AID	Name	Type	Comment
2628	Selectivity Counterscreen Cell Adhesion Assay for Inhibitors of Platelet Integrin alphallb-beta3	confirmatory	Secondary assay: Selectivity of alphaIIbbeta3 inhibitors measured by inhibition of the alphaVbeta3 receptor
2634	Platelet Adhesion Secondary Assay for Inhibitors of Platelet Integrin alphallb-beta3	confirmatory	Secondary assay 2: Inhibition of the binding of human platelets to fibrinogen
2639	Platelet Aggregation Secondary Assay for Inhibitors of Platelet Integrin alphallb-beta3	confirmatory	Secondary assay 3: Inhibition of platelet aggregation

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.

RUC-1, a compound containing a thiadiazolopyrimidinone core, inhibits alphaIIbbeta3 with ~10 uM IC50 in vitro (Blue et al., 2008) and shows anti-thrombotic effects in vivo (Blue et al., 2009). Analogs of RUC-1 were synthesized and tested in platelet binding (AID 2634) and platelet aggregation (AID 2639) assays to assess their activity. Selectivity was determined by a cell-based alphaVbeta3 binding assay (AID 2628). This work identified RUC-1 analogs that were selective and more potent inhibitors of alphaIIbbeta3. A probe compound (CID 44820665; probe number ML165) was identified later.

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

Blue R, Kowalska MA, Hirsch J, Murcia M, Janczak CA, Harrington A, Jirouskova M, Li J, Fuentes R, Thornton MA, Filizola M, Poncz M, and Coller BS. Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel alphaIIb-specific alphaIIbbeta3 antagonist. Blood. 2009, 114:195-201

NCGC Assay Protocol Summary:

Please refer to AIDs 2634, 2639, 2628 for detailed assay protocols.

Compound Ranking:

This summary is written for the purposes of summarizing the probe activities from the project. MLSCN probes are given a score of 100. Molecules in the prior art are given a score of 80. Other, less active molecules in the same chemical series as the probe molecules are given a score of 50. Inconclusive compounds are given a score of 20. Inactive analogues from these series are given a score of 0.

Grant Number: 1 R03 MH083257-01