Bookmark and Share
BioAssay: AID 1650

A Cell Based Secondary Assay To Explore Cytotoxicity of West Nile Virus Anti-Viral Hit Compounds

This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the West Nile Virus anti-viral assay. In addition to profiling potential lead compounds for cytotoxicity, cytotoxicity can mask anti-viral efficacy of test compound, therefore it is important that cytotoxicity be explored at an early stage of an anti-viral project. Vero E6 cell line is one of the most prevalently used cell lines for antiviral screening such as West Nile virus screening (AID: 1621). ..more
_
   
 Tested Compounds
 Tested Compounds
All(600)
 
 
Active(183)
 
 
Inactive(417)
 
 
 Tested Substances
 Tested Substances
All(601)
 
 
Active(184)
 
 
Inactive(417)
 
 
 Related BioAssays
 Related BioAssays
AID: 1650
Data Source: Southern Research Specialized Biocontainment Screening Center (WNV_Cytotox)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-04-01
Modify Date: 2010-02-26

Data Table ( Complete ):           Active    All
BioActive Compounds: 183
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
1621A Cell Based Assay for the Identification of Lead Compounds with Anti-Viral Activity Against West Nile Virusconfirmatory
1635A Summary of Cell Based Assays for the Identification of Lead Compounds with Anti-Viral Activity Against West Nile Virussummary2
2238A Cell Based Assay for the Identification of Synthesized/Analog Compounds with Anti-Viral Activity Against West Nile Virusconfirmatory
2244A Cell Based Secondary Assay To Explore Cytotoxicity of West Nile Virus Anti-Viral Synthesized/Analog Compoundsconfirmatory
2024Titer reduction assay for the selected compounds from the DR study as a confirmatory assay (for 55 compounds)other
2065Titer reduction assay of West Nile Virus for synthesized compoundsother
2419A Cell Based Assay for the Identification of Probe Candidates with Anti-Viral Activity against West Nile Virusconfirmatory
2402A Cell Based Secondary Assay To Explore Cytotoxicity of West Nile Virus Anti-Viral Synthesized/Analog Compounds IIconfirmatory
2405A Cell Based Assay for the Identification of Probe candidate Compounds with Anti- Respiratory Syncytial virusconfirmatory
2409A Cell Based Assay for the Identification of Probe candidate Compounds with Anti- Venezuelan Equine Encephalitis virus.confirmatory
2414A Cell Based Secondary Assay To Explore Cytotoxicity of West Nile Virus Anti-Viral Synthesized/Analog Compounds in Vero 76 Cells.confirmatory
588371A Cell Based Assay for the Identification of Probe Candidates with Anti-Viral Activity against West Nile Virus (EC_1)confirmatory
588372A Cell Based Assay for the Identification of Compounds with Anti-Viral Activity against West Nile Virus (EC_2)confirmatory
588374A Cell Based Secondary Assay To Explore Cytotoxicity of West Nile Virus Anti-Viral Synthesized/Analog Compounds (extended characterization 1)confirmatory
623930A Cell Based Secondary Assay To Explore Cytotoxicity of West Nile Virus Anti-Viral Synthesized/Analog Compounds (extended characterization 2)confirmatory
623934A Cell Based Assay for the Identification of Compounds with Anti-Viral Activity against West Nile Virus (EC_3)confirmatory
Description:
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. Marintha Heil, Southern Research Institute
Award: 1R03 MH084847-01

This functional assay was developed for detection of compounds inhibiting Vero E6 cells viability as a secondary screen to the West Nile Virus anti-viral assay. In addition to profiling potential lead compounds for cytotoxicity, cytotoxicity can mask anti-viral efficacy of test compound, therefore it is important that cytotoxicity be explored at an early stage of an anti-viral project. Vero E6 cell line is one of the most prevalently used cell lines for antiviral screening such as West Nile virus screening (AID: 1621).

In the present assay, we treated Vero E6 cells with 601 compounds selected as hits in the West Nile Virus anti-viral assay for 72 hours over a 10 point 2-fold dilution series, ranging from 0.039uM to 20 uM. Following 72 hours of treatment, relative viable cell number was determined using a commercially available assay (Cell Titer Glo, Promega). Each plate contained 64 replicates of vehicle treated cells which served as negative controls.
Protocol
Cell Culture: Vero E6 cells were subcultured every 7 days in E-MEM with 10% fetal bovine serum and 2 mM glutamine (complete growth medium), incubated at 37 degrees C in 5% carbon dioxide, and maintained for no more than 20 passages after ATCC receiving.

Cell Plating: Fifteen uL of complete growth medium containing 3000 cells were dispensed per well, while stirring. Plates were incubated at 37 C for 24h prior to compound addition.

Compound Dosing/Plating: Carrier control / compounds were diluted in complete growth medium to prepare a 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume). These plates were incubated briefly while awaiting mock virus addition.

Mock virus addition: At the time of virus addition to the anti-viral plates, 10uL of complete media was added to the cytotoxicity plates. These plates were returned to the incubator at 37 C, 5% CO2 for 96h.

Endpoint/Detection: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature 10 min. Thirty uL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Envision microplate reader with an integration time of 0.1 s in luminescence mode.

Data Analysis: Sixty-four control wells containing cells treated with DMSO vehicle and were included on each assay plate. Because controls matched those of the antiviral plates, no cytotoxicity control was added to the assay plates, thus no Z calculations were performed. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med Ctrl Drug)/(Med Cell Ctrl - Med Ctrl Drug). The normalized % viability was plotted against the tested concentrations of 0.039uM to 20 uM. The CC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0, respectively and allowing extrapolation to identify weakly active compounds.
Comment
Outcome: Compounds that showed <70% cell viability for at least one concentration were defined as "Active". If the % viability at all doses was >70%, the compound was defined as "Inactive".

Scoring: SRBCSC uses a three-tier scoring system where scores of 0-40 apply to primary screen data, 41-80 indicates confirmatory screen data, and 81-100 is reserved for confirmatory data on resynthesized compounds. Inactive compounds at any screening level receive a score of 0. In this confirmatory screen, "Active" compounds were scored based on CC50 results on a tier of 40-80 with compounds failing confirmation scoring 0.
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1CC50 ModifierString
2CC50*FloatμM
3CC50 Std Dev ModifierString
4CC50 Std DevFloat
5CC50 Hill SlopeFloat
6CC50 Normalized Chi2Float
7% Cell Viability @ 20 uM (20μM**)Float%
8% Cell Viability @ 10 uM (10μM**)Float%
9% Cell Viability @ 5 uM (5μM**)Float%
10% Cell Viability @ 2.5 uM (2.5μM**)Float%
11% Cell Viability @ 1.25 uM (1.25μM**)Float%
12% Cell Viability @ 0.625 uM (0.625μM**)Float%
13% Cell Viability @ 0.313 uM (0.313μM**)Float%
14% Cell Viability @ 0.156 uM (0.156μM**)Float%
15% Cell Viability @ 0.078 uM (0.078μM**)Float%
16% Cell Viability @ 0.039 uM (0.039μM**)Float%
17Max % ViabilityMaximum % Viability observed across all concentrations.Float%
18Max Viability ConcConcentration at which Maximum % Viability was observed.FloatμM
19Min % ViabilityMinimum % Viability observed across all concentrations.Float%
20Min Viability ConcConcentration at which Minimum % Viability was observed.FloatμM

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03 MH084847-01

Data Table (Concise)
PageFrom: