A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV)
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, more ..
BioActive Compounds: 409
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 a 384-well cell-based assay that measures 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 5% fetal bovine serum (FBS) at 37oC in a humidified 5% CO2 atmosphere. Cells were passaged as needed and harvested from flasks using 0.25% 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.
Infectious material - Frozen Infected Virus Cell Preparation: Two vials of RSV (strain Long) containing 1 x 107 pfu/mL was thawed using an Eppendorf thermomixer for 13 min at 15 degrees C, with shaking at 350 rpm. Two mL of the virus stock was added to a T-225 flask containing 3.0 x 108 HEp-2 cells in 30 mL Complete DMEM/F12. The cells were incubated for 18 - 20 h at 37 degrees C, 5% C02, 90% relative humidity. The medium was aspirated and the cells washed with 10 mL PBS without Mg2+ or Ca2+. Cells were harvested from flasks using 0.25% trypsin-EDTA. The cells were resuspended in a freezing medium of 95% fetal calf serum and 5% DMSO to a final cell density of 2 x 106 cells/mL. One mL aliquots of this virus infected cell suspension were dispensed to cryovials and cells were rate frozen to -80 degrees C. Frozen infected cells were then transferred to -150 degrees C for long term storage.
Dose Response Compound Preparation: For dose response screening, compounds or carrier control (DMSO) were diluted to 6x in Complete DMEM/F12 and 5ul was dispensed to assay plates (3% DMSO). Test compounds were serially diluted in a plate to plate matrix or "stacked plate" matrix. All 320 compounds in a source plate were diluted together resulting in a 10 point dose response dilution series. It was visualized as a serial dilution series proceeding vertically through a stack of plates with the high dose plate on top and the low dose plate on the bottom. (final plate well concentration ranging from 50 uM to 0.097 uM and a final DMSO concentration of 0.5%).
Control Drug: The positive control drug for this assay, ribavirin  (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 35uM. All wells contained 0.5% DMSO.
Preparation of HEp-2 cells: Cells were harvested and resuspended to 80,000 cells per ml in Complete DMEM/F12.
Frozen infected HEp-2 cells: Cells were thawed in a room temperature water bath with gentle agitation. The tube was inverted 5-10. Cells were diluted to 80,000 cells per ml by adding the contents of the vial to 24 ml of cold (4 degrees C) media.
Assay Set up: Twenty five ul of uninfected HEp-2 cells were plated in the cell control wells. Frozen infected cells were combined with uninfected HEp-2 cells at a 1:100 ratio. Twenty five ul of the cell mixture was 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.
Endpoint Read: Following the six day incubation period, the assay plates were equilibrated to room temperature for 30 min and an equal volume (30 uL) of Cell Titer-Glo reagent (Promega Inc.) was added to each well using a WellMate (Matrix, Hudson, NH) and plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a Perkin Elmer EnvisionTM multi-label reader (PerkinElmer, Wellesley, MA) with an integration time of 0.1 s.
Data Analysis: 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 overall Z score for the campaign were >= 0.7. 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). Eight ribavirin positive control wells were included on each plate for quality control purposes, but were not used in Z' calculations.
Using the criteria of 3 standard deviations greater than the mean, a hit cutoff of 22.3% Inhibition was used and resulted in 7,583 active compounds in the primary screen. Of those, only 2,465 were pursued in the confirmatory screen. 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 22.3% Inhibition was used and resulted in 7,583 active compounds in the primary screen. Of those, only 2,465 were pursued in the confirmatory screen. Of the compounds tested in the confirmatory screen, 409 compounds showed at least 50% inhibition and were considered active. Compounds that exceeded the activity criteria in the primary screen, but were not tested in the confirmatory assay are 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)