Jespers, WillemVerdon, GrégoryAzuaje Guerrero, Jhonny AlbertoMajellaro, MaríaKeränen, HenrikGarcía Mera, XerardoCongreve, MilesDeflorian, FrancescaDe Graaf, ChrisZhukov, AndreiDoré, Andrew S.Mason, Jonathan S.Åqvist, JohanCooke, Robert M.Sotelo Pérez, EddyGutiérrez de Terán, Hugo2021-01-262021-01-262020W. Jespers, G. Verdon, J. Azuaje, M. Majellaro, H. Keränen, X. García-Mera, M. Congreve, F. Deflorian, C. de Graaf, A. Zhukov, A. S. Doré, J. S. Mason, J. Åqvist, R. M. Cooke, E. Sotelo, H. Gutiérrez-de-Terán, Angew. Chem. Int. Ed. 2020, 59, 165361433-7851http://hdl.handle.net/10347/24309We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X‐ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2AAR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2AAR were experimentally determined and investigated through a cycle of ligand‐FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X‐ray crystallography of the A2AAR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2AAR, an emerging target in immuno‐oncologyeng© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co.KGaA. This is an open access article under the terms of the CreativeCommons AttributionLicense, which permits use, distribution and reproduction in any medium, provided the original work is properly citedAtribución 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/Adenosine receptorsBiophysical mapping (BPM)Free energy perturbation (FEP)G protein-coupled receptor (GPCR)journal article10.1002/anie.2020037881521-3773open access