High‐throughput screening assay for the quantification of Cer d18:1/16:0, d18:1/24:0, d18:1/24:1, d18:1/18:0, d18:1/14:0, d18:1/20:0, and d18:1/22:0 in HepG2 cells using RapidFire mass spectrometry

2020 ◽  
Vol 34 (5) ◽  
Author(s):  
Sreekanth Dittakavi ◽  
Lavanya Mahadevan ◽  
Devaraj V. Chandrashekar ◽  
Ravi Kanth Bhamidipati ◽  
Juluri Suresh ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Hepg2 Cells ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

Development of a high-throughput screening assay for stearoyl-CoA desaturase using rat liver microsomes, deuterium labeled stearoyl-CoA and mass spectrometry

2008 ◽  
Vol 627 (1) ◽  
pp. 105-111 ◽  
Author(s):  
Patricia Soulard ◽  
Meg McLaughlin ◽  
Jessica Stevens ◽  
Brendan Connolly ◽  
Rocco Coli ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rat Liver ◽  
High Throughput ◽  
High Throughput Screening ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Stearoyl Coa Desaturase

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

2020 ◽  
Vol 25 (9) ◽  
pp. 1064-1071
Author(s):  
Maikel Izquierdo ◽  
De Lin ◽  
Sandra O’Neill ◽  
Martin Zoltner ◽  
Lauren Webster ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Trypanosoma Cruzi ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Signal To Noise Ratio ◽  
Screening Method ◽  
Cellular Functions ◽  
Screening Assay ◽  
High Throughput Screening Assay

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin–based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)–based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

High-performance mass spectrometry as a drug discovery tool: a high-throughput screening assay to identify RNA-binding ligands

2001 ◽  
Author(s):  
Kristin A. Sannes-Lowery ◽  
Jared J. Drader ◽  
Richard H. Griffey ◽  
Steven A. Hofstadler
Keyword(s):  
Mass Spectrometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
High Performance ◽  
Rna Binding ◽  
Screening Assay ◽  
High Throughput Screening Assay

scholarly journals High-Throughput Screening Assay for Sphingosine Kinase Inhibitors in Whole Blood Using RapidFire® Mass Spectrometry

2011 ◽  
Vol 16 (2) ◽  
pp. 272-277 ◽  
Author(s):  
Maureen K. Highkin ◽  
Matthew P. Yates ◽  
Olga V. Nemirovskiy ◽  
William A. Lamarr ◽  
Grace E. Munie ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Whole Blood ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Mass Spectrometry Technology ◽  
Sphingosine 1 Phosphate ◽  
Mass Spectrometry System

To facilitate discovery of compounds modulating sphingosine-1-phosphate (S1P) signaling, the authors used high-throughput mass spectrometry technology to measure S1P formation in human whole blood. Since blood contains endogenous sphingosine (SPH) and S1P, mass spectrometry was chosen to detect the conversion of an exogenously added 17-carbon-long variant of sphingosine, C17SPH, into C17S1P. The authors developed procedures to achieve homogeneous mixing of whole blood in 384-well plates and for a method requiring minimal manipulations to extract S1P from blood in 96- and 384-well plates prior to analyses using the RapidFire® mass spectrometry system.

An efficient high-throughput screening assay against nuclear trans-port

2012 ◽  
Vol 34 (7) ◽  
pp. 927-934
Author(s):  
Min LUO ◽  
Quan-Cang ZHANG ◽  
Zhi-Gang LU
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Assay ◽  
High Throughput Screening Assay

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.

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