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.

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.

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.

VEEV culture: VEEV TC-83 was used for screening. The VEEV stock was prepared in Vero76 cells using an initial stock obtained from Dr. Chung.

Compound Dosing/Plating: The positive control was MPA at 10uM final well concentration. The compounds were diluted in complete growth medium to 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume).

Single Dose Compound Preparation: The MLSMR library was plated at 20uM single dose concentration.

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 25uM and extending to .05uM over a 10-plate 1:2 serial dilution pattern. DMSO and compounds were diluted in assay media to 6x and 5uL was dispensed to assay plates. The final DMSO in the assay for all screening concentrations was 0.25%.

Virus Addition: VEEV stock was diluted in the culture media to 6.44 pfu/ml. (MOI 4e-5)

VEEV and Cell Plating: 3,000 cells/well alone or with VEEV virus at the previously indicated dilution(180,000 cells/ml) were plated in 25 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 in an actively humidified incubator with 5.0% CO2 at 37C for 72h and 95% 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 Perkin Elmer Envision multi-label reader.

Data was analyzed using ActivityBase software (IDBS, Inc, Guilford, UK). Thirty-two control wells containing cells only and twenty-four 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. The mean Z prime for the campaign was >/= 0.8. 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).

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 requested and 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 not tested in the confirmatory assay 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

Multiplexing: No

BSL: BSL2

Screening Concentration: 20

Screening Concentration Range Max: 25

Screening Concentration Range Min: 0.5

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

Grant Number: 1 R03 MH087448-01A1