BioActive Compounds: 7
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. Marintha Heil, Southern Research Institute
Award: 1R03 MH084847-01
West Nile virus (WNV) is a mosquito borne infectious agent that causes febrile illness and occasionally encephalitis. Outbreaks had been reported in Africa, Asia, and Europe since 1937. In 1999, the first case of WNV was detected in New York City from which time the virus spread rapidly west resulting in the largest epidemics of neuorinvasive WNV disease ever reported in the US. Currently, there is no vaccine for human use or antivirals that are available to treat WNV infections, leading to patient care which is mainly supportive. Hence this program will provide chemical probes from which to optimize leads for treatment of this emerging disease.
In the present assay, we tested total 27 analogs synthesized in SAR studies based on previously observed activity the West Nile Virus anti-viral assay. The assay measures cytopathic effect (CPE) induced in Vero E6 cells by WNV infection using cell viability as the end point. The screen for inhibitors of CPE caused by WNV infection involves challenging 3,000 cells with WNV virus at a multiplicity of infection (MOI) of 0.1 in the presence of the compounds. Cells were treated with compounds in a dose response manner over a 10 point 2-fold dilution series, ranging from 0.391 uM to 200 uM.
The WNV virus strain NY-99 stock was prepared from Vero E6 cells at the SRI BSL-3 facility. The assay includes both cell and virus controls and cell survival was measured 96 h post virus addition using CellTiterGlo viability reagent (Promega). Percent cell viability was calculated by using mean luminescence values of the virus-infected cells in the presence of compound divided by the uninfected cell control x 100. The percent cell viability was taken as an assessment of antiviral activity.
Cell Culture: Vero E6 cells were cultured in Eagles modified MEM (E-MEM) with 2 mM L-glutamine, 10% FBS and 100 U of penicillin, 100 ug of streptomycin per mL (culture media). The cells are maintained at 37C, 5.0% CO2 to 100% confluence being passaged every seven days. For cell plating, cells were detached from flask bottom by using Trypsin-EDTA solution and then re-suspended in a growth media
WNV culture: WNV strain NY-99 was used for screening. The WNV stock was prepared in Vero E6 cells using an initial stock obtained from Dr. Heil. Briefly, Vero E6 cells were grown in two T-150 plates to 90% confluence. After the supernatant was removed from the culture, 2ml of WNV at 10E7 pfu/ml was then added to the culture media. Virus was adsorbed to cells for 2 hours at 37C and then the fresh culture media was replaced. After four days incubation, the supernatant was harvested and the cell debris pelleted by low speed centrifugation. The cell debris was then spun down at 3,000 rpm for 10 minutes, and the supernatant was aliquoted (1ml per tube) and stored at -80C. These virus stocks were titrated in Vero E6 cells using agarose overlay plaque method and the titer was 8.3E7 pfu/ml.
Cell Plating: Once controls and compounds had been dispensed to the assay plates, 3,000 cells/well were plated in 15 uL using a Matrix WellMate. Plates were incubated overnight at 37C, 5.0% CO2 and high humidity. Then the plates were transferred to a BSL-3 containment laboratory.
Compound Dosing/Plating: Carrier control / compounds were diluted in complete growth medium to prepare a 6X concentrated dosing solution which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume). These plates were incubated briefly while awaiting mock virus or virus addition.
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 200uM. A total of ten concentrations were generated for the dose response curve with the lowest concentrations of 0.391 uM. Carrier control (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 0.5%.
Virus Addition: WNV stock was diluted in the culture media to 60,000 pfu/ml and 10 uL was added to the compound wells and the virus control wells (viral MOI of 0.1 ~ 0.2). Media only (mock virus) was added to the cell control wells. All additions were done using a Matrix WellMate housed in a class II Biosafety Cabinet within the BSL-3 laboratory. The plates were incubated in an actively humidified incubator with 5.0% CO2 at 37C for 96 h.
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 with an integration time of 0.1 s. This step was also performed within the BSL-3 facility.
Data Analysis: Thirty two control wells containing cells only (Cell control) , twenty four wells containing cells and virus (Virus control) 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. And also eight wells containing cells, virus and control drug, Mycophenolic Acid at 5 uM were included on each assay plate. The percent inhibition of virus induced cytopathic effect (CPE) was calculated as: 100*(Cmpd Lum - Median Virus Ctrl)/(Median Cell Ctrl - Med Virus Ctrl). The normalized % CPE inhibition was plotted against the tested compound concentrations. The IC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Outcome: Compounds that showed >=30% CPE inhibition for at least one concentration were defined as Active. If Inhibition at all doses was <30%, the compound was defined as Inactive.
Score: Because of the inherent error in all high throughput screens including the fallacy of over-interpreting single dose data, the following tiered scoring system has been implemented at SRSBSC. Compounds in the single dose primary screen are scored on a scale of 0-40, based on % CPE inhibition. Active compounds in the confirmatory screen are scored based on EC50 results on a tier of 40-80 with compounds failing confirmation scoring 0. Synthesized/Analog active compounds are also scored based on EC50 results and fall into the most reliable tier where actives will be scored from 80-100. Inactive compounds show a score of 0.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)