Single Stabilizing Point Mutation Enables High-Resolution Co-Crystal Structures of the Adenosine A_2A Receptor with Preladenant Conjugates

The G protein-coupled adenosine A_2A receptor (A_2A AR) is an important new (potential) drug target in immuno-oncology, and for neurodegenerative diseases. Preladenant and its derivatives belong to the most potent A_2A AR antagonists displaying exceptional selectivity. While crystal structures of the human A_2A AR have been solved, mostly using the A_2A -StaR2 protein that bears 9 point mutations, co-crystallization with Preladenant derivatives has so far been elusive. We developed a new A_2A AR construct harboring a single point mutation (S91^3.39 K) which renders it extremely thermostable. This allowed the co-crystallization of two novel Preladenant derivatives, the polyethylene glycol-conjugated (PEGylated) PSB-2113, and the fluorophore-labeled PSB-2115. The obtained crystal structures (2.25 Å and 2.6 Å resolution) provide explanations for the high potency and selectivity of Preladenant derivatives. They represent the first crystal structures of a GPCR in complex with PEG- and fluorophore-conjugated ligands. The applied strategy is predicted to be applicable to further class A GPCRs.