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BioAssay: AID 624449

A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (6)

Venezuelan Equine Encephalitis Virus is a mosquito transmitted virus effecting both humans and horses. The last major outbreak in 1995, reported an estimated 70,000 - 100,000 humans infected and a similar number of known horse infections. In humans the symptoms present as fever, headache, and encephalitis. Although the mortality rate is below 1%, the neurological disease is apparent in >/= 14% more ..
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 Tested Compounds
 Tested Compounds
All(7)
 
 
Active(4)
 
 
Inactive(3)
 
 
 Tested Substances
 Tested Substances
All(7)
 
 
Active(4)
 
 
Inactive(3)
 
 
 Related BioAssays
 Related BioAssays
AID: 624449
Data Source: Southern Research Specialized Biocontainment Screening Center (VEE_AV_06)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-08-01
Hold-until Date: 2013-07-13
Modify Date: 2013-07-13

Data Table ( Complete ):           Active    All
BioActive Compounds: 4
Depositor Specified Assays
Show more
AIDNameTypeComment
588727A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83confirmatoryPrimary and Confirmatory Screen (TC-83)
602241A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (2)confirmatoryConfirmatory Screen (TC-83)
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)confirmatoryConfirmatory Screen (TC-83)
624063A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV TC-83 (4)confirmatoryConfirmatory Screen (TC-83)
624284A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (5)confirmatoryConfirmatory Screen (TC-83)
602242A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526confirmatoryConfirmatory Screen (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 (Vero76) Counter Screen
602240Vero 76 Cytoxicity Assay for VEEV Compounds (2)confirmatoryCytotoxicity (Vero76) Counter Screen
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)confirmatoryCytotoxicity (Vero76) Counter Screen
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)confirmatoryCytotoxicity (Vero 76) counter screen
624295Vero 76 Cytoxicity Assay for VEEV Compounds (6)confirmatoryCytotoxicity (Vero 76) counter screen
602307Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain)otherVirus titer reduction assay TC-83
624286Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (3)otherVirus titer reduction assay TC-83
602411Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (2) Trinidad donkey strainotherVirus titer reduction assay (WT-Trinidad Donkey)
602470A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) AssayconfirmatoryRespiratory Syncytial Virus (RSV) Counter Screen
602483HEp2 Cytoxicity Assay for VEEV Compounds (1)confirmatoryCytotoxicity (Hep-2) counter screen
623935A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)confirmatoryChikungunya Virus Counter Screen
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)confirmatoryChikungunya VirusToxicity Screen
588723A Cell-based HTS to discover molecules that inhibit VEEV, encephalitic alphavirus - Project Summarysummary
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)other
651734A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (7)confirmatory
651735Vero 76 Cytoxicity Assay for VEEV Compounds (8)confirmatory
651738A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (2)confirmatory
651874A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (2)confirmatory
651883Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (strain Trinidad donkey) (03)other
651884A Cell-Based Counter Screen testing Compounds that Inhibit VEEV TC-83, in strain KU V3526 (2)confirmatory
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 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 a mosquito transmitted virus effecting both humans and horses. The last major outbreak in 1995, reported an estimated 70,000 - 100,000 humans infected and a similar number of known horse infections. In humans the symptoms present as fever, headache, and encephalitis. Although the mortality rate is below 1%, the neurological disease is apparent in >/= 14% of patients. There is no FDA approved vaccine or therapeutic and supportive care is limited. Thus, prophylaxis and efficacious treatments are critical to minimizing the impact of the transmissible disease on human and equines.

The US Army has been developing vaccines for VEEV as they appreciate the impact of the disease on soldiers as well as its potential use as a bioweapon. The vaccines, which are comprised of attenuated live virus, are still in the investigational new drug (IND) stage and are only available through the Special Immunization Program at United State Army Medical Research Institute of Infectious Diseases (USAMRIID) for protecting personnel working with the virus. A few other vaccine candidates are in the IND stage, such as formalized killed TC-83 vaccine and the live attenuated V3526 vaccine. Again those vaccines have not been FDA-approved due to lack of efficacy and adverse effects seen during clinical trials.

The assay provider has developed and validated a 384-well cell-based assay that measures CPE induced in Vero 76 by VEEV infection, using a luminescent-based detection system for signal endpoint. The overall goal of this project is to discover novel compounds with anti-VEEV replication efficacy and minimal cytotoxicity in vitro.
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 50 mL of heat-inactivated FBS (Gibco, Cat. No. 10082147 ) was added to 500mL of Dulbecco's Modified Eagle Medium (Gibco, Cat. No. 11995-073).

Virus : TC-83 strain was obtained from Dr. Brett Beitzel from United State Army Research Medical Research Institute for Infectious diseases and amplified in BHK C-21 cell line once.

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. Viruses, TC-83 strain, 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). Four ribavirin positive control wells were included on each plate for quality control purposes. To quantify the viral cytopathic effect, 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.

Of the compounds tested, those that showed 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 the IC50 of active compounds is used for purified/synthesized compounds to indicate a high level of confidence in the results. Inactive compounds are given a score of 0.
Categorized Comment
BAO: version: 1.4b1090

BAO: bioassay specification: assay stage: secondary: alternate confirmatory

BAO: bioassay specification: assay biosafety level: BSL2

BAO: assay format: cell based format

BAO: bioassay specification: assay measurement type: endpoint assay

BAO: format detail: reagent: inducer: none

BAO: meta target: molecular target: protein target: enzyme: transferase: kinase

BAO: meta target: biological process target: viral reproduction

BAO: meta target detail: binding reporter specification: interaction: protein:small molecule

BAO: assay design: viability reporter:atp content

BAO: detection technology: luminescence: chemiluminescence

Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Average IC50 ModifierString
2Average IC50*FloatμM
3IC50 Modifier Rep 1String
4IC50 Rep 1FloatμM
5IC50 Rep 2FloatμM
6% Inhibition @ 25 uM Rep 1 (25μM**)Float%
7% Inhibition @ 12.5 uM Rep 1 (12.5μM**)Float%
8% Inhibition @ 6.25 uM Rep 1 (6.25μM**)Float%
9% Inhibition @ 3.13 uM Rep 1 (3.13μM**)Float%
10% Inhibition @ 1.56 uM Rep 1 (1.56μM**)Float%
11% Inhibition @ 0.78 uM Rep 1 (0.78μM**)Float%
12% Inhibition @ 0.39 uM Rep 1 (0.39μM**)Float%
13% Inhibition @ 0.2 uM Rep 1 (0.2μM**)Float%
14% Inhibition @ 25 uM Rep 2 (25μM**)Float%
15% Inhibition @ 12.5 uM Rep 2 (12.5μM**)Float%
16% Inhibition @ 6.25 uM Rep 2 (6.25μM**)Float%
17% Inhibition @ 3.13 uM Rep 2 (3.125μM**)Float%
18% Inhibition @ 2.5 uM Rep 2 (2.5μM**)Float%
19% Inhibition @ 1.56 uM Rep 2 (1.56μM**)Float%
20% Inhibition @ 1.25 uM Rep 2 (1.25μM**)Float%
21% Inhibition @ 0.78 uM Rep 2 (0.78μM**)Float%
22% Inhibition @ 0.63 uM Rep 2 (0.63μM**)Float%
23% Inhibition @ 0.39 uM Rep 2 (0.39μM**)Float%
24% Inhibition @ 0.31 uM Rep 2 (0.31μM**)Float%
25% Inhibition @ 0.195 uM Rep 2 (0.195μM**)Float%
26% Inhibition @ 0.156 uM Rep 2 (0.156μM**)Float%
27% Inhibition @ 0.08 uM Rep 2 (0.08μM**)Float%
28% Inhibition @ 0.04 uM Rep 2 (0.04μM**)Float%
29% Inhibition @ 0.02 uM Rep 2 (0.02μM**)Float%
30VerificationString

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

Data Table (Concise)
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