scholarly journals A Highly Specific Cell-Based High-Throughput Screening Assay for Ligands of Cyclic Adenosine Monophosphate Receptor Protein in Gram-Negative Bacteria

2013 ◽  
Vol 11 (6) ◽  
pp. 382-387 ◽  
Author(s):  
Hongxia Wang ◽  
Anisia J. Silva ◽  
Lynn Rasmussen ◽  
E. Lucile White ◽  
Jorge A. Benitez
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Receptor Protein ◽  
Specific Cell ◽  
Gram Negative Bacteria ◽  
Screening Assay ◽  
Gram Negative ◽  
High Throughput Screening Assay

scholarly journals High-Throughput Screening Assay for Inhibitors of TonB-Dependent Iron Transport

2015 ◽  
Vol 21 (3) ◽  
pp. 316-322 ◽  
Author(s):  
Mathew Hanson ◽  
Lorne D. Jordan ◽  
Yan Shipelskiy ◽  
Salete M. Newton ◽  
Phillip E. Klebba
Keyword(s):  
Outer Membrane ◽  
High Throughput ◽  
High Throughput Screening ◽  
Iron Transport ◽  
Iron Acquisition ◽  
Metabolic Inhibitors ◽  
Screening Assay ◽  
Gram Negative ◽  
Microtiter Plates ◽  
High Throughput Screening Assay

The TonB-dependent Gram-negative bacterial outer membrane protein FepA actively transports the siderophore ferric enterobactin (FeEnt) into the periplasm. We developed a high-throughput screening (HTS) assay that observes FeEnt uptake through FepA in living Escherichia coli, by monitoring fluorescence quenching that occurs upon binding of FeEnt, and then unquenching as the bacteria deplete it from solution by transport. We optimized the labeling and spectroscopic methods to screen for inhibitors of TonB-dependent iron uptake through the outer membrane. The assay works like a molecular switch that is on in the presence of TonB activity and off in its absence. It functions in 96-well microtiter plates, in a variety of conditions, with Z factors of 0.8–1.0. TonB-dependent iron transport is energy dependent, and the inhibitory effects of the metabolic inhibitors carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, azide, cyanide, and arsenate on FeEnt uptake were readily detected by the assay. Because iron acquisition is a determinant of bacterial pathogenesis, HTS with this method may identify inhibitors that block TonB function and constitute novel therapeutics against infectious disease caused by Gram-negative bacteria.

scholarly journals Optimization of a Luminescence-Based High-Throughput Screening Assay for Detecting Apyrase Activity

2016 ◽  
Vol 22 (1) ◽  
pp. 94-101 ◽  
Author(s):  
John R. Veloria ◽  
Ashwini K. Devkota ◽  
Eun Jeong Cho ◽  
Kevin N. Dalby
Keyword(s):  
High Throughput ◽  
Herbicide Resistance ◽  
High Throughput Screening ◽  
Detection System ◽  
Cost Effective ◽  
Adenosine Monophosphate ◽  
Adenosine Diphosphate ◽  
Screening Assay ◽  
High Throughput Screening Assay ◽  
Automated Screening

Apyrase is a calcium-activated enzyme that catalyzes the conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP), adenosine monophosphate (AMP), and Pi. It is currently used in studies involving cancer and platelet aggregation in humans, as well as herbicide resistance in plants. Inhibitors of apyrase are being investigated for their use to suppress tumors and combat herbicide resistance. Only a few inhibitors of apyrase have been reported, many of which were identified through automated screening using a 96-well plate format and colorimetric phosphate detection. However, these screens have had limitations, including large volumes, inconsistent reproducibility, high incidence of false hits, and lack of higher-throughput compatibility. A luciferin/luciferase-based detection system has been reported to examine potential inhibitors of apyrase; however, these reactions were performed in tubes with the assay completion in seconds, which necessitate the development of a high-throughput screening (HTS)–compatible format for screening. Therefore, a more cost-effective biochemical assay that improved the limitations of the previous assay formats using a commercially available luminescence-based detection system was developed. This new robust mix-and-read platform incorporates a low-volume luminescence-based protocol, formatted for use in 384-well microplates. This new format provides a simple and cost-effective method to screen for apyrase inhibitors and will facilitate larger HTS efforts to identify potent inhibitors of apyrase.

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 A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

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