Screening of Non-Purine Ligands Binding to Adenosine Receptors A1, A2A, A2B, and A3

Adenosine receptors (ARs) are G-protein coupled receptors (GPCRs) which have been shown to have therapeutic potential. ARs are made up of four subtypes, A1 , A2A , A2B , and A3 . A3 AR ligands have previously been indicated to lower inflammation, prevent or treat cancer, and produce antihyperalgesic effects in many preclinical pain models, specifically for neuropathy. We have previously published in animal models that ferulic acid dimer, a non-purine compound with the 3-methoxy phenyl moiety, has non-opioid antinociceptive properties through primarily binding to A3 ARs. Current literature suggests that the antinociceptive properties of a natural compound, incarvillateine, which contains the 3-methoxy phenyl moiety is mediated through ARs, however there is a lack of sufficient evidence of in vitro binding of such compounds at ARs. We thus hypothesized that compounds with the 3-methoxy phenyl moiety show binding to ARs, with an affinity in the submicromolar range. For this study, we performed fluorescent competitive binding assays using cells transfected with ARs: Chinese Hamster Ovary (CHO) cells for A1 or A3 receptors and Human Embryonic Kidney (HEK) cells for A2A or A2B receptors. The compounds tested included trans cinnamic acid analogs and curcumin analogs. The compounds were synthesized using cavitand-mediated photoisomerization and cavitand-mediated photodimerization. Our fluorescent binding protocol employed the separation method, in which the unbound ligand was removed after equilibrium was reached, via PBS wash. The binding assays performed suggested that 3-methoxy cinnamic acid dimer was the most effective in binding to A3 ARs, while the monomer did not bind. There was very little to no binding observed at the other receptors. Other analogs of trans cinnamic acid and curcumin did not show binding at any of the ARs so far. In conclusion, 3-methoxy cinnamic acid dimer binds to G-inhibitory coupled adenosine receptor, A3 . Future studies will investigate structure-activity-relationship of 3-methoxy phenyl dimers to demonstrate why dimerization leads to superior binding compared to similar monomeric compounds. © FASEB.