scholarly journals High-Throughput Screening of a 100,000-Compound Library for Inhibitors of Influenza A Virus (H3N2)

2008 ◽  
Vol 13 (9) ◽  
pp. 879-887 ◽  
Author(s):  
William E. Severson ◽  
Michael McDowell ◽  
Subramaniam Ananthan ◽  
Dong-Hoon Chung ◽  
Lynn Rasmussen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Influenza A ◽  
Moderate Activity ◽  
Compound Library ◽  
Highly Active ◽  
Virus Life Cycle ◽  
Influenza Activity ◽  
Compound Concentration ◽  
Viral Cytopathic Effect

Using a highly reproducible and robust cell-based high-throughput screening (HTS) assay, the authors screened a 100,000-compound library at 14- and 114-µM compound concentration against influenza strain A/Udorn/72 (H3N2). The “hit” rates (>50% inhibition of the viral cytopathic effect) from the 14- and 114-µM screens were 0.022% and 0.38%, respectively. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen at the lower concentration yielded 3 compounds, which displayed moderate activity (SI50 = 10-49). Intriguingly, the screen at the higher concentration revealed several additional hits. Two of these hits were highly active with an SI50 > 50. Time of addition experiments revealed 1 compound that inhibited early and 4 other compounds that inhibited late in the virus life cycle, suggesting they affect entry and replication, respectively. The active compounds represent several different classes of molecules such as carboxanilides, 1-benzoyl-3-arylthioureas, sulfonamides, and benzothiazinones, which have not been previously identified as having antiviral/anti-influenza activity. ( Journal of Biomolecular Screening 2008:879-887)

scholarly journals Discovery of Novel Benzoquinazolinones and Thiazoloimidazoles, Inhibitors of Influenza H5N1 and H1N1 Viruses, from a Cell-Based High-Throughput Screen

2010 ◽  
Vol 16 (1) ◽  
pp. 73-81 ◽  
Author(s):  
Joseph A. Maddry ◽  
Xi Chen ◽  
Colleen B. Jonsson ◽  
Subramaniam Ananthan ◽  
Judith Hobrath ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Influenza A ◽  
Active Compounds ◽  
Influenza Activity ◽  
A Cell ◽  
Molecular Libraries ◽  
Viral Cytopathic Effect

A highly reproducible and robust cell-based high-throughput screening (HTS) assay was adapted for screening of small molecules for antiviral activity against influenza virus strain A/Vietnam/1203/2004 (H5N1). The NIH Molecular Libraries Small Molecule Repository (MLSMR) Molecular Libraries Screening Centers Network (MLSCN) 100,000-compound library was screened at 50 µM. The “hit” rate (>25% inhibition of the viral cytopathic effect) from the single-dose screen was 0.32%. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen yielded 5 active compounds (SI value >3). One compound showed an SI50 value of greater than 3, 3 compounds had SI values ranging from greater than 14 to 34, and the most active compound displayed an SI value of 94. The active compounds represent 2 different classes of molecules, benzoquinazolinones and thiazoloimidazoles, which have not been previously identified as having antiviral/anti-influenza activity. These molecules were also effective against influenza A/California/04/2009 virus (H1N1) and other H1N1 and H5N1 virus strains in vitro but not H3N2 strains. Real-time qRT-PCR results reveal that these chemotypes significantly reduced M1 RNA levels as compared to the no-drug influenza-infected Madin Darby canine kidney cells.

High Throughput Screening of a 100,000 Compound Library for Inhibitors of Influenza A Virus (H3N2)

2007 ◽  
Vol 74 (3) ◽  
pp. A62-A62
Author(s):  
W SEVERSON ◽  
M MCDOWELL ◽  
L RASUMUSSEN ◽  
M SOSA ◽  
S ANANTHAN ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
High Throughput ◽  
High Throughput Screening ◽  
Influenza A ◽  
Compound Library

scholarly journals Development of a Simple Assay Protocol for High-Throughput Screening of Mycobacterium tuberculosis Glutamine Synthetase for the Identification of Novel Inhibitors

2005 ◽  
Vol 10 (7) ◽  
pp. 725-729 ◽  
Author(s):  
Upasana Singh ◽  
Vinita Panchanadikar ◽  
Dhiman Sarkar
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Glutamine Synthetase ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Coli Strain ◽  
Compound Library ◽  
Essential Enzyme ◽  
Assay Protocol

Mycobacterium tuberculosis glutamine synthetase (GS) is an essential enzyme involved in the pathogenicity of the organism. The screening of a compound library using a robust high-throughput screening (HTS) assay is currently thought to be the most efficient way of getting lead molecules, which are potent inhibitors for this enzyme. The authors have purified the enzyme to a >90% level from the recombinant Escherichia coli strain YMC21E, and it was used for partial characterization as well as standardization experiments. The results indicated that the Kmof the enzyme for L-glutamine and hydroxylamine were 60 mM and 8.3 mM, respectively. The Km for ADP, arsenate, and Mn2+ were 2 [.proportional]M, 5 [.proportional]M, and 25 [.proportional]M, respectively. When the components were adjusted according to their Km values, the activity remained constant for at least 3 h at both 25° C and 37° C. The Z′ factor determined in microplate format indicated robustness of the assay. When the signal/noise ratios were determined for different assay volumes, it was observed that the 200-[.proportional]l volume was found to be optimum. The DMSO tolerance of the enzyme was checked up to 10%, with minimal inhibition. The IC50 value determined for L-methionine S-sulfoximine on the enzyme activity was 3 mM. Approximately 18,000 small molecules could be screened per day using this protocol by a Beckman Coulter HTS setup.

scholarly journals Development of a High-Throughput Cell-Based Assay for Superoxide Production in HL-60 Cells

2010 ◽  
Vol 15 (4) ◽  
pp. 388-397 ◽  
Author(s):  
Patricia M. Seitz ◽  
Rona Cooper ◽  
Gregory J. Gatto ◽  
Fernando Ramon ◽  
Thomas D. Sweitzer ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Superoxide Production ◽  
Test Set ◽  
Cellular Processes ◽  
Detection Assay ◽  
Z Value ◽  
Compound Concentration ◽  
Compound Test ◽  
Early Drug

Superoxide affects many normal and pathogenic cellular processes, and the detection of superoxide produced by cells is therefore of interest for potential therapeutic applications. To develop a high-throughput cell-based assay for the detection of extracellular superoxide production that could be run in a 384-well or 1536-well format, 2 luminescent reagents, Lucigenin and Diogenes, and one fluorescent reagent, Oxyburst Green BSA, were tested. HL-60 cells, which had been differentiated to a neutrophil-like phenotype with DMSO and frozen in large batches, were used in assays. All 3 superoxide detection reagents performed well statistically in terms of IC50 reproducibility and met a desired Z′ value requirement of >0.4. When tested against a 1408-compound test set at 5 or 10 µM compound concentration, a higher hit rate was obtained with the 2 luminescent reagents compared with that obtained with the fluorescent Oxyburst Green BSA reagent. The Oxyburst Green BSA assay was ultimately chosen for compound profiling and high-throughput screening activities. This 1536 superoxide detection assay using cryopreserved differentiated HL-60 cells represents a shifting paradigm toward the utilization of more therapeutically relevant cells in early drug development activities.

scholarly journals High-Throughput Screening Method of Inhibitors that Block the Interaction between 2 Helical Regions of HIV-1 gp41

2005 ◽  
Vol 10 (1) ◽  
pp. 13-19 ◽  
Author(s):  
Bong-Suk Jin ◽  
Won-Kyu Lee ◽  
Kwangseog Ahn ◽  
Myung Kyu Lee ◽  
Yeon Gyu Yu
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Method ◽  
Assay Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Compound Library ◽  
Elisa Method ◽  
Helical Bundle ◽  
Fusion Inhibitors ◽  
Hiv 1

The HIV-1 envelope glycoprotein transmembrane subunit, gp41, mediates the fusion of viral and target cell membranes. The 2 helical regions in the ectodomain of gp41, the N-helix and the C-helix, form a helical bundle complex that has been suggested as a fusion-active conformation. Previously, an enzyme-linked immunosorbent assay (ELISA) method had been established to measure the interaction of 2 helical regions of gp41. In this study, the ELISA method was modified to apply high-throughput screening (HTS) of an organic compound library. A few compounds had been identified to prevent the interaction between 2 helical regions of gp41, and they were further shown to inhibit the gp41-mediated viral infection. In addition, they specifically quenched the fluorescence of tryptophan in the N-helix region, indicating that these compounds bound to the N-helix rather than the C-helix of gp41. These results suggested that this assay method targeting gp41 could be used for HTS of HIV fusion inhibitors. ( Journal of Biomolecular Screening 2005:13-19)

scholarly journals A Fluorescence-based High Throughput-Screening assay for the SARS-CoV RNA synthesis complex

2020 ◽  
Author(s):  
Cecilia Eydoux ◽  
Veronique Fattorini ◽  
Ashleigh Shannon ◽  
Thi-Tuyet-Nhung Le ◽  
Bruno Didier ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
High Throughput ◽  
High Throughput Screening ◽  
Rna Synthesis ◽  
Drug Repositioning ◽  
List Type ◽  
Emerging Viruses ◽  
Screening Assay ◽  
Highly Active ◽  
High Throughput Screening Assay

AbstractThe Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) emergence in 2003 introduced the first serious human coronavirus pathogen to an unprepared world. To control emerging viruses, existing successful anti(retro)viral therapies can inspire antiviral strategies, as conserved viral enzymes (eg., viral proteases and RNA-dependent RNA polymerases) represent targets of choice. Since 2003, much effort has been expended in the characterization of the SARS-CoV replication/transcription machinery. Until recently, a pure and highly active preparation of SARS-CoV recombinant RNA synthesis machinery was not available, impeding target-based high throughput screening of drug candidates against this viral family. The current Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic revealed a new pathogen whose RNA synthesis machinery is highly (>96% aa identity) homologous to SARS-CoV. This phylogenetic relatedness highlights the potential use of conserved replication enzymes to discover inhibitors against this significant pathogen, which in turn, contributes to scientific preparedness against emerging viruses. Here, we report the use of a purified and highly active SARS-CoV replication/transcription complex (RTC) to set-up a high-throughput screening of Coronavirus RNA synthesis inhibitors. The screening of a small (1,520 compounds) chemical library of FDA-approved drugs demonstrates the robustness of our assay and will allow to speed-up drug repositioning or novel drug discovery against the SARS-CoV-2.Principle of SARS-CoV RNA synthesis detection by a fluorescence-based high throughput screening assayHighlights- A new SARS-CoV non radioactive RNA polymerase assay is described- The robotized assay is suitable to identify RdRp inhibitors based on HTS

Overcoming Problems of Compound Storage in DMSO: Solvent and Process Alternatives

2009 ◽  
Vol 14 (6) ◽  
pp. 708-715 ◽  
Author(s):  
Timothy J. Waybright ◽  
John R. Britt ◽  
Thomas G. McCloud
Keyword(s):  
Water Absorption ◽  
High Throughput ◽  
High Throughput Screening ◽  
Time Course ◽  
Molar Concentration ◽  
Dmso Solution ◽  
Compound Library ◽  
Plate Production ◽  
The Common ◽  
Drug Solution

The common practice of preparing storage libraries of compounds in 100% DMSO solution well in advance of bioassay brings with it difficulties that affect the accuracy of the data obtained. This publication presents a series of studies done on a subset of compounds that are difficult to bioassay because they precipitate from DMSO solution. These compounds are members of a frequently used, diverse compound library of the sort commonly used in the high-throughput screening (HTS) environment. Experiments were performed to determine the concentration of drug in solution above the precipitate, observe the time course and effect of various mixtures of solvents upon precipitation, measure the viscosity of cosolvents to determine compatibility with HTS, determine water absorption rates for various solvent combinations, and investigate resolubilization techniques to ensure proper drug solution for HTS. Recommendations are made on how to best maximize the probability that problem compounds will remain in solution, be accurately transferred during assay plate production, and, as a result, be accurately bioassayed at the specified molar concentration. ( Journal of Biomolecular Screening 2009:708-715)

Synthesis and evaluation of phenoxymethylbenzamide analogues as anti-trypanosomal agents

MedChemComm ◽  
2015 ◽  
Vol 6 (3) ◽  
pp. 403-406 ◽  
Author(s):  
Alexandra Manos-Turvey ◽  
Emma E. Watson ◽  
Melissa L. Sykes ◽  
Amy J. Jones ◽  
Jonathan B. Baell ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Compound Library

The synthesis of a compound library based on a high throughput screening hit led to the discovery of several potent anti-trypanosomal agents.

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