Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Centers Network (MLPCN)
Assay Provider: Dr. William Severson, Southern Research Institute
Grant number: 1 R03 MH082403-01A1

Assay Rationale and Summary: Currently, there are no commercially available vaccines to protect humans against Respiratory syncytial virus (RSV). RSV is associated with substantial morbidity and mortality and is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age. Nevertheless, severe lower respiratory tract disease may occur at any age, especially among the elderly or among those with compromised cardiac, pulmonary, or immune systems. The existing therapies for the acute infection are ribavirin and the prophylactic humanized monoclonal antibody (Synagis from MedImmune) that is limited to use in high risk pediatric patients. The economic impact of RSV infections due to hospitalizations and indirect medical costs is greater than $ 650 million annually. The assay provider has developed and validated an HTS assay that measures cytopathic effect (CPE) induced in HEp-2 cells by RSV infection, using a luminescent-based detection system for signal endpoint. We anticipate that the proposed studies utilizing the Molecular Libraries Probes Production Network (MLPCN) HTS resources will generate multiple scaffolds targeting various junctures in the RSV viral lifecycle. These may be furthered developed into probes to construct novel single or combination therapeutics.

Cell Culture: HEp-2 cells (ATCC CCL-23, American Tissue Culture Type) were maintained as adherent cell lines in Optimem 1 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: 50 mL Pen/Strep/Glutamine (Gibco, Cat. No. 10378) was added to four liters of room temperature DMEM/F12 (Sigma, Cat. No. D6434) and the pH adjusted to 7.5 using 1N NaOH. The medium was sterile filtered through a 0.2 um filter 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 (CDMEM/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) fast freeze using 100% Ethanol dry ice for five minutes 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 6.0E+07 pfu/ml.

Dose Response Compound Preparation: For dose response screening, compounds or carrier control (DMSO) were diluted to 6x 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 50 uM to 0.39 uM and a final DMSO concentration of 0.5%). Twenty ul of each dilution was dispensed to assay plates (3% DMSO) in triplicate.
Control Drug: The positive control drug for this assay, ribavirin [1] (No. 196066, MP Biomedicals, Solon, OH) was solubilized in DMSO. It was diluted and added to the assay plates as described for test compounds. Final concentration for ribavirin was 100uM. All wells contained 0.5% DMSO.

Preparation of HEp-2 cells: Cells were harvested and resuspended to 178,000 cells per ml in Complete DMEM/F12.

Assay Set up: Sixty ul of HEp-2 cells (10,595 cells/well) forty ul of media and 20 ul of 3% DMSO were plated in the cell control wells. Sixty ul of HEp-2 cells (10,595 cells/well), forty ul of a 1:500 dilution of virus (viral MOI = 0.45) and 20 ul of compound or ribavirin control drug were added to the virus control and compound wells. All cell plating was conducted using a Matrix WellMate and cells were maintained at room temperature with stirring during the plating process. The assay plates were incubated for six days at 37 degrees C, 5% CO2 and 90% relative humidity.

Data Analysis: Eight control wells containing cells only and 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. 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.

Compounds that demonstrated at least 50% inhibition and 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 initial 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. In assays using purified and synthesized compounds a scale of 81-100 based on the IC50 result is used to denote a high level of confidence in both the substance and the data. Compounds that did not confirm activity were given the score of 0.

Grant Number: 1R03 MH084847-01