Structure-guided design of a high-affinity platelet integrin α _IIbβ ₃ receptor antagonist that disrupts Mg ²⁺ binding to the MIDAS

An integrin found on platelets, α _IIbβ ₃ mediates platelet aggregation, and α _IIbβ ₃ antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg ²⁺) located in the b subunit metal ion - dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the α _IIb subunit and a carboxyl group that coordinates the MIDAS Mg ²⁺ in the β ₃ subunits. They induce conformational changes in the β 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 α _IIbβ ₃ (RUC-1) that binds exclusively to the α _IIβ subunit; here, we report the structure-based design and synthesis of RUC-2, a RUC-1 derivative with a ∼100-fold higher affinity. RUC-2 does not induce major conformational changes in β ₃as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2-α _IIbβ ₃ headpiece complex in 1 mM calcium ion (Ca ²⁺)/5 mM Mg ²⁺ at 2.6 Å revealed that RUC-2 binds to α _IIb the way RUC-1 does, but in addition, it binds to the β ₃ MIDAS residue glutamic acid 220, thus displacing Mg ²⁺ from the MIDAS. When the Mg ²⁺ concentration was increased to 20 mM, however, Mg ²⁺ 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 Mg ²⁺ concentration. Thus, RUC-2 inhibits ligand binding by a mechanism different from that of all other α _IIbβ ₃ antagonists and may offer advantages as a therapeutic agent.