scholarly journals Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen

2021 ◽  
Author(s):  
Shufeng Liu ◽  
Chao-Kai Chou ◽  
Wells W Wu ◽  
Binquan Luan ◽  
Tony T Wang
Keyword(s):  
High Throughput ◽  
Structural Protein ◽  
Compound Libraries ◽  
Cell Clones ◽  
Replication Systems ◽  
Functional Analyses ◽  
High Throughput Screens ◽  
First Time ◽  
Large Compound ◽  
Level 2

The development of antivirals against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been hampered by the lack of efficient cell-based replication systems that are amenable to high-throughput screens in biosafety level 2 laboratories. Here we report that stable cell clones harboring autonomously replicating SARS-CoV-2 RNAs without S, M, E genes can be efficiently derived from the baby hamster kidney (BHK-21) cell line when a pair of mutations were introduced into the non-structural protein 1 (Nsp1) of SARS-CoV-2 to ameliorate cellular toxicity associated with virus replication. In a proof-of-concept experiment we screened a 273-compound library using replicon cells and identified three compounds as novel inhibitors of SARS-CoV-2 replication. Altogether, this work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. IMPORTANCE: SARS-CoV-2 replicon systems that have been reported up to date were unsuccessful in deriving stable cell lines harboring non-cytopathic replicons. The transient expression of viral sgmRNA or a reporter gene makes it impractical for industry-scale screening of large compound libraries using these systems. Here, for the first time, we derived stable cell clones harboring the SARS-CoV-2 replicon. These clones may now be conveniently cultured in a standard BSL-2 laboratory for high throughput screen of compound libraries. This achievement represents a ground-breaking discovery that will greatly accelerate the pace of developing treatments for COVID-19.

scholarly journals Demonstrating Enhanced Throughput of RapidFire Mass Spectrometry through Multiplexing Using the JmjD2d Demethylase as a Model System

2013 ◽  
Vol 19 (2) ◽  
pp. 278-286 ◽  
Author(s):  
Melanie Leveridge ◽  
Rachel Buxton ◽  
Argyrides Argyrou ◽  
Peter Francis ◽  
Bill Leavens ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Enzymatic Activity ◽  
High Throughput ◽  
Fold Increase ◽  
Model System ◽  
Analysis Time ◽  
Sample Handling ◽  
High Throughput Screens ◽  
Pooled Samples ◽  
Large Compound

Using mass spectrometry to detect enzymatic activity offers several advantages over fluorescence-based methods. Automation of sample handling and analysis using platforms such as the RapidFire (Agilent Technologies, Lexington, MA) has made these assays amenable to medium-throughput screening (of the order of 100,000 wells). However, true high-throughput screens (HTS) of large compound collections (>1 million) are still considered too time-consuming to be feasible. Here we propose a simple multiplexing strategy that can be used to increase the throughput of RapidFire, making it viable for HTS. The method relies on the ability to analyze pooled samples from several reactions simultaneously and to deconvolute their origin using “mass-tagged” substrates. Using the JmjD2d H3K9me3 demethylase as a model system, we demonstrate the practicality of this method to achieve a 4-fold increase in throughput. This was achieved without any loss of assay quality. This multiplex strategy could easily be scaled to give even greater reductions in analysis time.

scholarly journals Development and Validation of a High-Throughput Screen for Inhibitors of SARS CoV and Its Application in Screening of a 100,000-Compound Library

2006 ◽  
Vol 12 (1) ◽  
pp. 33-40 ◽  
Author(s):  
William E. Severson ◽  
Nice Shindo ◽  
Mindy Sosa ◽  
Thomas Fletcher ◽  
E. Lucile White ◽  
...  
Keyword(s):  
High Throughput ◽  
Cytopathic Effect ◽  
High Throughput Screen ◽  
Compound Library ◽  
Hit Rate ◽  
Compound Libraries ◽  
Potential Inhibitors ◽  
Development And Validation ◽  
Large Compound

The authors have developed a high-throughput screen (HTS) that allows for the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries. The luminescent-based assay measures the inhibition of SARS CoV–induced cytopathic effect (CPE) in Vero E6 cells. The assay was validated in 96-well plates in a BSL3 containment facility. The assay is sensitive and robust, with Z values > 0.6, signal to background (S/B) > 16, and signal to noise (S/N) > 3. The assay was further validated with 2 different diversity sets of compounds against the SARS CoV. The “hit” rate for both libraries was approximately 0.01%. The validated HTS assay was then employed to screen a 100,000-compound library against SARS CoV. The hit rate for the library in a single-dose format was determined to be approximately 0.8%. Screening of the 3 libraries resulted in the identification of several novel compounds that effectively inhibited the CPE of SARS CoV in vitro—compounds which will serve as excellent lead candidates for further evaluation. At a 10-μM concentration, 3 compounds with selective indexes (SI50) of > 53 were discovered.

scholarly journals Bio-instructive materials on-demand – combinatorial chemistry of peptoids, foldamers, and beyond

2021 ◽  
Author(s):  
Claudine Herlan ◽  
Dominik Feser ◽  
Ute Schepers ◽  
Stefan Bräse
Keyword(s):  
High Throughput ◽  
Combinatorial Chemistry ◽  
Medicinal Chemistry ◽  
Materials Science ◽  
Modular Design ◽  
Rapid Synthesis ◽  
On Demand ◽  
Compound Libraries ◽  
Large Compound

Combinatorial chemistry allows for the rapid synthesis of large compound libraries for high throughput screenings in biology, medicinal chemistry, or materials science. Especially compounds from a highly modular design are...

scholarly journals High-Throughput Mass Spectrometry Screening for Inhibitors of Phosphatidylserine Decarboxylase

2007 ◽  
Vol 12 (5) ◽  
pp. 628-634 ◽  
Author(s):  
Chris D. Forbes ◽  
Joshuaine G. Toth ◽  
Can C. Özbal ◽  
William A. Lamarr ◽  
Jennifer A. Pendleton ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Catalytic Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Lipid Synthesis ◽  
High Quality ◽  
Compound Libraries ◽  
Z Value ◽  
Large Compound ◽  
Mass Spectrometry Assay

A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries. The high-throughput mass spectrometry assay directly measures phosphatidylserine and phosphatidylethanolamine using the RapidFire™ platform at a rate of 1 sample every 7.5 s. The assay is robust, with an average Z′ value of 0.79 from a screen of 9920 compounds. Of 60 compounds selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry permitted a high-quality screen to be performed for an otherwise intractable target. ( Journal of Biomolecular Screening 2007:628-634)

scholarly journals High-throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2 Mac1 Macrodomain

2021 ◽  
Author(s):  
Morgan A Dasovich ◽  
Junlin Zhou ◽  
Ajit G Thomas ◽  
Jack A Goodman ◽  
Robert Lyle McPherson ◽  
...  
Keyword(s):  
Antiviral Therapy ◽  
High Throughput ◽  
Activity Assay ◽  
Selective Inhibitors ◽  
Antiviral Therapies ◽  
Compound Libraries ◽  
Promising Target ◽  
High Throughput Assay ◽  
Large Compound ◽  
The Way

Macrodomains are a class of conserved ADP-ribosylhydrolases expressed by viruses of pandemic concern, including coronaviruses and alphaviruses. Viral macrodomains are critical for replication and virus-induced pathogenesis; therefore, these enzymes are a promising target for antiviral therapy. However, no potent or selective viral macrodo-main inhibitors currently exist, in part due to the lack of a high-throughput assay for this class of enzymes. Here, we developed a high-throughput ADP-ribosylhydrolase assay using the SARS-CoV-2 macrodomain Mac1. We performed a pilot screen which identified dasatinib and dihydralazine as ADP-ribosylhydrolase inhibitors. Importantly, dasatinib does not inhibit MacroD2, the closest Mac1 homolog in humans. Our study demonstrates the feasibility of identifying selective inhibitors based on ADP-ribosylhydrolase activity, paving the way for screening large compound libraries to identify improved macrodomain inhibitors and explore their potential as antiviral therapies for SARS-CoV-2 and future viral threats.

High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes

2021 ◽  
pp. 247255522110262
Author(s):  
Jonathan Choy ◽  
Yanqing Kan ◽  
Steve Cifelli ◽  
Josephine Johnson ◽  
Michelle Chen ◽  
...  
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening Strategy ◽  
Time Resolved ◽  
Protein Levels ◽  
Increased Risk ◽  
Compound Screening ◽  
High Throughput Screens ◽  
Screening Library

High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes.

An overview of analytical methods for monitoring bacterial transglycosylation

2014 ◽  
Vol 6 (19) ◽  
pp. 7590-7596 ◽  
Author(s):  
Bart Blanchaert ◽  
Erwin Adams ◽  
Ann Van Schepdael
Keyword(s):  
High Throughput ◽  
Analytical Methods ◽  
High Throughput Screens

This review highlights the fluorescence and radioactively labeled assays and high-throughput screens for the search for antibiotics targeting bacterial transglycosylation.

Efficient Modulation of Catalytic Performance of Electrocatalytic Nitrogen Reduction with Transition Metal Anchored N/O-codoped Graphene by Coordination Engineering

2021 ◽  
Author(s):  
Xiaolin Wang ◽  
Li-Ming Yang
Keyword(s):  
Nitrogen Fixation ◽  
Transition Metal ◽  
High Throughput ◽  
First Principles ◽  
High Throughput Screening ◽  
Catalytic Performance ◽  
Nitrogen Reduction ◽  
Highly Efficient ◽  
First Time

We for the first time report the discovery of a series of highly efficient electrocatalysts, i.e., transition metal anchored N/O-codoped graphene, for nitrogen fixation via high-throughput screening combined with first-principles...

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