Application of high-throughput screening to identify a novel αIIb-specific small-molecule inhibitor of αIIbβ3-mediated platelet interaction with fibrinogen

Small-molecule alpha IIb beta 3 antagonists competitively block ligand binding by spanning between the D224 in alpha IIb and the MIDAS metal ion in beta 3. They variably induce conformational changes in the receptor, which may have undesirable consequences. To identify alpha IIb beta 3 antagonists with novel structures, we tested 33 264 small molecules for their ability to inhibit the adhesion of washed platelets to immobilized fibrinogen at 16 mu M. A total of 102 compounds demonstrated 50% or more inhibition, and one of these (compound 1, 265 g/mol) inhibited ADP-induced platelet aggregation (IC50: 13 +/- 5 mu M), the binding of soluble fibrinogen to platelets induced by mAb AP5, and the binding of soluble fibrinogen and a cyclic RGD peptide to purified alpha IIb beta 3. Compound 1 did not affect the function of GPIb, alpha 2 beta 1, or the other beta 3 family receptor alpha V beta 3. Molecular docking simulations suggest that compound 1 interacts with alpha IIb but not beta 3. Compound 1 induced partial exposure of an alpha IIb ligand-induced binding site (LIBS), but did not induce exposure of 2 03 LIBS. Transient exposure of purified alpha IIb beta 3 to eptifibatide, but not compound 1, enhanced fibrinogen binding ("priming"). Compound 1 provides a prototype for small molecule selective inhibition of alpha IIb beta 3, without receptor priming, via targeting alpha IIb.