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BioAssay: AID 504647

Assay for Inhibitors of Influenza NS1 Protein Function: Influenza virus replication assay in MDCK cells infected with virus A/PR/8/34

Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill more ..
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 Tested Compounds
 Tested Compounds
All(95)
 
 
Active(23)
 
 
Inactive(72)
 
 
 Tested Substances
 Tested Substances
All(95)
 
 
Active(23)
 
 
Inactive(72)
 
 
 Related BioAssays
 Related BioAssays
AID: 504647
Data Source: NCGC (NS100053)
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2011-04-12
Modify Date: 2011-04-14

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 23
Related Experiments
AIDNameTypeComment
2326qHTS Assay for Inhibitors of Influenza NS1 Protein FunctionConfirmatorydepositor-specified cross reference: Primary screen assay.
2336Summary Assay for Inhibitors of Influenza NS1 Protein FunctionSummarydepositor-specified cross reference: Summary assay for project.
492980Assay for Inhibitors of Influenza NS1 Protein Function: Hit ValidationConfirmatorydepositor-specified cross reference: Hit validation from primary screen.
602450qHTS Assay for Inhibitors of Influenza NS1 Protein Function: Viral Replication TCID50 SAR for ProbeOthersame project related to Summary assay
602452qHTS Assay for Inhibitors of Influenza NS1 Protein Function: SAR Using NS1 and pYES strainsOthersame project related to Summary assay
602454qHTS Assay for Inhibitors of Influenza NS1 Protein Function: Viral Replication TCID50Othersame project related to Summary assay
602456qHTS Assay for Inhibitors of Influenza NS1 Protein Function: RT-PCROthersame project related to Summary assay
623997qHTS Assay for Inhibitors of Influenza NS1 Protein Function: Mammalian Cell ViabilityOthersame project related to Summary assay
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH084878-01A1
Assay Submitter (PI): Daniel Engel

Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill global needs. In addition their potential usefulness against newly emergent strains is not known. Efforts are needed to develop novel agents against influenza virus, including broad-spectrum agents. Identification of small molecules that inhibit NS1 function either directly or by interfering with specific cellular pathways may be a key to increasing our defense against the virus.

We have miniaturized and optimized a cell based assay in which NS1 from influenza A is expressed in the budding yeast S. cerevisiae. NS1 is a multi-functional protein that counters the host innate immune response and facilitates viral versus cellular gene expression. Expression of NS1 causes a pronounced slow growth phenotype in yeast due to its intrinsic molecular activities. Small molecules that suppress the slow growth phenotype can be identified by a straightforward growth recovery assay using optical density (OD) as the measurement. The same yeast strain not expressing NS1 was used as positive control in the yeast growth recovery assay. The present experiment attempted to confirm the activity of compounds observed in the primary screen.

This assay confirms the activity of compounds from the primary screen by measuring their affect on viral replication.
Protocol
Protocol details are given in Basu et al. J. Vir. 83(4):1881-1891 2009. Titers of influenza virus stocks were determined by 50% tissue culture infective dose (TCID50) analysis on MDCK cells, maintained in Iscove medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. All media and sera were from Invitrogen. The viruses were propagated in 10-day-old embryonated chicken eggs at 37C. Confluent cell monolayers were infected at a multiplicity of infection (MOI) of 0.1 for 48 h in the presence or absence of 5uM compound. Compounds were added at the beginning of infection and were present throughout the infection. After 48 h virus titers were determined by standard TCID50 analysis.
Comment
Vehicle control gave a TCID50 of 8.5. Compounds that reduced TCID50 by 1 unit or greater were considered active and given a score of 80. Compounds with less activity were considered inactive and given a score of 0.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1TCID50 (5μM**)This endpoint dilution assay quantifies the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells. Host cells are plated and serial dilutions of the virus are added. After incubation, the percentage of cell death (i.e. infect ed cells) is manually observed and recorded for each virus dilution, and results are used to mathematically calculate a TCID50 result.FloatLog Dilution
2Cell viability (5μM**)Percent viable cells remaining after compound incubation.Float%

** Test Concentration.
Additional Information
Grant Number: MH084878-01A1

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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