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.