A Titer Reduction Assay to evaluate Selected Compounds as new Inhibitors of Respiratory Syncytial Virus (RSV)
Respiratory syncytial virus (RSV) 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 those with compromised cardiac, pulmonary, or immune systems. The economic impact of RSV infections due to hospitalizations and indirect medical costs more ..
BioActive Compounds: 51
Southern Research's Specialized Biocontainment Screening Center (SRSBSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Assay Provider: Dr. William Severson, Southern Research Institute
Award: 1R03 MH082403-01
Respiratory syncytial virus (RSV) 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 those with compromised cardiac, pulmonary, or immune systems. The economic impact of RSV infections due to hospitalizations and indirect medical costs is > $ 650 million annually. The only FDA approved therapies are ribavirin and the prophylactic humanized monoclonal antibody (Synagis(R) from MedImmune). Hence, this program will provide chemical probes for optimization to supplement existing chemotherapeutics.
We identified 2,465 compounds that showed > 22% inhibition in the primary cell-based HT screen. The hits were evaluated by measuring their antiviral activity, cell toxicity and selectivity in dose-response experiments. The inhibitory activity of the compounds were confirmed in dose-response experiments and verified in a titer reduction assay which measured the difference in viral titer between non-treated and treated cells. A compound that inhibits viral replication results in a reduction in progeny virus compared to virus cell controls. The titer reduction assay involves challenging HEp-2 cells (~80% confluent) with RSV Long strain at a multiplicity of infection (MOI) of 20 in the presence of compounds. The assay measures the progeny viral titer using the TCID50 method. The progeny virus was serially diluted 10-fold and the suspension transferred to fresh HEp-2 cells in 384-well plates. The plates were developed 6 days post infection using Cell Titer Glo to evaluate infection in each well and the TCID50 values were calculated by the Reed & Muench method.
Compounds Screened: 51 compounds were selected for TCID50 analysis based on the criteria of activity: an efficacy EC50 value of <10 uM and with toxicity to efficacy SI50 of >3. These compounds were resupplied from the University of Kansas Chemistry center for a dose-response confirmatory assay.
Cell Culture: HEp-2 cells were cultured in Optimem 1 with 2 mM L-glutamine, and 10% FBS (culture media). The cells are maintained at 37C, 5.0% CO2 to 100% confluence being passaged every three days. For cell plating, cells were detached from flask bottom by using 0.05% Trypsin-EDTA solution and then re-suspended in a growth media.
RSV culture: RSV strain Long 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. 0.1 mL of infected media per T175 for 1.5 hours, wash, and replenish with 25mL media for a 2 day incubation. The 0.1 mL of infected media originated from a three day culture in a 6 well plate.
After two days incubation at 37C, 5% C02 and high humidity, the supernatant was harvested and the cell debris pelleted by centrifuging at 1,000 rpm for 5 minutes at 18C. Trehalose and FBS were added to a final concentration of 10% each and the supernatant was aliquoted (1 ml per tube) and stored at -80C. These virus stocks were titrated in HEp-2 cells using an agarose overlay plaque method and the titer was 1.0E11 pfu/ml.
Cell Plating: HEp-2 cells were seeded at 70% confluence in a 12-well plate in a volume of 1mL and incubated for overnight at 37C with 5% CO2. Plates were incubated overnight at 37C, 5.0% CO2 and high humidity.
Control and Drug Preparation: Carrier Control consisted of DMSO diluted in assay media to 0.5% and 1000uL was dispensed to both cell and virus control wells of 12- well tissue culture treated plates. Test compounds were diluted in media to be at target concentration with a DMSO concentration of 0.5%.
Virus Addition: RSV stock was diluted in the culture media to 2.0E08 pfu/ml and 100 uL was added to the test wells and the virus control wells (viral MOI of 20). Media only (mock virus) was added to the cell control wells. All additions were performed in a class II Biosafety Cabinet. The plates were incubated in an actively humidified incubator with 5.0% CO2 at 37C for 48 h.
Titration of Progeny viruses
Titer of progeny viruses produced from the cell was measured by TCID50 assay in 384-well plate format with 4 wells per dilution of virus. 10 ul of 10-fold serial dilutions of progeny virus containing medium from respective samples (drug treated or untreated) were transferred to infect fresh Hep-2 cells in a 384-well format. The cell plates were incubated at 37C, 5% CO2, and high humidity for an additional 6 days. The Cell Titer Glo assay was used to determine viability of the cells. The assay plates were equilibrated to room temperature for 10 minutes and then an equal volume of Cell Titer-Glo reagent (Promega Inc.) was added to each well. Plates were incubated for 10 min at room temperature and luminescence was measured using a multi-label plate reader with an integration time of 0.1 s. A well showing luminescence signal less than mean of non infected control signal minus 5 times of standard deviation of the control was regarded as positive for infection.
Of the 51 compounds selected for dose response testing, all showed more than 10 fold reduction in the progeny titer (<-1 of log reduction) for at least one concentration, and were defined as Active. Scoring for this assay was based on the decreasing shift if virus titer relative to the control.
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 subsequent testing of purchased compounds, those showing activity are scored on a scale of 41-80, in this case, based on the maximum observed decrease in titer reduction. Compounds that did not confirm as actives are given the score of 0. Synthesized compounds are rated on the highest tier of 81-100.
Possible artifacts in this assay include, but are not limited to, compounds that precipitate or are cytotoxic.
** Test Concentration.
Data Table (Concise)