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BioAssay: AID 493090

An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (5)

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 ..
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 Tested Compounds
 Tested Compounds
All(5)
 
 
Active(1)
 
 
Inactive(4)
 
 
 Tested Substances
 Tested Substances
All(5)
 
 
Active(1)
 
 
Inactive(4)
 
 
 Related BioAssays
 Related BioAssays
AID: 493090
Data Source: Southern Research Specialized Biocontainment Screening Center (RSV_Tox_5)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-01-25
Hold-until Date: 2012-01-21
Modify Date: 2012-01-24

Data Table ( Complete ):           Active    All
BioActive Compound: 1
Depositor Specified Assays
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AIDNameTypeProbeComment
2391A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV)confirmatory Primary and confirmatory screen.
2410An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV)confirmatory Cytotoxicity of primary screen hits.
449732A Titer Reduction Assay to evaluate Selected Compounds as new Inhibitors of Respiratory Syncytial Virus (RSV)other TCIC50 of selected compounds
488972A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using purified and synthesized compoundsconfirmatory Confirmatory screen of purified and synthesized compounds (2).
488976An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using purified and synethesized compoundsconfirmatory Cytotoxicity screen of screen of purified and synthesized compounds (2).
492966A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (3)confirmatory Confirmatory screen of purified and synthesized compounds (3).
492968An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (3)confirmatory Cytotoxicity screen of screen of purified and synthesized compounds (3).
493016A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (4)confirmatory Confirmatory screen of purified and synthesized compounds (4).
493015An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (4)confirmatory Cytotoxicity screen of screen of purified and synthesized compounds (4).
2440A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory Syncytial Virus (RSV) - Summarysummary Summary of project.
504509An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (6)confirmatory
504526A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (6)confirmatory
504655A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (7)confirmatory
504674An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (7)confirmatory
504818An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (8)confirmatory
504820A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (8)confirmatory
504823A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (9)confirmatory
504825An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (10)confirmatory
504826An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (9)confirmatory
504827A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (10)confirmatory
504829A Time of Addition Assay to evaluate New Inhibitors of Respiratory syncytial virus (RSV)other1
504830A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV)other
540264A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (11)confirmatory
540266An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (11)confirmatory
540320A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) (3)other
540337A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (12)confirmatory
540338An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (12)confirmatory
588454An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (13)confirmatory
588455A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (13)confirmatory
588483An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (14)confirmatory
588485A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (14)confirmatory
588713A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) (4)other
588791A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) (5)other
602159A Time of Addition Assay to evaluate New Inhibitors of Respiratory syncytial virus (RSV) (2)other1
Description:
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.
Protocol
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.

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 media and 20 ul of compound or ribavirin control drug were added to the 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 10 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: 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.
Comment
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.

Compounds showing viability of less than 70% at any concentration are considered "Active" in this assay.

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. In screens using purified and synthesized compounds a score of 81-100 based on the CC50 value is used to indicate a high level of confidence is the results. Compounds that do not show activity received a score of 0.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1CC50 ModifierString
2CC50*FloatμM
3% Cell Viability @ 50 uM (50μM**)Float%
4% Cell Viability @ 25 uM (25μM**)Float%
5% Cell Viability @ 12.5 uM (12.5μM**)Float%
6% Cell Viability @ 6.25 uM (6.25μM**)Float%
7% Cell Viability @ 3.125 uM (3.125μM**)Float%
8% Cell Viability @ 1.563 uM (1.563μM**)Float%
9% Cell Viability @ 0.781uM (0.781μM**)Float%
10% Cell Viability @ 0.391 uM (0.391μM**)Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03 MH084847-01

Data Table (Concise)
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