Characterization of Dual-Acting A₃ Adenosine Receptor Positive Allosteric Modulators That Preferentially Enhance Adenosine-Induced Gα_i3 and Gα_oA Isoprotein Activation

The A₃ adenosine receptor (A₃AR) is a promising therapeutic target for inflammatory diseases, cancer, and chronic neuropathic pain, with agonists already in advanced clinical trials. Here we report an in-depth comparison of the pharmacological properties and structure–activity relationships of existing and expanded compound libraries of 2-substituted 1H-imidazo­[4,5-c]­quinolin-4-amine and 4-amino-substituted quinoline derivatives that function as A₃AR positive allosteric modulators (PAMs). We also show that our lead compound from each series enhances adenosine-induced A₃AR signaling preferentially toward activation of Gα_i3 and Gα_oA isoproteins, which are coexpressed with the A₃AR in immune cells and spinal cord neurons. Finally, utilizing an extracellular/intracellular chimeric A₃AR approach composed of sequences from a responding (human) and a nonresponding (mouse) species, we provide evidence in support of the idea that the imidazoquinolin-4-amine class of PAMs variably interacts dually with the orthosteric ligand binding site as well as with a separate allosteric site located within the inner/intracellular regions of the receptor. This study has advanced both structural and pharmacological understanding of these two classes of A₃AR PAMs, which includes leads for future pharmaceutical development.