Crystal structure of adenosine A receptor in complex with clinical candidate Etrumadenant reveals unprecedented antagonist interaction.

The G protein-coupled adenosine A receptor (AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an AAR antagonist with ancillary blockade of the AAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the AAR in complex with Etrumadenant, obtained with differently thermostabilized AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T88 with the cyano group of Etrumadenant. T88 is mutated in most AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the AAR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed AAR crystallization construct that is devoid of ligand binding site mutations.