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

Abstract

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 (A3R), 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 A3R, especially by selective agonists like N6-methyladenosine (m6A) and namodenoson, remained elusive. Here, we identified N6-isopentenyl adenosine (i6A), a novel A3R-selective ligand, via comprehensive modified adenosine library screening. Cryo-EM analyses of A3R-Gi signaling complexes with two nonselective and three selective agonists revealed the structural basis for A3R activation. We further conducted structure-guided engineering of m6A-insensitive A3R, which would greatly facilitate future discoveries of the physiological functions of the selective activation of A3R by modified adenosines. Our results clarify the selective activation of adenosine receptors, providing the basis for future drug discovery.

2-Substituted (N)-Methanocarba A3 Adenosine Receptor Agonists: In Silico, In Vitro, and In Vivo Characterization

“…AR binding affinities were determined at three subtypes of hARs using standard radioligand inhibition assays, and mAR affinity was determined for some analogues for comparison (Table ). ,, The affinities represented a greater range than previously reported 2-arylethynyl (N)-methanocarba 5′-methylamides such as 2b , 3a , and 4 – 8 . − Consistently, homologous 2-arylethynyl derivatives prepared here that contain a 2-chlorophenylethynyl moiety ( 3b – 3d ) displayed potent hA 3 AR affinities. Among the 2-aryl derivatives, the following compounds showed the highest hA 3 AR affinity ( K i , nM): 9 , 3.28; 14 , 4.82; 23 , 2.44; 27 , 10.1; 34 , 8.66.…”

“…The A 3 AR, a G protein-coupled receptor (GPCR) of Class A, has been modeled extensively using homology modeling and molecular dynamics (MD) simulation − prior to the very recent report of experimental structures. − We and others have investigated the structure–activity relationship (SAR) of adenosine-derived A 3 AR agonists, and recently structure-guided approaches have been used to arrive at highly selective agonists ,− , Two prototypical A 3 AR agonists 1a and 1b (Figure ) containing a 5′- N -methylcarboxamido-ribose moiety are in advanced clinical trials (Phases 2 and 3). ,− However, greater A 3 AR selectivity can be achieved by replacing the furanose ring of adenosine analogues with a North (N) methanocarba (bicyclo[3.1.0]­hexyl) ring system. ,− , This ring system enforces a rigid conformation that approximates the receptor-preferred conformation, particularly in the A 3 AR case. The enhanced affinity is consistent with an entropic cost in the binding of the corresponding ribose analogue, which can attain either a (N) or South (S) conformation, either in solution or when receptor bound .…”