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BioAssay: AID 651884

A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526 (2)

Venezuelan equine encephalitis virus is one of the Alphavirus species in the family of Togaviridae characterized by a non-segmented, positive sense strand RNA virus. VEEV, endemic to North, Central and South America, is an example of an arbovirus passing from a mosquito vector through rodent hosts that may result in epizootic subtypes causing disease in equines and humans. While there are more ..
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 Tested Compounds
 Tested Compounds
All(4)
 
 
Active(4)
 
 
 Tested Substances
 Tested Substances
All(4)
 
 
Active(4)
 
 
 Related BioAssays
 Related BioAssays
AID: 651884
Data Source: Southern Research Specialized Biocontainment Screening Center (VEE_AV3526_02)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-12-06
Hold-until Date: 2013-12-01
Modify Date: 2013-12-02

Data Table ( Complete ):           Active    All
BioActive Compounds: 4
Depositor Specified Assays
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AIDNameTypeComment
588727A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83confirmatoryPrimary and Confirmatory Assays
602241A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (2)confirmatoryConfirmatory (TC-83)
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)confirmatoryConfirmatory (TC-83)
624063A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV TC-83 (4)confirmatoryConfirmatory (TC-83)
624284A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (5)confirmatoryConfirmatory (TC-83)
624449A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (6)confirmatoryConfirmatory (TC-83)
651734A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (7)confirmatoryConfirmatory (TC-83)
602242A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526confirmatoryCounter (v3586)
602484A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (1)confirmatoryConfirmatory (Trinidad Donkey)
588719Vero 76 Cytoxicity Assay for VEEV CompoundsconfirmatoryCytotoxicity counter screen (Vero 76)
602240Vero 76 Cytoxicity Assay for VEEV Compounds (2)confirmatoryCytotoxicity counter screen (Vero 76)
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)confirmatoryCytotoxicity counter screen (Vero 76)
624069Vero 76 Cytoxicity Assay for VEEV Compounds (5)confirmatoryCytotoxicity counter screen (Vero 76)
624295Vero 76 Cytoxicity Assay for VEEV Compounds (6)confirmatoryCytotoxicity counter screen (Vero 76)
624450Vero 76 Cytoxicity Assay for VEEV Compounds (7)confirmatoryCytotoxicity counter screen (Vero 76)
602307Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain)otherVirus Titer Reduction (TC-83)
624286Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (3)otherVirus Titer Reduction (TC-83)
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)otherVirus Titer Reduction (TC-83)
602411Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (2) Trinidad donkey strainotherVirus Titer Reduction (Trinidad Donkey)
602470A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) AssayconfirmatoryRSV
602483HEp2 Cytoxicity Assay for VEEV Compounds (1)confirmatoryCytotoxicity counter screen (Hep-2)
623935A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)confirmatoryCHIKV
651738A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (2)confirmatoryCHIKV
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)confirmatoryCytotoxicity counter screen (Vero 76)
588723A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus - Project Summarysummary
651886Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (5)other
651917A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (8)confirmatory
651930Vero 76 Cytoxicity Assay for VEEV Compounds (9)confirmatory
651932A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assay (2)confirmatory
651934A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (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: Dong Hoon Chung, University of Louisville
Award: 1 R03 MH087448-01A1

Venezuelan equine encephalitis virus is one of the Alphavirus species in the family of Togaviridae characterized by a non-segmented, positive sense strand RNA virus. VEEV, endemic to North, Central and South America, is an example of an arbovirus passing from a mosquito vector through rodent hosts that may result in epizootic subtypes causing disease in equines and humans. While there are various subtypes, human infection follows a similar route of acute symptoms of fever, headache, lymphopenia, myalgia and malaise but the severity may increase to encephalitis. Fatalities occur in ~1% of the patients that present with disease. Although recorded natural epidemics are rare, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases have classified it as a category B priority biodefense agent in part due to its history as a biological weapon and the ease with which it may be cultured, manipulated and aerosolized.

Treatment of VEEV is limited to supportive care since there are no drugs available, although two non-FDA-approved vaccines are available for at risk populations. The live attenuated vaccine, TC-83, is produced in Central and South America for use in horses, while an inactivated form of VEE, C-84, is licensed for use in horses in the United States. Neither the TC-83 nor the C-84 vaccine is FDA approved for human use in the United States, although military and laboratory personnel may receive vaccination through the USAMRIID Special Immunization Program. The severity of the side effects and the inability to provide complete disease protection by the vaccine necessitate additional work to develop efficacious prophylactics.
Protocol
Cell Culture: Vero 76 (CRL-1587, ATCC) were purchased from ATCC and maintained in 37C incubator with 5% CO2. The cells were cultured in a complete media (Minimum Essential Media with Earle's salt and 10% fetal bovine serum). Cells were passaged once a week and harvested from flasks using 0.05% trypsin-EDTA.

Assay Media - Preparation of Complete DMEM media: 5mL Pen/Strep (Gibco, Cat. No. 15149) and 50mL of heat-inactivated FBS (Gibco, Cat. No. 10082147 ) was added to 500mL of Dulbecco's Modified Eagle Medium (Gibco, Cat. No. 11995-073).

Virus : V3526 strain. Virus was rescued from BHK C-21 cells that were transfected with infectious V3526 RNA. The recued virus was amplified in BHK C-21 cells once and then used as a stock virus.

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

Control Drug: The positive control drug for this assay, mycophenolic acid was solubilized in DMSO. It was diluted and added to the assay plates as described for test compounds. Final concentration for ribavirin was 10uM. All wells contained 0.25% DMSO.

Assay Set up: Vero 76 cells were plated in 96-well plates at a density of 15,000 cells per well in a volume of 45uL of DMEM complete media. The cells were grown for 24 hours prior to testing in a 5% CO2, 37C cell culture incubator. V3526 VEEV virus, was diluted in cell culture medium to be 750 pfu/ 15uL ( 0.05 MOI) and then added to the plates at a volume of 15uL per well. The plates were incubated for 48 hours in a 37C incubator with 5% CO2 .

Endpoint Read: Following the two day incubation period, the assay plates were equilibrated to room temperature for 10 min and an equal volume (90uL) of Cell Titer-Glo reagent (Promega Inc.) was added to each well using a Microflow (Biotek,VT) and plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Synergy4 Multimode plate reader (Biotek,VT) with an integration time of 0.2 s.

Data Analysis: Results are reported as percent (%) CPE inhibition and were calculated using the following formula: % CPE inhibition = 100*(Test Cmpd - Med Virus)/(Med Cells - Med Virus). IC50s were calculated for each substance using the 4 parameter Levenburg-Marquardt algorithm with the minimum and maximum parameters locked at 0 and 100, respectively.
Comment
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.

Compounds showing at least 30% inhibition at any tested dose were considered active. 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 inhibitory activity where a score of 40 corresponds to 100% inhibition. In the confirmatory dose response screen, active compounds were scored on a scale of 41-80 based on the IC50 result while compounds that did not confirm as actives were given the score of 0. A scale of 81-100 based on IC50 result is used for synthesized and purified compounds to indicate a high level of confidence in the data and compound quality.
Categorized Comment
Phenotypic Screen: Yes

Multiplexing: No

BSL: BSL2

Screening Concentration Range Max: 50

Screening Concentration Range Min: 0.78

Assay Format: Cell-based

Assay Type: Viability/Toxicity

Assay Method: End-point

Assay Detection: Bio-luminescence

Used for Hit Validation?: No

Used during SAR?: Yes

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50 Rep 1*FloatμM
2IC50 Rep 2FloatμM
3% Inhibition @ 25 uM Rep 1 (25μM**)Float%
4% Inhibition @ 12.5 uM Rep 1 (12.5μM**)Float%
5% Inhibition @ 6.25 uM Rep 1 (6.25μM**)Float%
6% Inhibition @ 3.125 uM Rep 1 (3.125μM**)Float%
7% Inhibition @ 1.563 uM Rep 1 (1.563μM**)Float%
8% Inhibition @ 0.781 uM Rep 1 (0.781μM**)Float%
9% Inhibition @ 0.625 uM Rep 1 (0.625μM**)Float%
10% Inhibition @ 0.391 uM Rep 1 (0.391μM**)Float%
11% Inhibition @ 0.313 uM Rep 1 (0.313μM**)Float%
12% Inhibition @ 0.195 uM Rep 1 (0.195μM**)Float%
13% Inhibition @ 0.156 uM Rep 1 (0.156μM**)Float%
14% Inhibition @ 0.078 uM Rep 1 (0.078μM**)Float%
15% Inhibition @ 0.039 uM Rep 1 (0.039μM**)Float%
16% Inhibition @ 0.02 uM Rep 1 (0.02μM**)Float%
17% Inhibition @ 0.01 uM Rep 1 (0.01μM**)Float%
18% Inhibition @ 0.005 uM Rep 1 (0.005μM**)Float%
19% Inhibition @ 25 uM Rep 2 (25μM**)Float%
20% Inhibition @ 12.5 uM Rep 2 (12.5μM**)Float%
21% Inhibition @ 6.25 uM Rep 2 (6.25μM**)Float%
22% Inhibition @ 3.125 uM Rep 2 (3.125μM**)Float%
23% Inhibition @ 1.563 uM Rep 2 (1.563μM**)Float%
24% Inhibition @ 0.781 uM Rep 2 (0.781μM**)Float%
25% Inhibition @ 0.625 uM Rep 2 (0.625μM**)Float%
26% Inhibition @ 0.391 uM Rep 2 (0.391μM**)Float%
27% Inhibition @ 0.313 uM Rep 2 (0.313μM**)Float%
28% Inhibition @ 0.195 uM Rep 2 (0.195μM**)Float%
29% Inhibition @ 0.156 uM Rep 2 (0.156μM**)Float%
30% Inhibition @ 0.078 uM Rep 2 (0.078μM**)Float%
31% Inhibition @ 0.039 uM Rep 2 (0.039μM**)Float%
32% Inhibition @ 0.02 uM Rep 2 (0.02μM**)Float%
33% Inhibition @ 0.01 uM Rep 2 (0.01μM**)Float%
34% Inhibition @ 0.005 uM Rep 2 (0.005μM**)Float%
35VerificationString

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

Data Table (Concise)
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