The alphaIIbbeta3 receptor plays a vital role in hemostasis and thrombosis, where receptor deficiency causes the hemorrhagic disorder, Glanzmann thrombasthenia, and uncontrolled receptor activation causes thrombosis and blood vessel occlusion. Small molecule inhibitors of alphaIIbbeta3, tirofiban and eptifibatide, have undesirable side effects thought to arise from conformational changes in more ..
Target
Tested Compounds:
Depositor Specified Assays
| AID | Name | Type | Comment |
|---|---|---|---|
| 2663 | Inhibitors of Platelet Integrin alphallb-beta3: Summary | summary |
Description:
NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1 R03 MH083257-01
Assay Provider: Barry Coller, Rockefeller University
NCGC Assay Overview:
The alphaIIbbeta3 receptor plays a vital role in hemostasis and thrombosis, where receptor deficiency causes the hemorrhagic disorder, Glanzmann thrombasthenia, and uncontrolled receptor activation causes thrombosis and blood vessel occlusion. Small molecule inhibitors of alphaIIbbeta3, tirofiban and eptifibatide, have undesirable side effects thought to arise from conformational changes in alphaIIbbeta3. These changes transiently activate the receptor to cause spontaneous platelet aggregation and thrombus formation. The goal of this project is to identify new alphaIIbbeta3 antagonists that do not activate the receptor or initiate conformational changes that expose novel epitopes. This assay was used to determine the selectivity of alphaIIbbeta3 inhibitors by measuring their ability to block the alphaVbeta3 receptor. The assay detects the binding of cells expressing human alphaVbeta3 to collagen (Blue et al., 2008). Cells were added to vitronectin-coated plates and after incubation and washing, bound cells were detected by measuring endogenous alkaline phosphatase (AP) activity. Inhibitors of alphaVbeta3 block cell binding to collagen and result in a decrease in AP activity.
NIH Chemical Genomics Center [NCGC]
MLPCN Grant: 1 R03 MH083257-01
Assay Provider: Barry Coller, Rockefeller University
NCGC Assay Overview:
The alphaIIbbeta3 receptor plays a vital role in hemostasis and thrombosis, where receptor deficiency causes the hemorrhagic disorder, Glanzmann thrombasthenia, and uncontrolled receptor activation causes thrombosis and blood vessel occlusion. Small molecule inhibitors of alphaIIbbeta3, tirofiban and eptifibatide, have undesirable side effects thought to arise from conformational changes in alphaIIbbeta3. These changes transiently activate the receptor to cause spontaneous platelet aggregation and thrombus formation. The goal of this project is to identify new alphaIIbbeta3 antagonists that do not activate the receptor or initiate conformational changes that expose novel epitopes. This assay was used to determine the selectivity of alphaIIbbeta3 inhibitors by measuring their ability to block the alphaVbeta3 receptor. The assay detects the binding of cells expressing human alphaVbeta3 to collagen (Blue et al., 2008). Cells were added to vitronectin-coated plates and after incubation and washing, bound cells were detected by measuring endogenous alkaline phosphatase (AP) activity. Inhibitors of alphaVbeta3 block cell binding to collagen and result in a decrease in AP activity.
Protocol
NCGC Assay Protocol Summary:
CS1 cells stably expressing normal human alphaVbeta3 were suspended in binding buffer (138 mM NaCl, 12 mM NaHCO3, 10 mM HEPES, 2.7 mM KCl, 0.4 mM NaH2PO4, 0.1% glucose, 0.35% BSA, 1 mM MgCl2, pH 7.4) and the cell counts were adjusted to 2E6/mL. Polystyrene 96-well microtiter plates (Nunc) were coated with 5 ug/ml vitronectin for 1 hour, and blocked with HBMT for at least 1 hour. Cells were treated with compounds or control solutions for 15 minutes at 37 C before being added to the microtiter wells. After adhering for 1 hour at 37 C, cells were removed by washing 3 times with binding buffer. Adherent cells were quantified by their endogenous acid phosphatase activity on p-nitrophenyl phosphate as described in Law et al., 1999.
Reference
Blue R, Murcia M, Karan C, Jirouskova M, and Coller BS. Application of high-throughput screening to identify a novel alphaIIb-specific small- molecule inhibitor of alphaIIbbeta3-mediated platelet interaction with fibrinogen. Blood. 2008, 111:1248-56
Law DA, Nannizzi-Alaimo L, Ministri K, Hughes PE, Forsyth J, Turner M, Shattil SJ, Ginsberg MH, Tybulewicz VL, Phillips DR. Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets. Blood. 93:2645-2652.
CS1 cells stably expressing normal human alphaVbeta3 were suspended in binding buffer (138 mM NaCl, 12 mM NaHCO3, 10 mM HEPES, 2.7 mM KCl, 0.4 mM NaH2PO4, 0.1% glucose, 0.35% BSA, 1 mM MgCl2, pH 7.4) and the cell counts were adjusted to 2E6/mL. Polystyrene 96-well microtiter plates (Nunc) were coated with 5 ug/ml vitronectin for 1 hour, and blocked with HBMT for at least 1 hour. Cells were treated with compounds or control solutions for 15 minutes at 37 C before being added to the microtiter wells. After adhering for 1 hour at 37 C, cells were removed by washing 3 times with binding buffer. Adherent cells were quantified by their endogenous acid phosphatase activity on p-nitrophenyl phosphate as described in Law et al., 1999.
Reference
Blue R, Murcia M, Karan C, Jirouskova M, and Coller BS. Application of high-throughput screening to identify a novel alphaIIb-specific small- molecule inhibitor of alphaIIbbeta3-mediated platelet interaction with fibrinogen. Blood. 2008, 111:1248-56
Law DA, Nannizzi-Alaimo L, Ministri K, Hughes PE, Forsyth J, Turner M, Shattil SJ, Ginsberg MH, Tybulewicz VL, Phillips DR. Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets. Blood. 93:2645-2652.
Comment
Compound Ranking:
Two compounds were tested in this assay and both were inactive and were assigned a score of 0.
Two compounds were tested in this assay and both were inactive and were assigned a score of 0.
Result Definitions
| TID | Name | Description | Histogram | Type | Unit | |
|---|---|---|---|---|---|---|
| Outcome | The BioAssay activity outcome | Outcome | ||||
| 1 | Activity | Indicates type of activity observed: active or inactive. | String | |||
| 2 | Potency* | The concentration of sample yielding half-maximal inhibition. |  | Float | μM | |
| 3 | Compound QC | NCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling' | String |
* Activity Concentration.
Additional Information
Grant Number: 1 R03 MH083257-01
Data Table (Concise)
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