Unraveling Integrin Antagonists' Target-Recognition Mechanisms

Abstract

Background: Pharmaceutical blockade of the platelet αIIbβ3 integrin receptor has reduced mortality from cardiovascular disease. However, gaps in mechanistic understanding limit clinical efficacy and delay new drug development.Objectives: Integrating surface plasmon resonance (SPR) and dynamic force spectroscopy (DFS), we aim to measure the strength of integrin:ligand bonds, determining their weakest link and identifying new therapeutic intervention routes.Methods: cHarGD, a cyclic peptide structurally similar to eptifibatide, a widely used antiplatelet drug, served as a model ligand, one readily coupled to biosensors and AFM tips. SPR provided kinetic, equilibrium, and transition state thermodynamic parameters for αIIbβ3:cHarGD complex formation, while DFS measured their mechanical stability. cHarGA, lacking the aspartate required αIIbβ3 binding, served as a negative control.Results: SPR demonstrated that integrin binding to immobilized cHarGD was rapid (kon ∼ 7 × 103 L/mol-sec at 25 oC), readily reversible (koff ∼ 10−2 sec−1), and specific (100-fold smaller signals with cHarGA). Eyring and van't Hoff analyses indicated that after overcoming an entropic barrier (ΔGao‡ 12 kcal/mol), both enthalpy and entropy favored assembly of the αIIbβ3:cHarGD complex (ΔG° - 8 kcal/mol). Preliminary DFS experiments (12 nN/sec loading rate) indicated that the rupture force of cHarGD:αIIbβ3 is about 300 pN. In control experiments, where the tip was functionalized with cHarGA or albumin, lower rupture forces of 225 pN and 170 pN were observed.Conclusions: Our SPR data indicate that entropy plays a major role in target recognition by integrin antagonists, a property shared by ∼25% of drug:receptor interactions. Our DFS data suggest that integrin:ligand interactions are stabilized by multivalent contacts between clustered receptors and pharmaceutical inhibitors.This study will provide the first complete picture of the landscape for integrin:ligand interactions, using temperature and force as thermodynamic variables to determine the energetics and nm scale on which bond disruption occurs.