X-Ray Crystallography of G Protein-coupled Receptors

Abstract

G protein-coupled receptors (GPCRs) represent the largest family of integral membrane proteins and are widely appreciated as highly important drug targets. While routine drug discovery is largely based on high-throughput screening campaigns to identify potential lead compounds, structure-based drug design (SBDD) can substantially support the optimization route of the identified lead. However, high-resolution structural information of the desired target, for example by employing X-ray crystallography, is a prerequisite for SBDD. Crystal structure elucidation of proteins by X-ray crystallography is nowadays routinely applied for water-soluble proteins but still remains a challenge for membrane proteins and specifically for GPCRs. <br /> Herein, we aimed to establish a structural biology platform for membrane proteins in the Pharmaceutical Institute at the University of Bonn. The successful implementation was demonstrated by the elucidation of new high-resolution crystal structures of the adenosine A_2A receptor (A2AAR) in complex with relevant ligands. The A_2AAR belongs to the adenosine receptor family of GPCRs that consists of four different members. A drug that blocks the A_2AAR is already established for the treatment of Parkinson’s Disease, and others are intensively investigated as novel checkpoint inhibitors in the field of immuno-oncology with several ongoing clinical trials. <br /> In total, seven new crystal structures of the A2AAR could be resolved, in complex with six A2AAR antagonists and one partial agonist (LUF5834). These comprise five previously unpublished A_2AAR antagonists that were developed in-house, including a fluorescence-labeled ligand, a novel dual A_2AAR antagonist/MAO-B inhibitor and a xanthine-based dual A_2A/A2BAR antagonist. Additionally, one structure was obtained with a dual A_2A/A_2AAR antagonist that was developed by a pharmaceutical company and is currently under clinical investigation (Etrumadenant). Our structures revealed important determinants for A_2AAR antagonist binding of new scaffolds including novel interactions, e.g., a direct hydrogen bond between A_2AAR antagonists PSB-21417 and Etrumadenant with amino acid T88. Our ligands are currently being further optimized using the newly obtained structural information. <br /> This achievement was facilitated by the discovery of an extraordinarily stable A_2AAR crystallization construct that only harbored a single mutation in the transmembrane domains (S91K) and that was proven to be superior to previously established and highly-mutated A_2AAR constructs.