scholarly journals A robust bacterial assay for high-throughput screening of human 4-hydroxyphenylpyruvate dioxygenase inhibitors

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Jessie Neuckermans ◽  
Alan Mertens ◽  
Dinja De Win ◽  
Ulrich Schwaneberg ◽  
Joery De Kock
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Metabolic Disorders ◽  
Cost Effective ◽  
Dependent Manner ◽  
Screening System ◽  
Screening Assay ◽  
E Coli ◽  
High Throughput Screening Assay ◽  
Hereditary Tyrosinemia

Abstract Hereditary tyrosinemia type 1 (HT1) and alkaptonuria (AKU) are inherited metabolic disorders caused by defective enzymes involved in tyrosine catabolism. Nitisinone, an ex-herbicide and member of the β-triketone family, is therapeutically applied to prevent accumulation of toxic metabolites in patients by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPD). Here, we developed a colorimetric bacterial whole-cell screening system that allows quantifying the inhibitory effects of human HPD inhibitors in a high-throughput and a robust fashion. The principle of our screening system is based on the degradation of tyrosine through 4-hydroxyphenylpyruvate into homogentisate by human HPD expressed in E. coli and subsequent production of a soluble melanin-like pigment. With the aim to optimise the assay, we tested different E. coli strains, expression and reaction temperatures, and time-points for supplementing the substrate. We found that in our assay the addition of prototypical β-triketone HPD inhibitors decreases pigment production in a dose-dependent manner with increasing inhibitor concentrations. In addition, plate uniformity, signal variability and spatial uniformity assessment showed that we have developed a robust high-throughput screening assay that is simple to use, cost-effective and enables identification and evaluation of novel therapeutic human HPD inhibitors for the treatment of tyrosine-related metabolic disorders.

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.

scholarly journals Development of a High-Throughput Screening Assay for Inhibitors of Small Ubiquitin-Like Modifier Proteases

2013 ◽  
Vol 18 (5) ◽  
pp. 621-628 ◽  
Author(s):  
Wei Yang ◽  
Liangli Wang ◽  
Wulf Paschen
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Dependent Manner ◽  
Screening Assay ◽  
Sumo Protease ◽  
Sumo Conjugation ◽  
Sumo Proteases ◽  
High Throughput Screening Assay ◽  
Lysine Residues ◽  
Protease Substrate

Small ubiquitin-like modifier (SUMO1–3) is a small group of proteins that are ligated to lysine residues in target proteins. SUMO conjugation is a highly dynamic process, as SUMOylated proteins are rapidly deconjugated by SUMO proteases. SUMO conjugation/deconjugation plays pivotal roles in major cellular pathways and is associated with a number of pathological conditions. It is therefore of significant clinical interest to develop new strategies to screen for compounds to specifically interfere with SUMO conjugation/deconjugation. Here, we describe a novel high-throughput screening (HTS)–compatible assay to identify inhibitors of SUMO proteases. The assay is based on AlphaScreen technology and uses His-tagged SUMO2 conjugated to Strep-tagged SUMO3 as a SUMO protease substrate. A bacterial SUMOylation system was used to generate this substrate. A three-step purification strategy was employed to yield substrate of high quality. Our data indicated that this unique substrate can be readily detected in the AlphaScreen assays in a dose-dependent manner. Cleavage reactions by SUMO protease with or without inhibitor were monitored based on AlphaScreen signals. Furthermore, the assay was adapted to a 384-well format, and the interplate and interday variability was evaluated in eight 384-well plates. The average Z′ factor was 0.83 ± 0.04, confirming the suitability for HTS applications.

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.

Sign in / Sign up

Export Citation Format

Share Document