A Cell-Based Confirmatory Screen for Compounds that Inhibit VEEV, TC-83 (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 ..
BioActive Compounds: 5
Depositor Specified Assays
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.
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.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Using the criteria of 3 standard deviations greater than the mean, a hit cutoff of 13.69% Inhibition was used to define activity in the primary screen. Of the compounds available for testing in the confirmatory screen, those that showed at least 30% inhibition at any tested dose were considered active. Compounds that exceeded the activity cutoff in the primary screen, but were available for confirmatory testing were designated with the outcome of inconclusive.
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.
Phenotypic Screen: Yes
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?: Yes
Used during SAR?: Yes
* Activity Concentration. ** Test Concentration.
Data Table (Concise)