Cell growth conditions and media
HEp-2 cells (ATCC CCL-23, American Tissue Culture Type) were maintained as adherent cell lines in Opti-MEM1® (Invitrogen) with 2 mM L-glutamine and 10% fetal bovine serum (FBS) at 37°C in a humidified 5% CO2 atmosphere. Cells were passaged as needed and harvested from flasks using 0.25% trypsin-EDTA.
Preparation of Complete DMEM/F12 was as follows: 50 mL Pen/Strep/Glutamine (Gibco) was added to four liters of room temperature DMEM/F12 (Sigma) and the pH adjusted to 7.5 using 1 N NaOH. The medium was sterile filtered (0.2 μm) and 10 mL of HI-FBS (Gibco) was added per 500 mL of media.
Human respiratory syncytial virus (hRSV) strain Long (ATCC VR-26) was used for assays. Virus was serially diluted and a dilution of 1:10 was used to amplify the seed stock. Briefly, a TCID50 format of 10-fold serial dilutions (from 10-1 to 10-7) was used to dilute the virus. HEp-2 cells grown in a 384-well plate were infected with hRSV. Plates were incubated at 37°C, 5% CO2, and 90% relative humidity for four days. Supernatant from the 384-well plate’s highest viral dilution was used to infect a single well of HEp-2 cells in a 6-well plate format containing approximately 2.4 × 106 cells/well. A two mL volume of Complete-Opti-MEM1 (C-Opt1) (Gibco) containing 10% FBS per well was removed and replaced with 100 μL of C-Opt1. Virus culture supernatant from the 384-well TCID50 was added to 100 μl C-Opt1 and incubated at 37°C, 5% CO2, and 90% relative humidity for 1.5 h rotating every 30 min to facilitate infection. The media was removed and replaced with 2 mL of C-Opt1 and incubated at 37°C, 5% CO2, and 90% relative humidity. After 72 h, the supernatant was removed and the cell debris pelleted by centrifugation at 300 × g, 5 min, at 18°C. One T-175, containing 4.78 × 106 HEp-2 cells was incubated overnight and used to amplify the virus. After 18 h, media was removed, cells were washed with 10 mL Complete-DMEM/F12 (2% FBS, 1.25% P/S/G, pH 7.5) and replenished with 4 mL C-DMEM/F12. A 100 μL sample of clarified hRSV was added to a T-175 and incubated for 1.5 h at 37°C, 5% CO2, and 90% relative humidity. The media was removed and replenished with 25 mL of C-DMEM/F12, and incubated at 37°C, 5% CO2, and 90% relative humidity for 48 h. The media was transferred to a 50 mL conical tube and cell debris pelleted at 300 × g, 5 min, at 18°C. Trehalose and FBS were added to a final concentration of 10% (v/v) each for preservation  and the supernatant was aliquoted (1 mL per tube), fast frozen in 100% EtOH/dry ice and stored at –150°C. Virus stocks titers were quantified in HEp-2 cells using an agarose overlay plaque method. The titer of the virus was 1.0 × 107 pfu/mL.
Infectious material: Frozen infected virus cell preparation
Preparation of the frozen hRSV-infected HEp-2 cells has been previously described . Briefly, a T-225 flask containing 3.0 × 108 HEp-2 cells in 30 mL Complete DMEM/F12, pH 7.5, was grown to 95% confluence. Two mL hRSV (strain Long) containing 1 × 107 pfu/mL was added to the flask and incubated for 18 – 20 h at 37°C, 5% C02, 90% relative humidity. After incubation, 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. Cells were centrifuged at 300 × g for 10 min and re-suspended in 95% FBS, 5% DMSO at a concentration of 2 × 106 cells/mL. The cells were determined to be at least 99% viable. The cells were aliquoted in 1 mL aliquots, rate frozen at –1°C/min to -80°C and stored at –150°C. Viability was also evaluated when thawed and determined to be at least 98.5%. We confirmed the percentile of infected cells in two ways; immunostaining and cell counting using FACS and a limited dilution methodology.
FACS analysis of frozen infected cells
Frozen hRSV-infected and un-infected HEp-2 cells (2 × 106 cells) were centrifuged at 300 × g for 5 min and the supernatant removed. Cell pellets (uninfected and hRSV-infected) were fixed in 1 mL of 4% paraformaldehyde for 15 min on ice. Cells were washed twice in 1 mL staining buffer (1X - Dulbecco's Phosphate Buffered Saline Solution [DPBS], 2% FCS) centrifuging at 300 × g for 5 min between washes. Cells were resuspended in 1 mL of staining buffer and 2 × 105 cells were aliquoted into 12 × 75 mm tissue culture tubes. Dilutions (1:5000, 1:2000, 1:1000, 1:500) of mouse monoclonal [4 clone blend] to hRSV(Abcam) was added to the cells, incubated for 30 min on ice and washed twice in 3.5 mL staining buffer. Secondary antibody (goat anti-mouse IgG/M conjugated to FITC (BD Pharmingen) was added, incubated for 30 min on ice and washed twice with 3.5 mL DPBS. Cells were resuspended in 0.4 mL DPBS and analyzed on a FACSCalibur flow cytometer. Controls included unstained cells, cells stained with either the primary or secondary antibody and uninfected cells stained with both antibody reagents.
Compound libraries and controls
The positive control drug for this assay, ribavirin (MP Biomedicals, Solon, OH) was solubilized in DMSO, diluted and added to the assay plates as described for test compounds. Final concentration for ribavirin was 35 μM. All wells contained 0.5% DMSO.
The MLSMR is a library of biologically relevant small organic molecules that has been utilized for HTS as part of the NIH Roadmap initiative, the Molecular Libraries Production Center Network (MLPCN). This library has been updated and expanded since the initiation of the program in 2005. Compounds were solubilized at 10 mM in DMSO and all compounds were diluted in assay media for a final concentration of 10 μM in the screen. The concentration of DMSO in each assay well, including all control wells was 0.5%.
For single dose screening in a 384 well plate format, compounds or carrier control (DMSO) were diluted to 6× in Complete DMEM/F12 using a Biomek FX and 5 μL was transferred to the assay plate. Cells were added to the plate in 25 uL of media using a Thermo/Matrix Wellmate. Final plate well concentration was 10 μM compound, 2,000 cells, and 0.5% DMSO in a total volume of 30 uL.
For dose response screening in a 384 well plate format, compounds or carrier control (DMSO) were diluted to 6× in Complete DMEM/F12 using a Biomek FX and 5 μL 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 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 μM to 0.097 μM and a final DMSO concentration of 0.5%).
Compounds or carrier control (DMSO) were diluted to 6x in C-DMEM/F12 and 5 μL was dispensed to 384-well assay plates (3% DMSO or 60 μM compound in 3% DMSO). Twenty five μL of uninfected HEp-2 cells were plated in the cell control wells. Frozen hRSV-infected cells were combined with uninfected HEp-2 cells at a 1:100 ratio. Twenty five μL 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°C, 5% CO2 and 90% relative humidity.
Following the six day incubation period, the assay plates were equilibrated to room temperature for 30 min. An equal volume (30 μL) of Cell Titer-Glo reagent (Promega Inc.) was added to each well using a WellMate (Matrix, Hudson, NH) and the plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a multi-label reader (Envision, PerkinElmer, Wellesley, MA) with an integration time of 0.1 s.
HTS data were analyzed using ActivityBase software (IDBS, Inc., Guildford, UK). Antiviral activity is described as percent CPE inhibition = 100*((luminescence compound well minus median luminescence virus control)/ (median luminescence cell control minus median luminescence virus control)). Percent viability = 100 * luminescence compound well/median luminescence cell control. An active compound, or “hit,” was defined as a compound that exhibited a % CPE inhibition of >22% without compromising cell viability. Two dose-response curves were calculated for each substance. One assessed % CPE inhibition at each dose (EC50); the other assessed cytotoxicity at each dose (CC50). EC50 values (for % CPE inhibition) and CC50 values were calculated using the 4-parameter Levenburg-Marquardt algorithm with parameter A locked at 0 and parameter B locked at 100. Standard deviation, normalized chi2, and Hill slope were used to evaluate the curves. Values were not extrapolated beyond the tested range of concentrations. The selective index (SI) was calculated as SI = CC50/EC50. The criteria for determining compound activity are based on its SI. Compounds with an SI value of >3 were defined as active, whereas compounds that exhibited an SI value less than 3 were defined as inactive.
Thirty-two control wells containing cells only and 24 wells containing cells and virus were included on each assay plate and used to calculate Z factors for each plate and to normalize the data on a per plate basis. Eight ribavirin positive control wells were included on each plate for quality control purposes but were not used in Z calculations.
The Z factor values were calculated from 1 minus (3*standard deviation of cell control plus 3* standard deviation of the virus control / [mean cell control signal minus mean virus control signal . The signal-to-background (S/B) was calculated from mean cell control signal divided by the mean virus control signal. The signal-to-noise (S/N) was calculated from mean cell control signal minus mean virus control signal divided by the standard deviation of the cell control signal minus the standard deviation of the virus control signal .
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. Ten μL of 10-fold serial dilutions of progeny virus containing medium from respective samples (drug treated or untreated) were used to infect fresh Hep-2 cells in a 384-well format. The cell plates were incubated at 37°C, 5% CO2, and high humidity for an additional 6 days. The Cell Titer Glo assay was used to determine viability of the cells. A well showing a luminescence signal less than the mean of the non-infected control signal minus five times the standard deviation of the control was regarded as positive for infection.
Plaque assays - compound preparation
Compounds or carrier control (DMSO) were diluted in Complete DMEM/F12 and 2 mL per well was dispensed to 6-well assay plates (final plate well concentration was 25 μM, ribavirin; 35 μM and final DMSO concentration 0.5%).
Preparation of HEp-2 cells and plaque assay setup
HEp-2 cells were harvested and resuspended to 500,000 cells per mL in Complete DMEM/F12 and seeded in 6 well tissue culture plates at 1,000,000 cells per well in 2 mL Complete Optimem1 and incubated 24 hours at 37°C, 5% CO2, 90% relative humidity. The media was aspirated from the wells, 0.5 mL hRSV Long strain (MOI of 0.1) diluted using C-DMEM/F12 was added and the plates incubated at 37°C, 5% CO2, rotating every 20 min. to facilitate infection. After 2 hours, the virus supernatant was aspirated and each well was washed with 3 mL of 1X PBS. Compounds were diluted in C-DMEM/F12 media to give a final concentration of 25 μM, added to assay plates and incubated at 37°C, 5% CO2 and 90% relative humidity. After 48 h, the supernatant (hRSV/compound/media; 1.6 mL) was removed, flash frozen on dry ice, and stored at –80°C.
HEp-2 cells in Complete Optimem1were seeded in 24 well tissue culture plates at 400,000 cells per well in 0.5 mL and incubated 24 h at 37°C, 5% CO2. The supernatant (hRSV/compound/ media) was removed from -80°C and thawed on ice. The supernatants were serially diluted in Complete DMEM/F12 media (10-1 to 10-4). The media was aspirated from the 24 well plates, 0.2 mL of each supernatant dilution was added to each well and the plates incubated at 37°C, 5% CO2, rotating every 20 min. to facilitate infection. After 2 h, each well was washed one time with 1X PBS followed by the addition of 0.5 mL 1% Avicel per well. The assay plates were incubated for six days at 37°C, 5% CO2 and 90% relative humidity.
Staining of virus plaques
Following the six day incubation period, the Avicel overlay was aspirated, washed with 0.5 mL of 1X PBS, and fixed with 0.5 mL of 4% paraformaldehyde per well. The assay plates were incubated at 4°C for 24 h. The paraformaldehyde was aspirated, each well washed with 1 mL deionized water, and stained with 1 mL of 0.05% neutral red with periodic shaking for 10 minutes at room temperature. The neutral red was aspirated and the plates briefly inverted without lids on paper towels for drying.