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BioAssay: AID 652250

Discovery of Novel Antagonists of Protease activated receptor 4: Single Point

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. PAR1 antagonism was efficacious for the prevention of cardiovascular events in two phase III clinical trials, though patients also had an increased risk of intracranial hemmorage. PAR4 is the low affinity thrombin receptor and thus inhibition of PAR4 may also prove to be efficacious for the prevention of cardiovascular events without the increased risk of bleeding. ..more
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 Tested Compounds
 Tested Compounds
All(191)
 
 
Probe(1)
 
 
Active(14)
 
 
Inactive(177)
 
 
 Tested Substances
 Tested Substances
All(191)
 
 
Probe(1)
 
 
Active(14)
 
 
Inactive(177)
 
 
AID: 652250
Data Source: Vanderbilt Specialized Chemistry Center (Protease activated receptor 4 (PAR4) Antagonist Single Point..)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2013-04-02
Hold-until Date: 2014-04-01
Modify Date: 2014-04-01

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: Chemical Probe: 1    Active: 14
Related Experiments
AIDNameTypeComment
652261ML354 Discovery of Novel Antagonists of Protease activated receptor 4Summarydepositor-specified cross reference
652253Discovery of Novel Antagonists of Protease activated receptor 4: CRC AssayConfirmatorysame project related to Summary assay
652255Discovery of Novel Antagonists of Protease activated receptor 4: PAR 1 Selectivity CRC AssayConfirmatorysame project related to Summary assay
686926ML354 Eurofin Panel Assay for PAR4 Antagonists Inhibitor (Probe Compound)Othersame project related to Summary assay
Description:
Assay Provider: Heidi Hamm
Assay Provider Affiliation: Vanderbilt University

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. PAR1 antagonism was efficacious for the prevention of cardiovascular events in two phase III clinical trials, though patients also had an increased risk of intracranial hemmorage. PAR4 is the low affinity thrombin receptor and thus inhibition of PAR4 may also prove to be efficacious for the prevention of cardiovascular events without the increased risk of bleeding.

There are no probes available to study the role of PAR4 in thrombosis and hemostasis in vivo. The current PAR4 antagonist YD-3 was first described as an antagonist in 2001 and no further characterization or optimization has been conducted. YD-3 has several drawbacks including, a lengthy synthetic scheme, and high lipophilicity making it unsuitable for chemical modification and having limited in vivo use. The field's understanding of the role of PAR4 in physiology is limited, because of the lack of a sufficient tool compound to study PAR4. An optimized compound will allow us to determine basic pharmacological information about PAR4 including receptor density, possible patient-to-patient variability, and mechanism of action for PAR4 antagonists and agonists, data which currently cannot be collected.
Protocol
Washed human platelets were prepared via standard procedure and suspended in Tyrode's buffer containing 0.1% BSA. Platelets were dye loaded for 1 hour with Fluo4-AM (Invitrogen, Eugene, OR) in calcium assay buffer (1X HBSS, 20 mM HEPES, 2.5 mM probenecid without calcium or magnesium). The calcium assay buffer containing dye is mixed with platelets to yield a final concentration of 2.5 mug/mL Fluo4-AM and 1.0x108 platelets/mL. 60 muL of dye loaded platelets were added to each well of a NUNC 384 well plate black optical bottom plate (Thermo, Rochester, NY). Fluorescence measurements were recorded in the Vanderbilt Center for Neuroscience Drug Discovery's Screening Center using a Functional Drug Screening System 6000, Hamamatsu (Hamamatsu, Japan). 10 muM of each compound was added in triplicate 6 minutes prior to the addition of 80 muM Protease activated receptor 4-activating peptide(PAR4-AP) (GL Biochem, Shanghai, China). 480:540 (ex:em) was measured each second for a total of 12 minutes at 37 oC. The final concentration of DMSO in the assay was 0.5%. The difference between basal and maximal relative fluorescence unit values (RFUs) for ex:em 480:540 was determined for each well. The change in RFU for DMSO treated control stimulated with 80 muM PAR4-AP was set to 100% for each donor. IC50 values were determined using GraphPad PRISM v.5.0 inhibitory sigmoidal dose response 'variable slope' parameter.
Compounds VU0099704-1, VU0478975-1, VU0469155-1, VU0469154-1, VU0469152-1, VU0476680-1, VU0476683-1, VU0476684-1, VU0476685-1 and VU0476689-1 demonstrated greater than 50% inhibition of 80 uM PAR4-AP calcium response, had resonable physiochemical properties and were thus subject to inhibitory CRC assays.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Single point Calcium % Max PAR4-AP mean (10μM**)See ProtocolFloat%
2Single point Calcium % Max PAR4-AP Stdev. (10μM**)See ProtocolFloat%
3CategoryString
4IC50_uM*See ProtocolFloatμM
5IC50_std_errSee ProtocolFloatμM

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 5P50HL081009-03

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