Structure-Guided Design of a High-Affinity Platelet Integrin αIIbβ3 Receptor Antagonist That Disrupts Mg2+ Binding to the MIDAS

A new drug blocks platelet aggregation by interacting with the integrin receptor differently from current drugs that cause adverse side effects. Free-Flowing Blood The αIIbβ3 integrin receptor, found on the surface of platelets, is central to the aggregation of platelets. This coagulation stops bleeding but, if unchecked, contributes to thrombosis, heart attacks, and some forms of stroke. Currently, there are three approved medications that block the integrin receptor and inhibit thrombosis, but they require intravenous administration and have the side effect of causing low platelet counts in some patients. The current study builds on previous research in which high-throughput screening identified a small molecule that could inhibit the receptor by binding to the αIIb subunit. An improved version of that compound, called RUC-2, also binds the αIIb subunit but, in addition, binds to the β3 subunit in an unprecedented way that prevents the binding of a vital Mg2+ ion to its usual site, preventing ligand binding and platelet aggregation. RUC-2 differs from drugs that were developed for oral therapy but failed during development as a result of increased mortality and low platelet counts. Unlike these drugs, RUC-2 does not induce the structural changes in the β3 subunit that may contribute to these adverse effects. RUC-2 could, therefore, have significant advantages over existing drugs, although this still needs to be tested directly in patients. An integrin found on platelets, αIIbβ3 mediates platelet aggregation, and αIIbβ3 antagonists are effective antithrombotic agents in the clinic. Ligands bind to integrins in part by coordinating a magnesium ion (Mg2+) located in the β subunit metal ion–dependent adhesion site (MIDAS). Drugs patterned on the integrin ligand sequence Arg-Gly-Asp have a basic moiety that binds the αIIb subunit and a carboxyl group that coordinates the MIDAS Mg2+ in the β3 subunits. They induce conformational changes in the β3 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 αIIbβ3 (RUC-1) that binds exclusively to the αIIb 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 β3 as judged by monoclonal antibody binding, light scattering, gel chromatography, electron microscopy, and a receptor priming assay. X-ray crystallography of the RUC-2–αIIbβ3 headpiece complex in 1 mM calcium ion (Ca2+)/5 mM Mg2+ at 2.6 Å revealed that RUC-2 binds to αIIb the way RUC-1 does, but in addition, it binds to the β3 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 αIIbβ3 antagonists and may offer advantages as a therapeutic agent.