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BioAssay: AID 1678

Broad Institute MLPCN Platelet Activation

Keywords: Platelet, activation, granule, secretion, arterial thrombosis, PAR1, SFLLRN, thrombin receptor ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Probe(3)
 
 
Active(3)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Probe(3)
 
 
Active(3)
 
 
 Related BioAssays
 Related BioAssays
AID: 1678
Data Source: Broad Institute (2016)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-04-13
Modify Date: 2011-03-04

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: Chemical Probe: 3    Active: 3
Related Experiments
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AIDNameTypeComment
1663MLPCN Platelet Activation -Dense Granule ReleaseScreeningdepositor-specified cross reference: 302517 hts compounds at singlepoint in Dense Granule Release measuring activity set 1
1889Luminescence Cell-Based Dose Confirmation HTS to Identify Inhibitors of Platelet Dense Granule ReleaseConfirmatorydepositor-specified cross reference: 1584 cherrypick compounds at dose in Dense Granule Release measuring activity set 1
1891Luminescence Biochemical Dose Response HTS to Identify Inhibitors of LuciferaseConfirmatorydepositor-specified cross reference: 1584 cherrypick compounds at dose in Luciferase Inhibition measuring activity set 1
2398Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule ReleaseConfirmatorydepositor-specified cross reference: 10 drypowder compounds at dose in Dense Granule Release measuring activity set 1
2509Fluorescence Cell-Based Screen to Identify Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on PlateletsScreeningdepositor-specified cross reference: 2 cherrypick compounds at singlepoint in PMA-Induced P-selectin measuring activity set 1
2511Fluorescence Cell-Based Dose Response to Confirm Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on PlateletsConfirmatorydepositor-specified cross reference: 8 drypowder compounds at dose in SFLLRN-induced P-Selectin measuring activity set 1
2518Fluorescence Cell-Based Dose Response to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated P-Selectin Induction on Platelets.Confirmatorydepositor-specified cross reference: 28 cherrypick compounds at dose in SFLLRN-induced P-Selectin measuring activity set 1
2519Luminescence Cell-Based Dose Response Followup to Identify Inhibitors of Platelet Dense Granule Release.Confirmatorydepositor-specified cross reference: 10 drypowder compounds at dose in Dense Granule Release measuring activity set 2
2522Fluorescence Cell-Based Screen to Identify Inhibitors of Calcium Ionophore-Mediated P-Selectin Induction on PlateletsScreeningdepositor-specified cross reference: 2 cherrypick compounds at singlepoint in Ionophore-induced P-selectin measuring activity set 1
2527Fluorescence Cell-Based Screen to Confirm Inhibitors of Calcium Ionophore-Mediated P-Selectin Induction on PlateletsScreeningdepositor-specified cross reference: 2 drypowder compounds at singlepoint in Ionophore-induced P-selectin measuring activity set 1
2529Fluorescence Cell-Based Screen to Confirm Inhibitors of Phorbol Myristate Acetate-Mediated P-Selectin Induction on PlateletsScreeningdepositor-specified cross reference: 2 drypowder compounds at singlepoint in PMA-Induced P-selectin measuring activity set 1
2645ELISA Cell-Based Screen to Identify Inducers of cAMP in PlateletsScreeningdepositor-specified cross reference: 16 cherrypick compounds at singlepoint in cAMP measuring activity set 1
2646ELISA Cell-Based Screen to Identify an Inducer of cAMP in PlateletsScreeningdepositor-specified cross reference: 1 drypowder compounds at singlepoint in cAMP measuring activity set 1
2655Fluorescence Cell-Based Screen to Identify an Inhibitor of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in PlateletsScreeningdepositor-specified cross reference: 2 drypowder compounds at singlepoint in SFFLRN-induced FITC phalloidin measuring activity set 1
2656Radioactive Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Dense Granule Release by PlateletsScreeningdepositor-specified cross reference: 7 cherrypick compounds at singlepoint in SFLLRN-induced 14C-serotonin measuring activity set 1
2657Fluorescence Cell-Based Screen to Identify Inhibitors of Thrombin Receptor-Activating Peptide SFLLRN-Mediated Actin Polymerization in PlateletsScreeningdepositor-specified cross reference: 2 cherrypick compounds at singlepoint in SFFLRN-induced FITC phalloidin measuring activity set 1
493031SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_SinglePoint_DryPowder_ActivityOtherdepositor-specified cross reference: 21 drypowder compounds at singlepoint in SFLLRN-induced P-Selectin measuring activity set 1
493068SFLLRN-induced P-Selectin Platelet Surface Expression Measured in Cell-Based System Using Flow Cytometry - 2016-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorydepositor-specified cross reference: 5 drypowder compounds at dose in SFLLRN-induced P-Selectin measuring activity set 3
493100Platelet granule secretion Measured in Cell-Based System Using Plate Reader - 2016-01_Inhibitor_Dose_DryPowder_ActivityConfirmatorydepositor-specified cross reference: 49 drypowder compounds at dose in Dense Granule Release measuring activity set 3
Description:
Broad Institute of MIT and Harvard, Cambridge MA

NIH Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Number 1R03DA026209-01

Internal Project ID: 2016

Keywords: Platelet, activation, granule, secretion, arterial thrombosis, PAR1, SFLLRN, thrombin receptor

Primary Collaborators:

Robert Flaumenhaft, Beth Israel Deaconess Medical Center, rflaumen@bidmc.harvard.edu

John Thomas, NHLBI, ThomasJ@nhlbi.nih.gov

Probes:
ML159, ML160, ML161


Project Overview:

The goal of this project is to identify small molecules that inhibit the activation of platelets by measuring the secretion of ATP-rich dense granules. The aim is to identify inhibitory compounds that act on the diverse mechanisms responsible for platelet activation. Probes demonstrating the specific ability to inhibit platelet activation will be further evaluated as potential antithrombic agents. Those potential probes will fall into the 3 attribute classes in order of priority:
1. Small molecule probes that are dense granule specific (Class 1)
2. Small molecule probes that are granule specific (Class 2)
3. Inhibitors of specific receptors or specific sets of receptors indicating MOA through certain G proteins or adapter proteins (Class 3)
Protocol
Primary Assay Summary:
Name: Dense Granule Release
Internal Assay ID: 2016-01

Assay: Dense granule release of platelet-rich plasma (PRP). Expired units of PRP obtained from various blood centers were plated in 384-well white assay plates (Aurora, 00030721) on average of 15,600,000 platelets/well in 20ul. PRP was exposed to 7.5uM compounds from the MLPCN collection or various doses of the positive control, Cilostazol (Sigma #C0737, Lot# 042K4704, BRD-K67017579-001-04-2 ) for 30 minutes prior to addition of the thrombin receptor (Par1) activator SFLLRN (5uM, Bachem, H-8365) and detection reagent CellTiter-Glo (Promega, G755) using a modified protocol, for measurement of ATP released from the dense granules. PBS is used in place of CellTiter-Glo Buffer thus preventing platelet lysis and a high background of ATP. Luminescence measurements are taken 15 minutes after reagent addition.

Outcome: A decrease in the luminescent signal will identify compounds that either inhibit the release of dense granules from the platelets, or inhibit the luciferase enzyme. Normalization of the HTS data across runs and calibration to the positive was performed as described below. The results are reported as % inhibition. Specificity for platelet inhibition will be determined in a secondary assay.


PubChem AID 1663
Number compounds evaluated: 302517
Active Compounds : Activity_Score >=50. Using this cutoff, the PubChem_Activity_Score for the active compounds is greater than the 99.93 percentile of the PubChem_Activity_Outcomeof the negative controls.
Number of active compounds: 661
Inconclusive Compounds: Activity_Score < 50 with at least one replicate activity score >=50.
Number of inconclusive compounds: 784


Dose Confirmation HTS - AID# 1889
Assay Biology/Type: Cell-Based (Platelets)
Readout Technology: Plate Reader/Luminescence.
Target Pathway: Platelets Activation.
Test concentration(s): 8-point Dose Titration (0.09, 0.18, 0.35, 0.70, 1.40, 2.81, 5.61 and 11.22 microM).
Activity criterion: EC50 <= 1 log over the highest tested concentration.
Substances evaluated: 1584
Substances active: 651


Dose Confirmation HTS - AID# 1891
Assay Biology/Type: Enzymatic (Luciferase).
Readout Technology: Plate Reader/Luminescence.
Target Pathway: NA.
Test concentration(s): 8-point Dose Titration (0.09, 0.18, 0.35, 0.70, 1.40, 2.81, 5.61 and 11.22 microM).
Activity criterion: EC50 <= 1 log over the highest tested concentration.
Substances evaluated: 1584
Substances active: 446
Categorized Comment - additional comments and annotations
From MLP Probe Report:
Probe count: 3
MLP Probe ML# for probe 1: ML159
PubChem Substance ID (SID) for probe 1: 87235625,91148452
PubChem Compound ID (CID) for probe 1: 26871482
IC50/EC50 (nM) for probe 1: 12262
Target for probe 1: Granule secretion
Disease relevance for probe 1: Bleeding and myocardial and cerebral infarctions
Anti-target for probe 1: Luciferase
Fold selectivity for probe 1: 9
NCBI Book chapter link for probe 1: http://www.ncbi.nlm.nih.gov/books/NBK55067/ (ID: 2476984)
Grant number for probe 1: DA026209-01
MLP Probe ML# for probe 2: ML160
PubChem Substance ID (SID) for probe 2: 87556782
PubChem Compound ID (CID) for probe 2: 824820
IC50/EC50 (nM) for probe 2: 361
Target for probe 2: Granule secretion
Disease relevance for probe 2: Bleeding and myocardial and cerebral infarctions
Anti-target for probe 2: Luciferase
Fold selectivity for probe 2: 310
NCBI Book chapter link for probe 2: http://www.ncbi.nlm.nih.gov/books/NBK55073/ (ID: 2477854)
Grant number for probe 2: DA026209-01
MLP Probe ML# for probe 3: ML161
PubChem Substance ID (SID) for probe 3: 87457110
PubChem Compound ID (CID) for probe 3: 1048267
IC50/EC50 (nM) for probe 3: 11200
Target for probe 3: Granule secretion
Disease relevance for probe 3: Bleeding and myocardial and cerebral infarctions
Anti-target for probe 3: Luciferase
Fold selectivity for probe 3: 10
NCBI Book chapter link for probe 3: http://www.ncbi.nlm.nih.gov/books/NBK55066/ (ID: 2476698)
Grant number for probe 3: DA026209-01
NCBI Book chapter title for probe 1: Chemical Genetic Analysis of Platelet Granule Secretion-Probe 1
NCBI Book chapter title for probe 2: Chemical Genetic Analysis of Platelet Granule Secretion-Probe 2
NCBI Book chapter title for probe 3: Chemical Genetic Analysis of Platelet Granule Secretion-Probe 3
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML_NUMML number assigned to the compound.String
Additional Information
Grant Number: 1R03DA026209-01

Data Table (Concise)
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