Structural insights into the agonist selectivity and structure-based engineering of the adenosine A3 receptor

Adenosine receptors, expressed across various tissues, play pivotal roles in physiological processes and are implicated in diverse diseases, including neurological disorders and inflammation, highlighting the therapeutic potential of receptor-selective agents. The Adenosine A3 receptor (A ₃ R), the last identified adenosine receptor, is also activated by breakdown products of post-transcriptionally modified tRNA and exhibits dual roles in neuron, heart, and immune cells, and is often overexpressed in tumors, making it a target for anticancer therapy. Despite extensive studies on the other adenosine receptors, the structure and activation mechanism of A ₃ R, especially by selective agonists like N ⁶ -methyladenosine (m ⁶ A) and namodenoson, remained elusive. Here, we identified N ⁶ -isopentenyl adenosine (i ⁶ A), a novel A ₃ R-selective ligand, via comprehensive modified adenosine library screening. Cryo-EM analyses of A ₃ R-G _i signaling complexes with two nonselective and three selective agonists revealed the structural basis for A ₃ R activation. We further conducted structure-guided engineering of m ⁶ A-insensitive A ₃ R, which would greatly facilitate future discoveries of the physiological functions of the selective activation of A ₃ R by modified adenosines. Our results clarify the selective activation of adenosine receptors, providing the basis for future drug discovery.