scholarly journals Label-Free High-Throughput Screening Assay for the Identification of Norepinephrine Transporter (NET / SLC6A2) Inhibitors

2021 ◽  
Author(s):  
Hubert Sijben ◽  
Wieke van Oostveen ◽  
Peter Hartog ◽  
Laura Stucchi ◽  
Giovanna Maresca ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Norepinephrine Transporter ◽  
Functional Assay ◽  
Substrate Uptake ◽  
Label Free ◽  
Screening Assay ◽  
Fluorescent Substrate ◽  
High Throughput Screening Assay ◽  
Wide Range

Abstract The human norepinephrine transporter (NET) is an established drug target for a wide range of neurological disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET inducibly expressed in a modified HEK293-JumpIn cell line. Three endogenous substrates of NET – norepinephrine (NE), dopamine (DA) and epinephrine (EP) – were each assessed in characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z’ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

scholarly journals Label-free high-throughput screening assay for the identification of norepinephrine transporter (NET/SLC6A2) inhibitors

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hubert J. Sijben ◽  
Wieke M. van Oostveen ◽  
Peter B. R. Hartog ◽  
Laura Stucchi ◽  
Andrea Rossignoli ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Norepinephrine Transporter ◽  
Functional Assay ◽  
Substrate Uptake ◽  
Label Free ◽  
Screening Assay ◽  
Fluorescent Substrate ◽  
High Throughput Screening Assay ◽  
Wide Range

AbstractThe human norepinephrine transporter (NET) is an established drug target for a wide range of psychiatric disorders. Conventional methods that are used to functionally characterize NET inhibitors are based on the use of radiolabeled or fluorescent substrates. These methods are highly informative, but pose limitations to either high-throughput screening (HTS) adaptation or physiologically accurate representation of the endogenous uptake events. Recently, we developed a label-free functional assay based on the activation of G protein-coupled receptors by a transported substrate, termed the TRACT assay. In this study, the TRACT assay technology was applied to NET expressed in a doxycycline-inducible HEK 293 JumpIn cell line. Three endogenous substrates of NET—norepinephrine (NE), dopamine (DA) and epinephrine (EP)—were compared in the characterization of the reference NET inhibitor nisoxetine. The resulting assay, using NE as a substrate, was validated in a manual HTS set-up with a Z′ = 0.55. The inhibitory potencies of several reported NET inhibitors from the TRACT assay showed positive correlation with those from an established fluorescent substrate uptake assay. These findings demonstrate the suitability of the TRACT assay for HTS characterization and screening of NET inhibitors and provide a basis for investigation of other solute carrier transporters with label-free biosensors.

scholarly journals Feasibility of Implementing Cell-Based Pathway Reporter Assays in Early High-Throughput Screening Assay Cascades for Antibody Drug Discovery

2012 ◽  
Vol 17 (6) ◽  
pp. 713-726 ◽  
Author(s):  
Alison J. Smith ◽  
Michael K. Hancock ◽  
Kun Bi ◽  
John Andrews ◽  
Paula Harrison ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Sample Type ◽  
Screening Assay ◽  
Assay Sensitivity ◽  
Specific Effects ◽  
High Throughput Screening Assay ◽  
Wide Range ◽  
Reporter Assays ◽  
Antibody Preparations

Implementing functional cell-based screens in early antibody discovery has become increasingly important to select antibodies with the desired profile. However, this is limited by assay tolerance to crude antibody preparations and assay sensitivity. The current study aims to address this challenge and identify routes forward. Two common types of high-throughput screening (HTS) antibody sample, derived from either phage display or hybridoma techniques, have been screened across a wide range of CellSensor beta-lactamase reporter assays in a variety of cell backgrounds to more extensively characterize assay tolerance. Pathway-, sample-, and cell background–specific effects were observed. Reporter assays for agonism were less affected by crude antibody preparations, with 8 of 21 sample tolerant, and the potential to implement an additional 8 assays by choosing the best-tolerated sample type. Antagonist mode assays exhibited more complexity, with potentiating as well as inhibitory effects. However, 5 of 24 antagonist assays were fully tolerant, with the potential to implement an additional 11 assays. Different subsets of assays were affected in agonist versus antagonist mode, and hybridoma sample sets were better tolerated overall. The study clearly demonstrates the potential to use cell-based reporter assays in biologics HTS, particularly if the method of antibody production is considered in the context of the required assay mode (agonist/antagonist).

scholarly journals High-Throughput Screening for Norepinephrine Transporter Inhibitors Using the FLIPRTetra

2007 ◽  
Vol 12 (3) ◽  
pp. 436-441 ◽  
Author(s):  
Ryan Wagstaff ◽  
Michael Hedrick ◽  
Jun Fan ◽  
Paul D. Crowe ◽  
Daniel DiSepio
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Radioligand Binding ◽  
Norepinephrine Transporter ◽  
Functional Screening ◽  
Monoamine Transporters ◽  
Binding Assays ◽  
Screening Assay ◽  
Fluorescent Substrate

Monoamine transporters regulate the concentration of neurotransmitters in the synapse following neurotransmission and are very important drug targets in the pharmaceutical industry. Because of the labor-intensive nature of functional uptake assays using radioactive substrates, high-throughput screening for monoamine transporter inhibitors has been limited to radioligand binding assays. In this article, the authors describe the development of a 384-well, high-throughput functional screening assay for norepinephrine transporter inhibitors using the FLIPRTetra and a recently identified fluorescent substrate, 4-(4-dimethylaminostyryl)- N-methyl-pyridinium (ASP+). ( Journal of Biomolecular Screening 2007:436-441)

scholarly journals A High-Throughput Screening Assay for the Identification of Flavivirus NS5 Capping Enzyme GTP-Binding Inhibitors

2011 ◽  
Vol 16 (8) ◽  
pp. 852-861 ◽  
Author(s):  
Brian J. Geiss ◽  
Hillary J. Stahla-Beek ◽  
Amanda M. Hannah ◽  
Hamid H. Gari ◽  
Brittney R. Henderson ◽  
...  
Keyword(s):  
Drug Development ◽  
High Throughput ◽  
High Throughput Screening ◽  
Antiviral Drug ◽  
Functional Assay ◽  
Screening Assay ◽  
Gtp Binding ◽  
High Throughput Screening Assay ◽  
Capping Enzyme

There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5′ end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE–guanosine triphosphate (GTP) and CE–RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE–GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE–GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development.

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.

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