A Counter Screen of Venezuelan Equine Encephalitis Virus (VEEV) Inhibitors in a Cell-Based Anti-Respiratory Syncytial Virus (RSV) Assay (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% more ..
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.
This assay is to test anti- Respiratory Syncytial Virus (RSV) activity of selected compounds from anti-VEEV screening in order to evaluate the specificity of the compounds for remotely related other viruses. RSV is a member of the family Paramyxoviridea, which is not phylogenically related with VEEV, alphavirus. We hypothesized that a compound targeting host cells would show a broad spectrum antiviral activity, conversely an alphavirus specific compound won't show the activity against RSV.
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.
RSV culture: Human respiratory syncytial virus (HRSV) strain Long (ATCC VR-26) was used for screening. The RSV stock was prepared in HEp-2 cells using an initial stock obtained from ATCC. Briefly, HEp-2 cells were grown in two T-175 flasks to 50% confluence in Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12), pH 7.5 with 2.5 mM L-glutamine, 2% FBS and 125 U of penicillin, 125 ug of streptomycin per ml. 0.2 ml of RSV was added to 25 ml of CDMEM/F12. After three days incubation at 37 degrees C, 5% C02 and high humidity, the supernatant was harvested and the cell debris pelleted by centrifuging at 1,000 rpm for 5 minutes at 18 degrees C. Trehalose and FBS were added to a final concentration of 10% each and the supernatant was aliquoted (1 ml per tube) and stored at -80 degrees C. These virus stocks were titrated in HEp-2 cells using an agarose overlay plaque method and the titer was 1.0 E7 pfu/ml.
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 25 uM to 0.005 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 received fifteen uL of RSV diluted in Complete DMEM/F12 media ( 40,000 pfu/mL, final 0.05 MOI). 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: Eight control wells containing cells only and eight wells containing cells and virus were included on each assay plate and used to calculate Z' value for each plate and to normalize the data on a per plate basis. 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). To quantify the viral cytopathic effect, IC50s were calculated for each substance showing at least 30% inhibition at any concentration 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.
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.
Phenotypic Screen: Yes
Screening Concentration Range Max: 25
Screening Concentration Range Min: 0.005
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)