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BioAssay: AID 2440

A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory Syncytial Virus (RSV) - Summary

This report summarizes the approaches used to discover small molecules inhibiting respiratory syncytial virus replication in a cell culture system with high specificity. 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 more ..
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AID: 2440
Data Source: Southern Research Specialized Biocontainment Screening Center (RSV_Summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-03-01
Modify Date: 2011-12-20
Depositor Specified Assays
Show more
AIDNameTypeProbeComment
2391A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV)confirmatory Primary and Confirmatory Screens for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
488972A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using purified and synthesized compoundsconfirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
492966A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (3)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
493016A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (4)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
493088A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (5)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
504526A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (6)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
504655A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (7)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
504820A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (8)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
504823A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (9)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
504827A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (10)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
540264A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (11)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
540337A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (12)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
588485A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (14)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
588455A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory syncytial virus (RSV) using synthesized compounds (13)confirmatory Confirmatory Screen for inhibitors of Respiratory Syncytial Virus induced cytopathic effect
2410An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV)confirmatory Counterscreen to identify cytotoxic compounds
488976An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using purified and synethesized compoundsconfirmatory Counterscreen to identify cytotoxic compounds
492968An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (3)confirmatory Counterscreen to identify cytotoxic compounds
493015An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (4)confirmatory Counterscreen to identify cytotoxic compounds
493090An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (5)confirmatory Counterscreen to identify cytotoxic compounds
504509An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (6)confirmatory Counterscreen to identify cytotoxic compounds
504674An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (7)confirmatory Counterscreen to identify cytotoxic compounds
504818An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (8)confirmatory Counterscreen to identify cytotoxic compounds
504826An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (9)confirmatory Counterscreen to identify cytotoxic compounds
504825An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (10)confirmatory Counterscreen to identify cytotoxic compounds
540266An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (11)confirmatory Counterscreen to identify cytotoxic compounds
540338An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (12)confirmatory Counterscreen to identify cytotoxic compounds
588454An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (13)confirmatory Counterscreen to identify cytotoxic compounds
588483An HTS Cytotoxicity Screen to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) using synethesized compounds (14)confirmatory Counterscreen to identify cytotoxic compounds
504829A Time of Addition Assay to evaluate New Inhibitors of Respiratory syncytial virus (RSV)other1 Time of Addition
449732A Titer Reduction Assay to evaluate Selected Compounds as new Inhibitors of Respiratory Syncytial Virus (RSV)other Plaque Reduction
504830A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV)other Plaque Reduction
540320A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) (3)other Plaque Reduction
588713A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) (4)other Plaque Reduction
588791A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV) (5)other Plaque Reduction
2440A Cell Based HTS Approach for the Discovery of New Inhibitors of Respiratory Syncytial Virus (RSV) - Summarysummary Summary AID
602159A Time of Addition Assay to evaluate New Inhibitors of Respiratory syncytial virus (RSV) (2)other1 Time of Addition
Description:
Data Source: Southern Research Specialized Biocontainment Screening Center (RSV Summary)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
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: 1 R03 MH082403-01A1

This report summarizes the approaches used to discover small molecules inhibiting respiratory syncytial virus replication in a cell culture system with high specificity. 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.

Two lines of experiments were provided to get possible scaffolds for the SAR; 1) to screen the current MLSMR compound library (>300,000 compounds), 2) to screen cytotoxic compounds out and select compounds specific for the virus in a dose response study. Additional titer reduction and time of addition studies were also performed.
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Summary of the Primary Screen for antiviral activity - AID 2391
Method: A cell based assay with CPE by an infectious virus
Test concentration: 10 microM
Activity criterion: >22.3% inhibition
Number compounds evaluated: 313,816
Number of active compounds: 7583

The protection of cytopathic effect from RSV infection in Hep-2 cells was measured as an antiviral effect of compounds screened. An end-point assay using the luminescence measuring ATP amount in a well was employed to measure the antiviral effect. A total of 313,816 compounds have been screened and Z values of the screen ranged between 0.6 and 0.9 with the median of 0.83. Seven thousand five hundred eighty-three compounds were evaluated as active with criteria of the average of negative control + 3 times of SD (22.3%) of whole screening. Two thousand four hundred sixty-five compounds were subjected to the dose response study to verify their activity and cytotoxicity.
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Confirmatory Screen for antiviral activity - AID 2391
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.097 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 2465
Number of active compounds: 409

Confirmatory Screen for antiviral activity - AID 488972
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 21
Number of active compounds: 3

Confirmatory Screen for antiviral activity - AID 492966
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 4
Number of active compounds: 1

Confirmatory Screen for antiviral activity - AID 493016
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 7
Number of active compounds: 3

Confirmatory Screen for antiviral activity - AID 493088
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 5
Number of active compounds: 3

Confirmatory Screen for antiviral activity - AID 504526
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 25
Number of active compounds: 10

Confirmatory Screen for antiviral activity - AID 504655
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 21
Number of active compounds: 5

Confirmatory Screen for antiviral activity - AID 504820
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 54
Number of active compounds: 20

Confirmatory Screen for antiviral activity - AID 504823
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 1.2 microM to 150 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 9
Number of active compounds: 9

Confirmatory Screen for antiviral activity - AID 504827
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 1.6 microM to 200 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 4
Number of active compounds: 4

Confirmatory Screen for antiviral activity - AID 540264
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 200 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 12
Number of active compounds: 9

Confirmatory Screen for antiviral activity - AID 540337
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 7
Number of active compounds: 1

Confirmatory Screen for antiviral activity - AID 588455
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 3
Number of active compounds: 3

Confirmatory Screen for antiviral activity - AID 588485
Method: A cell-based assay with CPE by an infectious virus in dose response format.
Test concentration: 0.39 microM to 50 microM
Activity criterion: Maximum % CPE Inhibition at any dose >=50%
Number of compounds evaluated: 5
Number of active compounds: 3


The same measurement as the primary screening was employed across multiple concentrations of compound. IC50 value was calculated.
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Secondary screen for cytotoxicity - AID 2410
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.097 microM to 50 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 2465
Number active: 2107

Secondary screen for cytotoxicity - AID 488976
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.391 microM to 50 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 21
Number active: 11

Secondary screen for cytotoxicity - AID 492968
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.391 microM to 50 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 4
Number active: 4

Secondary screen for cytotoxicity - AID 493015
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.391 microM to 50 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 7
Number active: 6

Secondary screen for cytotoxicity - AID 493090
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.391 microM to 50 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 5
Number active: 1

Secondary screen for cytotoxicity - AID 504509
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.391 microM to 50 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 25
Number active: 14

Secondary screen for cytotoxicity - AID 504674
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.391 microM to 50 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 21
Number active: 12

Secondary screen for cytotoxicity - AID 504818
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.391 microM to 50 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 54
Number active: 50

Secondary screen for cytotoxicity - AID 504826
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 1.2 microM to 150 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 9
Number active: 9

Secondary screen for cytotoxicity - AID 504825
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 1.6 microM to 200 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 4
Number active: 4

Secondary screen for cytotoxicity - AID 540266
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.39 microM to 200 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 19
Number active: 18

Secondary screen for cytotoxicity - AID 540338
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 1.17 microM to 200 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 7
Number active: 6

Secondary screen for cytotoxicity - AID 588454
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 1.56 microM to 200 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 3
Number active: 3

Secondary screen for cytotoxicity - AID 588483
Method: A cell-based assay with cell growth inhibition/cytotoxicity with compounds
Test concentration: 0.6 microM to 150 microM
Activity criterion: < 70% Cell Viability
Number compounds evaluated: 5
Number active: 3


Cytotoxicity of the compounds was evaluated to identify compounds that might be inappropriate for antiviral probes. The host cells, HEp-2 cells, were incubated for six days in a culture media containing different concentration of compounds. The cell viability was measured with an end-point assay using the luminescence measuring ATP amount in a well. Compounds causing cell viability to decrease to less than 70% viability at any concentration were deemed Active. The same measurement as the primary screening was employed with the exclusion of virus. CC50 values were calculated.

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Secondary screen for titer reduction - AID 449732
Method: A Titer Reduction Assay to evaluate Selected Compounds as new Inhibitors of Respiratory Syncytial Virus (RSV).
Test concentration: various between 2.81 microM and 50 microM
Activity criterion: <1 log reduction
Number compounds evaluated: 51
Number active: 51

Secondary screen for titer reduction - AID 504830
Method: A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV)
Respiratory Syncytial Virus (RSV).
Test concentration: 25 microM
Activity criterion: <1 log reduction
Number compounds evaluated: 4
Number active: 4

Secondary screen for titer reduction - AID 540320
Method: A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV)
Test concentration: 10 microM
Activity criterion: <1 log reduction
Number compounds evaluated: 15
Number active: 9

Secondary screen for titer reduction - AID 588713
Method: A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV)
Test concentration: 10 microM
Activity criterion: <1 log reduction
Number compounds evaluated: 4
Number active: 3

Secondary screen for titer reduction - AID 588791
Method: A Plaque Reduction Assay to evaluate New Inhibitors of Respiratory Syncytial Virus (RSV)
Test concentration: 10 microM
Activity criterion: <1 log reduction
Number compounds evaluated: 4
Number active: 3



The inhibitory activity of selected, available compounds showing antiviral activity and limited cytotoxicity were 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 with RSV Long strain in the presence of compounds. The assay measures the progeny viral titer using the TCID50 calculated by the Reed & Muench method.
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Secondary Time of Addition - AID 504829
Method: A Time of Addition Assay to evaluate New Inhibitors of Respiratory syncytial virus (RSV)
Test concentration: 25 microM
Activity criterion: >60% Cell viability at any timepoint of compound addition
Number compounds evaluated: 3
Number active: 3

Secondary Time of Addition - AID 602159
Method: A Time of Addition Assay to evaluate New Inhibitors of Respiratory syncytial virus (RSV)
Test concentration: 25 microM
Activity criterion: >60% Cell viability at any timepoint of compound addition
Number compounds evaluated: 3
Number active: 3 (one probe)


Compounds were added to plates at various times relative to viral infection of the cells with RSV Long strain. Following the six day incubation period, the percentage of viable cells was calculated for each of the compound addition time points to determine the timepoint at which compound addition no longer sustained cell viability above 60%.
Additional Information
Grant Number: 1 R03 MH082403-01A1

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