Bookmark and Share
BioAssay: AID 1891

Luminescence Biochemical Dose Response HTS to Identify Inhibitors of Luciferase

Keywords: Luciferase, luciferin, ATP, CellTiter-Glo, counterscreen, inhibitor, inhibition, platelets, dense granule secreation, ..more
_
   
 Tested Compounds
 Tested Compounds
All(1584)
 
 
Active(446)
 
 
Inactive(1138)
 
 
 Tested Substances
 Tested Substances
All(1584)
 
 
Active(446)
 
 
Inactive(1138)
 
 
 Related BioAssays
 Related BioAssays
AID: 1891
Data Source: Broad Institute (2016-02_INHIBITORS_DOSE-TITRATION_MLPCN-CHERRYPICK)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-07-28
Modify Date: 2009-09-11

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 446
Related Experiments
Show more
AIDNameTypeProbeComment
1663MLPCN Platelet Activation -Dense Granule ReleaseScreening depositor-specified cross reference: Primary HTS
1678Broad Institute MLPCN Platelet ActivationSummary3 depositor-specified cross reference: Summary of Project
2518Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.Confirmatory depositor-specified cross reference
1889Luminescence Cell-Based Dose Confirmation HTS to Identify Inhibitors of Platelet Dense Granule ReleaseConfirmatory same project related to Summary assay
2398Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule ReleaseConfirmatory same project related to Summary assay
2509Fluorescence Cell-Based Screen to Identify Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2511Fluorescence Cell-Based Dose Response to Confirm Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on PlateletsConfirmatory same project related to Summary assay
2519Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule Release.Confirmatory same project related to Summary assay
2522Fluorescence Cell-Based Screen to Identify Inhibitors of Calcium Ionophore-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2527Fluorescence Cell-Based Screen to Confirm Inhibitors of Calcium Ionophore-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2529Fluorescence Cell-Based Screen to Confirm Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on PlateletsScreening same project related to Summary assay
2645ELISA Cell-Based Screen to Identify Inducers of cAMP in PlateletsScreening same project related to Summary assay
2646ELISA Cell-Based Screen to Identify an Inducer of cAMP in PlateletsScreening same project related to Summary assay
2655Fluorescence Cell-Based Screen to Identify an Inhibitor of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in PlateletsScreening same project related to Summary assay
2656Radioactive Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Dense Granule Release by PlateletsScreening same project related to Summary assay
2657Fluorescence Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in PlateletsScreening same project related to Summary assay
493031SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_SinglePoint_DryPowder_ActivityOther same project related to Summary assay
493068SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatory same project related to Summary assay
493100Platelet granule secretion Measured in Cell-Based System Using Plate Reader - 2016-01_Inhibitor_Dose_DryPowder_ActivityConfirmatory same project related to Summary assay
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.
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.
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
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Binding
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1EC50 Qualifier'>', '=', or '<'String
2EC50*the concentration whereupon perceived activity reaches 50% of the maximumFloatμM
3EC50 Stderrthe standard error for the calculated EC50 valueFloatμM
4S0the fitted activity level at zero concentrationFloat%
5SInfthe fitted activity level at infinite concentrationFloat%
6Hill Slopethe slope at EC50Float
7# of Points Fitthe number of data points included in the plotInteger
8Max Activitythe maximum activity value observedFloat%
9Activity at 0.09uM (0.09μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
10Activity at 0.18uM (0.18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
11Activity at 0.35uM (0.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
12Activity at 0.70uM (0.7μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
13Activity at 1.40uM (1.4μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
14Activity at 2.81uM (2.81μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
15Activity at 5.61uM (5.61μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
16Activity at 11.22uM (11.22μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03DA026209-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
PageFrom: