Structural and pharmacological analysis of novel adenosine A2A receptor ligands to guide rational drug design

Extracellular adenosine signalling is mainly conferred through adenosine receptors, including the adenosine A2A receptor (A2AR), modulating a variety of different physiological responses through the human body. Because of its versatile role, A2AR represents a valuable drug target for neurodegenerative diseases and in immunotherapy for cancer. This work aims to characterise the receptor-ligand interactions for a selection of novel A2AR screening hits identified by Novartis, to uncover structure-activity relationships that can be exploited for lead identification and further ligand optimisation. To this end, a combination of pharmacological, in silico, and structural methods were employed. Four of the provided hit ligands were characterised as having nanomolar affinity and were selected for structural characterisation. The best hit, DEL-1, displayed a pIC50 of 7.3 ± 0.19 in competition binding assays and inhibited agonist-mediated receptor function with a pIC50 of 7.7 ± 0.18. A series of co-crystallisation trials were undertaken using differently stabilised receptor constructs, and adding the ligand at different points during the purification protocol. Additionally, in meso soaking experiments were explored. While these strategies were successful with the established antagonist ZM241385, no diffracting crystals were obtained with the novel compounds. In silico ligand docking studies predicted that DEL-1 and DEL-2 extend into a subordinate, potentially allosteric binding pocket, likely inducing different receptor conformations that might have hampered the success of the crystallographic studies. The performed pharmacological and docking studies with FOC-6 suggested ligand binding to a completely different receptor site, possibly a lipid pocket, that prevented the ZM241385–A2AR complex from forming well ordered crystals. Cryo-EM studies are ventured to characterise this interaction. Overall, this study forms a basis for rational chemical optimisation of the selected hits, which might lead to the discovery of novel, subtype selective or allosteric A2AR ligands and suggests novel strategies for structural characterisation of receptor ligand complexes.