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BioAssay: AID 651738

A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus) (2)

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 more ..
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 Tested Compounds
 Tested Compounds
All(13)
 
 
Active(1)
 
 
Inactive(12)
 
 
 Tested Substances
 Tested Substances
All(13)
 
 
Active(1)
 
 
Inactive(12)
 
 
 Related BioAssays
 Related BioAssays
AID: 651738
Data Source: Southern Research Specialized Biocontainment Screening Center (VEEhits_CHKVscreen02)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-31
Hold-until Date: 2013-10-24
Modify Date: 2013-10-24

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
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: Virus titer reduction assay TC-83
602411Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (2) Trinidad donkey strainOtherdepositor-specified cross reference: Virus titer reduction assay (WT-Trinidad Donkey)
602439Vero 76 Cytoxicity Assay for VEEV Compounds (3)Confirmatorydepositor-specified cross reference: Cytotoxicity (Vero76) Counter Screen
602455A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (3)Confirmatorydepositor-specified cross reference: Confirmatory Screen (TC-83)
602470A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) AssayConfirmatorydepositor-specified cross reference: Respiratory Syncytial Virus (RSV) Counter Screen
602483HEp2 Cytoxicity Assay for VEEV Compounds (1)Confirmatorydepositor-specified cross reference: Cytotoxicity (Hep-2) counter screen
602484A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, Trinidad Donkey (TrD) (1)Confirmatorydepositor-specified cross reference: confirmatory Trinidad Donkey
623935A Cell-Based Secondary Assay for Compounds that Inhibit VEEV, TC-83 and other Alphaviruses (Chikungunya virus)Confirmatorydepositor-specified cross reference: Chikungunya Virus Counter Screen
623936Vero 76 Cytoxicity Assay for VEEV Compounds (4)Confirmatorydepositor-specified cross reference: Cytotoxicity (Vero 76) counter screen
624063A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV TC-83 (4)Confirmatorydepositor-specified cross reference: Confirmatory Screen (TC-83)
624284A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (5)Confirmatorydepositor-specified cross reference: Confirmatory Screen (TC-83)
624286Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (3)Otherdepositor-specified cross reference: Virus titer reduction assay TC-83
624295Vero 76 Cytoxicity Assay for VEEV Compounds (6)Confirmatorydepositor-specified cross reference: Cytotoxicity (Vero 76) counter screen
624449A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (6)Confirmatorydepositor-specified cross reference: Confirmatory Screen (TC-83)
624450Vero 76 Cytoxicity Assay for VEEV Compounds (7)Confirmatorydepositor-specified cross reference: Cytotoxicity (Vero 76) counter screen
651578Virus Titer Reduction Secondary Screen for Compounds that Inhibit VEEV (TC-83 strain) (4)Otherdepositor-specified cross reference: Virus titer reduction assay TC-83
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
624069Vero 76 Cytoxicity Assay for VEEV Compounds (5)Confirmatorysame project related to Summary assay
651734A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (7)Confirmatorysame project related to Summary assay
651735Vero 76 Cytoxicity Assay for VEEV Compounds (8)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

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.

VEEV is a member of the alphavirus family, and there is a possibility that compounds that effectively inhibit VEEV may have broader activity against other alphaviruses. Southern Research's Specialized Biocontainment Screening Center (SRSBSC) has screened compounds that were effective against VEEV in a 96-well microplate assay for broader activity against an additional alphavirus (Chikungunya (CHIKV) virus).

SRSBSC has developed a 96-well cell-based assay that measures CPE induced in Vero 76 cells by CHIKV infection, using a luminescent-based detection system for signal endpoint. The overall goal of this project is to discover novel compounds with broad VEEV and CHIKV inhibition and minimal cytotoxicity in vitro.
Protocol
Cell Culture: Vero 76 cells obtained from ATCC (CRL-1587) were cultured and maintained in MEM-E (Invitrogen, 10370-088) with 10% Hi-FBS (Invitrogen 16000), 1% Penicillin/Streptomycin/L-glutamine (Invitrogen 10378-024) and 1% HEPES (Invitrogen 15630-080). The cells are maintained at 37C, 5.0% CO2 to 100% confluence being passaged 1:4 every 3-4 days. For cell plating, cells were detached from flask bottom by using Trypsin-EDTA solution and then re-suspended in a growth media. Cells were passaged no more than ten times after being thawed.

Compound Dosing/Plating: No positive control. The compounds were diluted in complete growth medium to 6X concentrated dosing solution which was dispensed into 96-well black clear-bottom tissue culture treated plates (25 uL volume).

Dose Response Compound Preparation: The compounds were tested in a dose response format using a 1:2 serial dilution with the highest concentrations starting at 100 uM and extending to 0.78 uM over a 8-dose 1:2 serial dilution pattern. DMSO and compounds were diluted in assay media to 4x and 25uL was dispensed to assay plates. The final DMSO in the assay for all screening concentrations was 0.25%.

Virus Addition: CHIKV stock was diluted in the culture media to 100 TCID50s/25 uL, and 25 uL were added to each test well.

Cell Plating and virus addition: 6,000 cells/well (120,000 cells/ml) were plated in 50 uL using a Matrix WellMate. All additions were done using a Matrix WellMate housed in a class II Biosafety Cabinet within the BSL-2 laboratory. The plates were incubated overnight in an actively humidified incubator with 5.0% CO2 at 37C for 18h and 95% humidity. Compounds (25 uL) were added after the cells had adhered to the plate, and CHIKV virus was added immediately after compound addition. The plates were incubated for 72 h in an actively humidified incubator with 5.0% CO2 at 37C for 72h and 95% humidity, and then endpoint reagent was added.

Endpoint Read: The assay plates were equilibrated to room temperature for 30 minutes and then 100 uL 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 Perkin Elmer Envision multi-label reader.

Data was analyzed using ActivityBase software (IDBS, Inc, Guilford, UK). Eight control wells containing cells only and eight wells containing cells and virus were included on each assay plate and used to calculate S/B values 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).
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 50% 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 - additional comments and annotations
From BioAssay Depositor:
BAO: assay design: viability reporter:atp content
BAO: assay format: cell based format
BAO: bioassay specification: assay biosafety level: BSL2 and BSL3
BAO: bioassay specification: assay measurement type: endpoint assay
BAO: bioassay specification: assay stage: secondary: alternate confirmatory
BAO: detection technology: luminescence: chemiluminescence
BAO: meta target detail: binding reporter specification: interaction: protein:small molecule
BAO: meta target: biological process target: viral reproduction
BAO: meta target: molecular target: protein target: enzyme: transferase: kinase
BAO: version: 1.4b1090
From PubChem:
Assay Format: Cell-based
Assay Cell Type: VERO 76
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1IC50*FloatμM
2% Inhibition @ 50 uM Rep 1 (50μM**)Float%
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 @ 5 uM Rep 1 (5μM**)Float%
7% Inhibition @ 3.13 uM Rep 1 (3.13μM**)Float%
8% Inhibition @ 2.5 uM Rep 1 (2.5μM**)Float%
9% Inhibition @ 1.56 uM Rep 1 (1.56μM**)Float%
10% Inhibition @ 1.25 uM Rep 1 (1.25μM**)Float%
11% Inhibition @ 0.78 uM Rep 1 (0.78μM**)Float%
12% Inhibition @ 0.63 uM Rep 1 (0.63μM**)Float%
13% Inhibition @ 0.39 uM Rep 1 (0.39μM**)Float%
14% Inhibition @ 0.31 uM Rep 1 (0.31μM**)Float%
15% Inhibition @ 0.156 uM Rep 1 (0.156μM**)Float%
16% Inhibition @ 0.078 uM Rep 1 (0.078μM**)Float%
17% Inhibition @ 0.039 uM Rep 1 (0.039μM**)Float%
18% Inhibition @ 25 uM Rep 2 (25μM**)Float%
19% Inhibition @ 12.5 uM Rep 2 (12.5μM**)Float%
20% Inhibition @ 6.25 uM Rep 2 (6.25μM**)Float%
21% Inhibition @ 5 uM Rep 2 (5μM**)Float%
22% Inhibition @ 3.13 uM Rep 2 (3.13μM**)Float%
23% Inhibition @ 2.5 uM Rep 2 (2.5μM**)Float%
24% Inhibition @ 1.56 uM Rep 2 (1.56μM**)Float%
25% Inhibition @ 1.25 uM Rep 2 (1.25μM**)Float%
26% Inhibition @ 0.78 uM Rep 2 (0.78μM**)Float%
27% Inhibition @ 0.63 uM Rep 2 (0.63μM**)Float%
28% Inhibition @ 0.39 uM Rep 2 (0.39μM**)Float%
29% Inhibition @ 0.31 uM Rep 2 (0.31μM**)Float%
30% Inhibition @ 0.195 uM Rep 2 (0.195μM**)Float%
31% Inhibition @ 0.156 uM Rep 2 (0.156μM**)Float%
32% Inhibition @ 0.078 uM Rep 2 (0.078μM**)Float%
33% Inhibition @ 0.039 uM Rep 2 (0.039μM**)Float%
34% Inhibition @ 0.02 uM Rep 2 (0.02μM**)Float%
35% Inhibition @ 0.01 uM Rep 2 (0.01μM**)Float%
36% Inhibition @ 0.005 uM Rep 2 (0.005μM**)Float%
37VerificationString

* 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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