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)

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

Grant Number: 1R03DA026209-01