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BioAssay: AID 623997

qHTS Assay for Inhibitors of Influenza NS1 Protein Function: Mammalian Cell Viability

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(40)
 
 
Active(1)
 
 
Inactive(32)
 
 
Inconclusive(7)
 
 
 Tested Substances
 Tested Substances
All(40)
 
 
Active(1)
 
 
Inactive(32)
 
 
Inconclusive(7)
 
 
AID: 623997
Data Source: NCGC (NS1700)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-04-04
Hold-until Date: 2013-04-03
Modify Date: 2013-04-03

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
2336Summary Assay for Inhibitors of Influenza NS1 Protein FunctionsummarySummary AID
Description:
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.

Hits resulting from this primary screen and other follow-up screens, were tested in four concentrations for this mammalian cell viability assay, using MDCK cells.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]

MLPCN Grant: MH084878
Assay Submitter (PI): Daniel Engel, University of Virgina
Protocol
Opaque-walled 96-well plates were seeded with 104 MDCK cells per well in 100 uL medium and incubated overnight at 37 deg C. The compound was added to triplicate wells for each concentration tested and incubated for 48 h at 37 deg C. The plate was equilibrated for 30 min at room temperature and 100 uL CellTiter Glo reagent (Promega) added according to the manufacturer's protocol. The plate was shaken for 2 min and then incubated for 10 min at room temperature. Luminescence was recorded in an LMAX 384 reader (Molecular Devices) with integration time of 1 s.
Comment
Compounds that showed a drop of viability below 50% at 10 uM are "active" (toxic); remaining compounds that showed a drop of viability below 50% at 100 uM are "inconclusive"; all other remaining compounds are "inactive"(non-toxic)."

"Active compounds are assigned a score of 90, "inconclusive" 50, and "inactive" 10.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Cell Viability at 5 uM Float%
2Cell Viability at 10 uM Float%
3Cell Viability at 50 uM Float%
4Cell Viability at 100 uM Float%
5Compound QC String
Additional Information
Grant Number: MH084878

Data Table (Concise)
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