A Novel High-Throughput Screening Assay for HCN Channel Blocker Using Membrane Potential-Sensitive Dye and FLIPR

2009 ◽  
Vol 14 (9) ◽  
pp. 1119-1128 ◽  
Author(s):  
Dmitry V. Vasilyev ◽  
Qin J. Shan ◽  
Yan T. Lee ◽  
Veronica Soloveva ◽  
Stanley P. Nawoschik ◽  
...  
Keyword(s):  
Membrane Potential ◽  
High Throughput ◽  
High Throughput Screening ◽  
Channel Blocker ◽  
Fluorescent Imaging ◽  
Fluorescence Signal ◽  
Hcn Channel ◽  
Plating Density ◽  
High Throughput Screening Assay ◽  
Cell Plating

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the wellto-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 ± 1500 FLIPR3 relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 µM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r2 = 0.56) between the respective IC50s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds. (Journal of Biomolecular Screening 2009:1119-1128)

scholarly journals Development of a High Throughput Screening Assay for Mitochondrial Membrane Potential in Living Cells

2002 ◽  
Vol 7 (4) ◽  
pp. 383-389 ◽  
Author(s):  
Shu-Gui Huang
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mitochondrial Membrane ◽  
Living Cells ◽  
Screening Assay ◽  
Mitochondrial Inner Membrane ◽  
Obesity And Diabetes ◽  
High Throughput Screening Assay

The mitochondrion plays a pivotal role in energy metabolism in eukaryotic cells. The electrochemical potential across the mitochondrial inner membrane is regulated to cope with cellular energy needs and thus reflects the bioenergetic state of the cell. Traditional assays for mitochondrial membrane potential are not amenable to high-throughput drug screening. In this paper, I describe a high-throughput assay that measures the mitochondrial membrane potential of living cells in 96- or 384-well plates. Cells were first treated with test compounds and then with a fluorescent potentiometric probe, the cationic-lipophilic dye tetramethylrhodamine methyl ester (TMRM). The cells were then washed to remove free compounds and probe. The amount of TMRM retained in the mitochondria, which is proportional to the mitochondrial membrane potential, was measured on an LJL Analyst fluorescence reader. Under optimal conditions, the assay measured only the mitochondrial membrane potential. The chemical uncouplers carbonylcyanide m-chlorophenyl hydrazone and dinitrophenol decreased fluorescence intensity, with IC50 values (concentration at 50% inhibition) similar to those reported in the literature. A Z' factor of greater than 0.5 suggests that this cell-based assay can be adapted for high-throughput screening of chemical libraries. This assay may be used in screens for drugs to treat metabolic disorders such as obesity and diabetes, as well as cancer and neurodegenerative diseases.

scholarly journals Validation of a Fluorescent Imaging Plate Reader Membrane Potential Assay for High-Throughput Screening of Glycine Transporter Modulators

2005 ◽  
Vol 10 (4) ◽  
pp. 365-373 ◽  
Author(s):  
Elfrida R. Benjamin ◽  
Joanne Skelton ◽  
Denise Hanway ◽  
Shakira Olanrewaju ◽  
Farhana Pruthi ◽  
...  
Keyword(s):  
Membrane Potential ◽  
High Throughput ◽  
High Throughput Screening ◽  
Ionic Composition ◽  
Fluorescent Imaging ◽  
Imaging Plate ◽  
Pharmacological Characterization ◽  
Glycine Transporter ◽  
Plate Reader ◽  
Small Molecule Modulators

A fluorescent imaging plate reader (FLIPR) membrane potential (Vm) assay was evaluated for pharmacological characterization and high-throughput screening (HTS) of rat glycine transporter type 2 (rGlyT2) in a stable rGlyT2-HEK cell line. Data show that glycine activation of rGlyT2 consistently results in a concentration-dependent Vm response on the FLIPR that is blocked by the potent and selective GlyT2 antagonist 4-benzyloxy-3,5-dimethoxy-N-[1-dimethylamino-cyclopentyl)methyl]-benz-amide (Org-25543). Agonist and antagonist pharmacologies match those reported using conventional [3H]glycine uptake assays and electrophysiology. The glycine response is dependent on buffer ionic composition consistent with GlyT2 physiology. Assay signal-to-background and coefficient of variation meets sufficient statistical criteria to conduct HTS. The results of a screen of the chemical inventory demonstrate that the assay is able to successfully identify and confirm GlyT2 inhibitors. The advantages of this assay are its homogeneity, compatibility with both 96- and 384-well formats, and lack of radioactivity usage. Thus, the authors conclude that a fluorescence-based Vm assay on FLIPR is a viable approach for identification and pharmacological profiling of small molecule modulators of the electrogenic transporter rGlyT2.

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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