A neoceptor approach to unraveling microscopic interactions between the human A2A adenosine receptor and its agonists

Strategically mutated neoceptors, e.g., with anionic residues in TMs 3 and 7 intended for pairing with positively charged amine-modified nucleosides, were derived from the antiinflammatory A(2A) adenosine receptor (AR). Adenosine derivatives functionalized at the 5', 2, and N-6 positions were synthesized. The T88D mutation selectively enhanced the binding of the chain-length-optimized 5'-(2-aminoethyl)uronamide but not 5'-(2-hydroxyethyl)uronamide, suggesting a critical role of the positively charged amine. Combination of this modification with the N-6(2-methylbenzyl) group enhanced affinity at the Q89D- and N181 D- but not the T88D-A(2A)AR. Amino groups placed near the 2- or N-6-position only slightly affected the binding to mutant receptors. The 5'-hydrazide MRS3412 was 670-and 161-fold enhanced, in binding and functionally, respectively, at the Q89D-A(2A)AR compared to the wild-type. Thus, we identified and modeled pairs of A(2A)AR-derived neoceptor-neoligand, which are pharmacologically orthogonal with respect to the native species.