Vögele, M.; Shahoei, R.; Petridis, L.; Li, J.; Lin, F.-Y.; Wang, L.; Springer, T. A.; Vendome, J.

Integrins are crucial cell adhesion receptors and attractive therapeutic targets, but developing oral small-molecule inhibitors has been challenging, at least in part due to inadvertent partial agonism caused by stabilization of the integrin's open, high-affinity state. To address this challenge, we present a computational approach using Absolute Binding Free Energy Perturbation (AB-FEP) calculations to predict whether a ligand will stabilize the open or closed integrin states, leveraging the difference between the ligand's binding free energy to the respective end states. Despite challenges posed by Ca and Mg ions, metal-coordinating residues in the binding pocket, and the subtlety of structural differences between states, AB-FEP achieves excellent classification performance on a set of known opening and closing ligands, significantly outperforming docking scores and MM-GBSA results. We also show a good correlation between AB-FEP binding free energy differences and experimental values. Furthermore, AB-FEP provides insights into intermediate integrin states and analysis of simulation trajectories confirmed the formation of a water-mediated hydrogen bond network with an ion in the binding pocket to be characteristic for closing ligands. This work demonstrates AB-FEP as a robust method for classifying integrin ligands and understanding their functional mechanisms, offering valuable guidance for designing safe and conformationally selective integrin therapeutics.