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BioAssay: AID 602483

HEp2 Cytoxicity Assay for VEEV Compounds (1)

This assay is designed to evaluate the response of HEp-2 cells to selected compounds from anti-VEEV screening ( AID: 588727) in order to begin developing a cytotoxicity profile. ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(1)
 
 
Inactive(2)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(1)
 
 
Inactive(2)
 
 
 Related BioAssays
 Related BioAssays
AID: 602483
Data Source: Southern Research Specialized Biocontainment Screening Center (VEE_HEp2_01)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-24
Hold-until Date: 2013-03-22
Modify Date: 2013-03-22

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compound: 1
Related Experiments
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AIDNameTypeComment
588719Vero 76 Cytoxicity Assay for VEEV CompoundsConfirmatorydepositor-specified cross reference: Cytotoxicity (Vero76) Counter Screen
588723A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus - Project SummarySummarydepositor-specified cross reference: Summary AID
588727A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83Confirmatorydepositor-specified cross reference: Primary and Confirmatory Screen (TC-83)
602240Vero 76 Cytoxicity Assay for VEEV Compounds (2)Confirmatorydepositor-specified cross reference: Cytotoxicity (Vero76) Counter Screen
602241A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (2)Confirmatorydepositor-specified cross reference: Confirmatory Screen (TC-83)
602242A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526Confirmatorydepositor-specified cross reference: Confirmatory Screen (v3586)
602307Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain)Otherdepositor-specified cross reference: Titer reduction assay TC-83
602411Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (2) Trinidad donkey strainOtherdepositor-specified cross reference: Titer reduction assay (WT-Trinidad Donkey)
623935A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)Confirmatorydepositor-specified cross reference
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)Confirmatorydepositor-specified cross reference
624063A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV TC-83 (4)Confirmatorydepositor-specified cross reference
624069Vero 76 Cytoxicity Assay for VEEV Compounds (5)Confirmatorydepositor-specified cross reference
624284A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (5)Confirmatorydepositor-specified cross reference
624286Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (3)Otherdepositor-specified cross reference
624295Vero 76 Cytoxicity Assay for VEEV Compounds (6)Confirmatorydepositor-specified cross reference
624449A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (6)Confirmatorydepositor-specified cross reference
624450Vero 76 Cytoxicity Assay for VEEV Compounds (7)Confirmatorydepositor-specified cross reference
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)Otherdepositor-specified cross reference
651734A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (7)Confirmatorydepositor-specified cross reference
651735Vero 76 Cytoxicity Assay for VEEV Compounds (8)Confirmatorydepositor-specified cross reference
651738A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (2)Confirmatorydepositor-specified cross reference
651874A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (2)Confirmatorydepositor-specified cross reference
651883Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (strain Trinidad donkey) (03)Otherdepositor-specified cross reference
651884A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526 (2)Confirmatorydepositor-specified cross reference
651886Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (5)Otherdepositor-specified cross reference
651917A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (8)Confirmatorydepositor-specified cross reference
651930Vero 76 Cytoxicity Assay for VEEV Compounds (9)Confirmatorydepositor-specified cross reference
651932A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assay (2)Confirmatorydepositor-specified cross reference
651934A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (3)Confirmatorydepositor-specified cross reference
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)Confirmatorysame project related to Summary assay
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)Confirmatorysame project related to Summary assay
602470A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) AssayConfirmatorysame project related to Summary assay
602484A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (1)Confirmatorysame project related to Summary assay
Description:
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dong Hoon Chung, University of Louisville
Award: 1 R03 MH087448-01A1
Introduction
This assay is designed to evaluate the response of HEp-2 cells to selected compounds from anti-VEEV screening ( AID: 588727) in order to begin developing a cytotoxicity profile.
Protocol
Cell Culture: HEp-2 cells (ATCC CCL-23, American Tissue Culture Type) were maintained as adherent cell lines in DMEM with 2 mM L-glutamine and 10% fetal bovine serum (FBS) at 37oC in a humidified 5% CO2 atmosphere. Cells were passaged as needed and harvested from flasks using 0.05% trypsin-EDTA.

Assay Media - Preparation of Complete DMEM/F12: DMEM/F12 (Invitrogen, Cat. No.11320) was supplemented with 5mL of Pen/Strep (Invitrogen, Cat. No. 10378016), 5 mL of 200mM glutamine(Invitrogen, Cat No.25030-081), and 10 mL of HI-FBS was added per 500 mL of media.

Dose Response Compound Preparation: For dose response screening, compounds or carrier control (DMSO) were diluted to 3x in Complete DMEM/F12. Test compounds were serially diluted 1:2 resulting in an 8 point dose response dilution series. (final plate well concentration ranging from 50 uM to 0.39 uM and a final DMSO concentration of 0.25%). Thirty ul of each dilution was dispensed to assay plates (0.75% DMSO) in triplicate.
Control Drug: The positive control drug for this assay, MPA was solubilized in DMSO.

Preparation of HEp-2 cells: Cells were harvested and resuspended to 267,000 cells per ml in Complete DMEM/F12.

Assay Set up: Forty five ul of HEp-2 cell suspension (12,000 cells/well) were plated in clear bottom black well 96-well plates and the plates were incubated in an incubator at 37oC in a humidified 5% CO2 atmosphere. The next day, thirty uL of drugging media (0.75% DMSO) were added to the each wells and the plates were incubated at 37oC for an hour. Each well then received fifteen uL of Complete DMEM/F12 media. For the cell control wells, Complete DMEM/F12 media were added instead of virus solution. Drug plating was conducted using a EVO100, 96MAC (Tecan) and virus was added using MicroFlo (Biotek). The assay plates were incubated for five days at 37 degrees C, 5% CO2 and 90% relative humidity.

Endpoint Read: The assay plates were equilibrated to room temperature for 30 minutes and then an equal volume of CellTiter-Glo reagent (Promega Inc.) was added to each well. Plates were incubated for 10 min at room temperature and luminescence was measured using a Synergy4 multi-label reader.

Data Analysis: 16 control wells containing cells treated with DMSO vehicle and were included on each assay plate. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med background)/(Med Cell Ctrl - Med background). The normalized % viability was plotted against the tested concentrations. 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" (toxic) and CC50 values were calculated. If the % viability at all doses was >70%, the compound was defined as "Inactive" (non-toxic).
The following tiered system has been implemented at Southern Research Institute for use with the PubChem Score: Compounds in the primary screen are scored on a scale of 0-40 based on % activity; a score of 40 corresponds to 100% activity. In the confirmatory dose response screen of primary screen hits, active compounds are scored on a scale of 41-80 based on IC50 result while compounds where activity was not confirmed are given the score 0. Confirmatory dose response and secondary screens of purified and/or resynthesized compounds, indicating the highest degree of confidence) are scored on a scale of 81-100 based on IC50 result. Inactive compounds are given the score 0.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay Detection: Bio-luminescence
Assay Format: Cell-based
Assay Method: End-point
Assay Type: Viability/Toxicity
BSL: BSL1
Multiplexing: No
Phenotypic Screen: Yes
Screening Concentration Range Max: 50
Screening Concentration Range Min: 0.04
Secondary Assay Sub-type: Counter-screen Assay
Used during SAR?: Yes
Used for Hit Validation?: No
From PubChem:
Assay Type: Toxicity
Assay Cell Type: HEp-2
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1CC50 *FloatμM
2% Cell Viability @ 50 uM (50μM**)Float%
3% Cell Viability @ 25 uM (25μM**)Float%
4% Cell Viability @ 12.5 uM (12.5μM**)Float%
5% Cell Viability @ 6.25 uM (6.25μM**)Float%
6% Cell Viability @ 3.125 uM (3.125μM**)Float%
7% Cell Viability @ 1.563 uM (1.563μM**)Float%
8% Cell Viability @ 0.782 uM (0.782μM**)Float%
9% Cell Viability @ 0.391 uM (0.391μM**)Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 MH087448-01A1

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