Keywords: Luciferase, luciferin, ATP, CellTiter-Glo, counterscreen, inhibitor, inhibition, platelets, dense granule secreation, ..more
BioActive Compounds: 446
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 1663 | MLPCN Platelet Activation -Dense Granule Release | screening | Primary HTS |
| 1678 | Broad Institute MLPCN Platelet Activation | summary | Summary of Project |
| 2518 | Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets. | confirmatory |
Description:
Keywords: Luciferase, luciferin, ATP, CellTiter-Glo, counterscreen, inhibitor, inhibition, platelets, dense granule secreation,
Assay Overview: Counter screen for luciferase inhibitors of Dense Granule Secretion. 20ul of 1.5uM ATP (Sigma, #A1852) in PBS is plated in 384-well white assay plates (Aurora, 00030721) and was exposed to the 1584 cherry-picked compounds chosen based on activity of the platelet dense granule release primary screen (AID1663) and structure to compounds with the highest activity, to provide some SAR data. A dose-response plate with the positive control Resveratrol (Sigma #5010, Lot#038K5202, BRD-K80738081, CID445154) was included in the run After compound addition, the detection reagent CellTiter-Glo (Promega, G755) was resuspend and diluted in PBS by the same protocol as for the primary screen. Luminescence measurements are taken immediately after reagent addition.
Expected Outcome: Compounds that inhibit luciferase from acting on the luciferin substrate contained within the CellTiter-Glo reagent will exhibit a decrease in luminescent signal. Normalization and dose-response curves will be fit using the Genedata Screener applications. The activity determination from this assay (IC50) will be compared to the activity determined from retest of the primary screen by calculation of the ratio of the two. The compounds showing a greater than 10-fold difference in activity will be further analyzed for the specific mechanism in which platelet activation is inhibited.
Assay Overview: Counter screen for luciferase inhibitors of Dense Granule Secretion. 20ul of 1.5uM ATP (Sigma, #A1852) in PBS is plated in 384-well white assay plates (Aurora, 00030721) and was exposed to the 1584 cherry-picked compounds chosen based on activity of the platelet dense granule release primary screen (AID1663) and structure to compounds with the highest activity, to provide some SAR data. A dose-response plate with the positive control Resveratrol (Sigma #5010, Lot#038K5202, BRD-K80738081, CID445154) was included in the run After compound addition, the detection reagent CellTiter-Glo (Promega, G755) was resuspend and diluted in PBS by the same protocol as for the primary screen. Luminescence measurements are taken immediately after reagent addition.
Expected Outcome: Compounds that inhibit luciferase from acting on the luciferin substrate contained within the CellTiter-Glo reagent will exhibit a decrease in luminescent signal. Normalization and dose-response curves will be fit using the Genedata Screener applications. The activity determination from this assay (IC50) will be compared to the activity determined from retest of the primary screen by calculation of the ratio of the two. The compounds showing a greater than 10-fold difference in activity will be further analyzed for the specific mechanism in which platelet activation is inhibited.
Protocol
Taken from 2016-02-W01-01
1. Reagents are prepared for the assay:
a. Frozen aliquots of 10mM ATP in PBS is thawed and diluted to 1.5uM in PBS. Enough reagent is prepared for the run (8ml/plate + extra dead volume for liquid handling).
b. Frozen aliquots of CellTiter-Glo (Promega) (CTG) in PBS is thawed and diluted in PBS 1:4. Enough reagent is prepared for the run (4ml/plate + extra dead volume for liquid handling).
2. A MultiDrop Combi (Thermo) is prepared with a standard cassette that is washed and primed with the reagent. Plates are filled with 20μl of 1.5uM ATP in PBS.
3. Compounds are added using the CyBi-Well (CyBio Inc.). 50nl are pinned using the 25nl pin array, which is programed for 2 seperate dips into the compound plate, seperated by a pin wash cycle.
4. During compound addition, a MultiDrop Combi is prepared for second reagent addition by washing cassette and priming with new reagent.
5. Upon completion of compound addition, 10μl of CTG (1:4) in PBS is added to every well.
6. Plates are transferend to an Envison Plate Reader (Perkin Elmer) to measure luminesence. The ultra sensitive detection is used, with the 1536 aperture in place, to decrease bleed-through from adjacent wells. Read time is 0.1s/well.
1. Reagents are prepared for the assay:
a. Frozen aliquots of 10mM ATP in PBS is thawed and diluted to 1.5uM in PBS. Enough reagent is prepared for the run (8ml/plate + extra dead volume for liquid handling).
b. Frozen aliquots of CellTiter-Glo (Promega) (CTG) in PBS is thawed and diluted in PBS 1:4. Enough reagent is prepared for the run (4ml/plate + extra dead volume for liquid handling).
2. A MultiDrop Combi (Thermo) is prepared with a standard cassette that is washed and primed with the reagent. Plates are filled with 20μl of 1.5uM ATP in PBS.
3. Compounds are added using the CyBi-Well (CyBio Inc.). 50nl are pinned using the 25nl pin array, which is programed for 2 seperate dips into the compound plate, seperated by a pin wash cycle.
4. During compound addition, a MultiDrop Combi is prepared for second reagent addition by washing cassette and priming with new reagent.
5. Upon completion of compound addition, 10μl of CTG (1:4) in PBS is added to every well.
6. Plates are transferend to an Envison Plate Reader (Perkin Elmer) to measure luminesence. The ultra sensitive detection is used, with the 1536 aperture in place, to decrease bleed-through from adjacent wells. Read time is 0.1s/well.
Comment
Dose Response Data Analysis:
32 Negative control (DMSO) and 32 positive control (Resveratrol , PubChem CID 445154) wells were included on every plate.
All wells tested were background subtracted using the median of the negative controls wells on the same plate and scaled using the median value of the positive control wells on the same plate. DMSO plates were run before and after the compound plate and Genedata's runwise additive correction was used with those as calibration plates to correct for plate effects.
EC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (v6.0.1). EC50 values were extrapolated up to 1 log
over the highest tested concentration.
PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)
PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration
32 Negative control (DMSO) and 32 positive control (Resveratrol , PubChem CID 445154) wells were included on every plate.
All wells tested were background subtracted using the median of the negative controls wells on the same plate and scaled using the median value of the positive control wells on the same plate. DMSO plates were run before and after the compound plate and Genedata's runwise additive correction was used with those as calibration plates to correct for plate effects.
EC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (v6.0.1). EC50 values were extrapolated up to 1 log
over the highest tested concentration.
PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)
PUBCHEM_ACTIVITY_OUTCOME
Activity_Outcome = 1 (inactive)
EC50 > 1 log over the highest tested concentration
Activity_Outcome = 2 (active)
EC50 <= 1 log over the highest tested concentration
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 | EC50 Qualifier | '>', '=', or '<' | String | |||
| 2 | EC50* | the concentration whereupon perceived activity reaches 50% of the maximum | | Float | μM | |
| 3 | EC50 Stderr | the standard error for the calculated EC50 value | | Float | μM | |
| 4 | S0 | the fitted activity level at zero concentration | | Float | % | |
| 5 | SInf | the fitted activity level at infinite concentration | | Float | % | |
| 6 | Hill Slope | the slope at EC50 | | Float | ||
| 7 | # of Points Fit | the number of data points included in the plot | | Integer | ||
| 8 | Max Activity | the maximum activity value observed | | Float | % | |
| 9 | Activity at 0.09uM (0.09μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 10 | Activity at 0.18uM (0.18μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 11 | Activity at 0.35uM (0.35μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 12 | Activity at 0.70uM (0.7μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 13 | Activity at 1.40uM (1.4μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 14 | Activity at 2.81uM (2.81μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 15 | Activity at 5.61uM (5.61μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % | |
| 16 | Activity at 11.22uM (11.22μM**) | The average measured activity of all accepted replicates at the specified concentration | | Float | % |
* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03DA026209-01
Data Table (Concise)
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