Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill more ..
Tested Compounds
Tested Compounds
Tested Substances
Tested Substances
Target
BioActive Compounds: 1073
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 504647 | Assay for Inhibitors of Influenza NS1 Protein Function: Influenza virus replication assay in MDCK cells infected with virus A/PR/8/34 | other | |
| 2336 | Summary Assay for Inhibitors of Influenza NS1 Protein Function | summary | |
| 492980 | Assay for Inhibitors of Influenza NS1 Protein Function: Hit Validation | confirmatory | |
| 485354 | qHTS of Yeast-based Assay for SARS-CoV PLP: Summary | summary |
Description:
NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH084878-01A1
Assay Submitter (PI): Daniel Engel
Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill global needs. In addition their potential usefulness against newly emergent strains is not known. Efforts are needed to develop novel agents against influenza virus, including broad-spectrum agents. Identification of small molecules that inhibit NS1 function either directly or by interfering with specific cellular pathways may be a key to increasing our defense against the virus.
We have miniaturized and optimized a cell based assay in which NS1 from influenza A is expressed in the budding yeast S. cerevisiae. NS1 is a multi-functional protein that counters the host innate immune response and facilitates viral versus cellular gene expression. Expression of NS1 causes a pronounced slow growth phenotype in yeast due to its intrinsic molecular activities. Small molecules that suppress the slow growth phenotype can be identified by a straightforward growth recovery assay using optical density (OD) as the measurement. The same yeast strain not expressing NS1 was used as positive control in the yeast growth recovery assay.
NIH Molecular Libraries Probe Centers Network [MLPCN]
MLPCN Grant: MH084878-01A1
Assay Submitter (PI): Daniel Engel
Influenza is a world-wide public health problem and emerging forms of the virus have the potential to cause a pandemic of equal or greater magnitude to the outbreaks recorded in 1918, 1957 or 1968. Vaccine development is proceeding and there also exist two classes of anti-influenza compounds. However these therapeutic modalities are neither fully effective nor widely enough available to fulfill global needs. In addition their potential usefulness against newly emergent strains is not known. Efforts are needed to develop novel agents against influenza virus, including broad-spectrum agents. Identification of small molecules that inhibit NS1 function either directly or by interfering with specific cellular pathways may be a key to increasing our defense against the virus.
We have miniaturized and optimized a cell based assay in which NS1 from influenza A is expressed in the budding yeast S. cerevisiae. NS1 is a multi-functional protein that counters the host innate immune response and facilitates viral versus cellular gene expression. Expression of NS1 causes a pronounced slow growth phenotype in yeast due to its intrinsic molecular activities. Small molecules that suppress the slow growth phenotype can be identified by a straightforward growth recovery assay using optical density (OD) as the measurement. The same yeast strain not expressing NS1 was used as positive control in the yeast growth recovery assay.
Protocol
The yeast S. cerevisiae control and NS1 expressing cells were kindly supplied by laboratory of Dr. Daniel Engel, University of Virginia. Yeast starter cultures were stored in single use aliquots at -80C.
Assay Reagents:
1. Yeast culture medium per liter: 1.2 g Yeast Nitrogen Base (w/out amino acids and Ammonium Sulphate), 5.0g Ammonium Sulphate, 0.77g -URA DO (drop-out) Supplement, adjust pH to 6.0. Adjust to 2% glucose prior to inoculating with yeast.
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2. Yeast assay medium: 1.2 g Yeast Nitrogen Base (w/out amino acids and Ammonium Sulphate), 5.0g Ammonium Sulphate, 0.77g -URA DO (drop-out) Supplement, adjust pH to 6.0. Adjust to 2% D-galactose prior to inoculating with yeast.
Assay Procedure:
1. Inoculate 100 mls of yeast culture medium with 1 ml of frozen yeast aliquote. Incubate overnight at 30C, 250RPM.
2. Harvest overnight culture at 4000 RPM and wash pellet with 15 mls H2O 3x.
3. Re-suspend washed pellet in 10 mls of yeast assay medium.
4. Prepare a 1:100 dilution of re-suspended culture in yeast assay medium.
5. Dispense 3.5 ul per well of reagents in step 4 in columns 2-48 in a black clear bottom 1536 well plate.
6. Dispense 3.5 ul per well of yeast assay medium in column 1 only.
7. Transfer 23 nl of compound per well.
8. Dispense 3.5 ul per well of yeast assay medium in columns 1-48.
9. Read plate using Viewlux protocol for optical density 600 (OD600) using excitation filter 600nm.
Assay Reagents:
1. Yeast culture medium per liter: 1.2 g Yeast Nitrogen Base (w/out amino acids and Ammonium Sulphate), 5.0g Ammonium Sulphate, 0.77g -URA DO (drop-out) Supplement, adjust pH to 6.0. Adjust to 2% glucose prior to inoculating with yeast.
.
2. Yeast assay medium: 1.2 g Yeast Nitrogen Base (w/out amino acids and Ammonium Sulphate), 5.0g Ammonium Sulphate, 0.77g -URA DO (drop-out) Supplement, adjust pH to 6.0. Adjust to 2% D-galactose prior to inoculating with yeast.
Assay Procedure:
1. Inoculate 100 mls of yeast culture medium with 1 ml of frozen yeast aliquote. Incubate overnight at 30C, 250RPM.
2. Harvest overnight culture at 4000 RPM and wash pellet with 15 mls H2O 3x.
3. Re-suspend washed pellet in 10 mls of yeast assay medium.
4. Prepare a 1:100 dilution of re-suspended culture in yeast assay medium.
5. Dispense 3.5 ul per well of reagents in step 4 in columns 2-48 in a black clear bottom 1536 well plate.
6. Dispense 3.5 ul per well of yeast assay medium in column 1 only.
7. Transfer 23 nl of compound per well.
8. Dispense 3.5 ul per well of yeast assay medium in columns 1-48.
9. Read plate using Viewlux protocol for optical density 600 (OD600) using excitation filter 600nm.
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators (NS1 inhibitors; NS1 is detrimental to yeast growth) are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent activators (NS1 inhibitors; NS1 is detrimental to yeast growth) are ranked higher than compounds that showed apparent inhibition.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Result Definitions
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| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| Score | The BioAssay activity ranking score |  | Integer | |||
| 1 | Phenotype | Indicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive. | String | |||
| 2 | Potency* | Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed. | | Float | μM | |
| 3 | Efficacy | Maximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves. | | Float | % | |
| 4 | Analysis Comment | Annotation/notes on a particular compound's data or its analysis. | String | |||
| 5 | Curve_Description | A description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control. | String | |||
| 6 | Fit_LogAC50 | The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units). | | Float | ||
| 7 | Fit_HillSlope | The Hill slope from a fit of the data to the Hill equation. | | Float | ||
| 8 | Fit_R2 | R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit. | | Float | ||
| 9 | Fit_InfiniteActivity | The asymptotic efficacy from a fit of the data to the Hill equation. | | Float | % | |
| 10 | Fit_ZeroActivity | Efficacy at zero concentration of compound from a fit of the data to the Hill equation. | | Float | % | |
| 11 | Fit_CurveClass | Numerical encoding of curve description for the fitted Hill equation. | | Float | ||
| 12 | Excluded_Points | Which dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations. | String | |||
| 13 | Max_Response | Maximum activity observed for compound (usually at highest concentration tested). | | Float | % | |
| 14 | Activity at 0.369 uM (0.369μM**) | % Activity at given concentration. | | Float | % | |
| 15 | Activity at 1.840 uM (1.84μM**) | % Activity at given concentration. | | Float | % | |
| 16 | Activity at 9.220 uM (9.22μM**) | % Activity at given concentration. | | Float | % | |
| 17 | Activity at 46.10 uM (46.1μM**) | % Activity at given concentration. | | Float | % | |
| 18 | Compound QC | NCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling' | String |
* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084878-01A1
Data Table (Concise)
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