An HTS Cytotoxicity Screen to evaluate 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: 2107
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.
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.
Assay Set up: Twenty five ul of uninfected HEp-2 cells were plated in the cell control wells. 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 #L) 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 were included on each assay to normalize the data on a per plate basis. Results are reported as percent (%) cell viability, calculated as: % cell viability = 100*(Test Cmpd/Median Cells). To quantify the cytotoxic effect of the compounds, CC50s using % cell viability 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.
From the primary screen for inhibition of CPE, 2,465 compounds were pursued for confirmatory and cytotoxicity testing. Of the compounds tested in this cytotoxicity screen, 2,107 showed a decrease in viability to less than 70%, and were considered active (toxic).
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 CC50 result while compounds that did not confirm as actives were given the score of 0.
Categorized Comment - additional comments and annotations
Assay Type: Toxicity
Assay Cell Type: HEp-2
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)