis and to test their susceptibility to commonly used TB drugs. Results Both planktonic and cording phenotypes were successfully monitored as fluorescent objects using the live-cell imaging system IncuCyte S3, allowing collection of data describing distinct characteristics of aggregate size and growth. The quantification of changes in total area of aggregates was used to define IC50 and MIC values of selected TB drugs which revealed that the cording phenotype grew more rapidly and displayed a higher susceptibility to rifampicin. In checkerboard approach, testing pair-wise combinations of sub-inhibitory concentrations of drugs, rifampicin, linezolid and pretomanid demonstrated superior growth inhibition of cording phenotype. Conclusions Our results emphasize the efficiency of using automated live-cell imaging and its potential in high-throughput whole-cell screening to evaluate existing and search for novel antimycobacterial drugs.

scholarly journals Development of a High-Throughput Screening Assay to Identify Inhibitors of the SARS-CoV-2 Guanine-N7-Methyltransferase Using RapidFire Mass Spectrometry

2021 ◽  
pp. 247255522110006
Author(s):  
Lesley-Anne Pearson ◽  
Charlotte J. Green ◽  
De Lin ◽  
Alain-Pierre Petit ◽  
David W. Gray ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Mutational Analysis ◽  
Nonstructural Protein ◽  
Translation Efficiency ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Starting Point ◽  
Approved Drugs ◽  
High Throughput Assay

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) represents a significant threat to human health. Despite its similarity to related coronaviruses, there are currently no specific treatments for COVID-19 infection, and therefore there is an urgent need to develop therapies for this and future coronavirus outbreaks. Formation of the cap at the 5′ end of viral RNA has been shown to help coronaviruses evade host defenses. Nonstructural protein 14 (nsp14) is responsible for N7-methylation of the cap guanosine in coronaviruses. This enzyme is highly conserved among coronaviruses and is a bifunctional protein with both N7-methyltransferase and 3′-5′ exonuclease activities that distinguish nsp14 from its human equivalent. Mutational analysis of SARS-CoV nsp14 highlighted its role in viral replication and translation efficiency of the viral genome. In this paper, we describe the characterization and development of a high-throughput assay for nsp14 utilizing RapidFire technology. The assay has been used to screen a library of 1771 Food and Drug Administration (FDA)-approved drugs. From this, we have validated nitazoxanide as a selective inhibitor of the methyltransferase activity of nsp14. Although modestly active, this compound could serve as a starting point for further optimization.

scholarly journals Stress-Based High-Throughput Screening Assays to Identify Inhibitors of Cell Envelope Biogenesis

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 808
Author(s):  
Maurice Steenhuis ◽  
Corinne M. ten Hagen-Jongman ◽  
Peter van Ulsen ◽  
Joen Luirink
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
Stress Responses ◽  
High Throughput Screening ◽  
Structural Integrity ◽  
Cell Envelope ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Compound Screening ◽  
Outer Membrane Biogenesis

The structural integrity of the Gram-negative cell envelope is guarded by several stress responses, such as the σE, Cpx and Rcs systems. Here, we report on assays that monitor these responses in E. coli upon addition of antibacterial compounds. Interestingly, compromised peptidoglycan synthesis, outer membrane biogenesis and LPS integrity predominantly activated the Rcs response, which we developed into a robust HTS (high-throughput screening) assay that is suited for phenotypic compound screening. Furthermore, by interrogating all three cell envelope stress reporters, and a reporter for the cytosolic heat-shock response as control, we found that inhibitors of specific envelope targets induce stress reporter profiles that are distinct in quality, amplitude and kinetics. Finally, we show that by using a host strain with a more permeable outer membrane, large-scaffold antibiotics can also be identified by the reporter assays. Together, the data suggest that stress profiling is a useful first filter for HTS aimed at inhibitors of cell envelope processes.

scholarly journals A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis

PLoS ONE ◽  
2015 ◽  
Vol 10 (6) ◽  
pp. e0129234 ◽  
Author(s):  
Lauren Forbes ◽  
Katherine Ebsworth-Mojica ◽  
Louis DiDone ◽  
Shao-Gang Li ◽  
Joel S. Freundlich ◽  
...  
Keyword(s):  
Small Molecules ◽  
High Throughput ◽  
Adenylate Kinase ◽  
High Throughput Screening ◽  
Cell Lysis ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Adenylate Kinase Release

scholarly journals Development of a Robust Cytopathic Effect-Based High-Throughput Screening Assay To Identify Novel Inhibitors of Dengue Virus

2012 ◽  
Vol 56 (6) ◽  
pp. 3399-3401 ◽  
Author(s):  
Kevin D. McCormick ◽  
Shufeng Liu ◽  
Jana L. Jacobs ◽  
Ernesto T. A. Marques ◽  
Nicolas Sluis-Cremer ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Natural Product ◽  
High Throughput ◽  
High Throughput Screening ◽  
Cytopathic Effect ◽  
Complex Molecule ◽  
Denv Infection ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Natural Product Library

ABSTRACTWe have developed a robust cytopathic effect-based high-throughput screening assay to identify inhibitors of dengue virus (DENV) infection. Screening of a small natural product library yielded 11 hits. Four of these were found to be potent inhibitors of DENV, although serotype differences were noted. Taken together, these data suggest that screening of larger and more complex molecule libraries may result in the identification of more potent and specific DENV inhibitors.

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