Structure-Guided Design Of A High-Affinity Platelet Integrin Alpha(Iib)Beta(3) Receptor Antagonist That Disrupts Mg2+ Binding To The Midas

An integrin found on platelets, alpha(IIb)beta(3) mediates platelet aggregation, and alpha(IIb)beta(3) antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the beta subunit metal ion-dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the alpha(IIb) subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the beta(3) subunits. They induce conformational changes in the beta(3) subunit that may have negative consequences such as exposing previously hidden epitopes and inducing the active conformation of the receptor. We recently reported an inhibitor of alpha(IIb)beta(3) (RUC-1) that binds exclusively to the alpha(IIb) subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a similar to 100-fold higher affinity. RUC-2 does not induce major conformational changes in beta(3) as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-alpha(IIb)beta(3) headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 angstrom revealed that RUC-2 binds to aIIb the way RUC-1 does, but in addition, it binds to the beta(3) MIDAS residue glutamic acid 220, thus displacing Mg2+ from the MIDAS. When the Mg2+ concentration was increased to 20 mM, however, Mg2+ was identified in the MIDAS and RUC-2 was absent. RUC-2's ability to inhibit ligand binding and platelet aggregation was diminished by increasing the Mg2+ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other alpha(IIb)beta(3) antagonists and may offer advantages as a therapeutic agent.