Engineering of A₃ Adenosine and P2Y Nucleotide Receptors and Their Ligands

Modification of the ribose moiety of nucleotides and nucleosides has provided new insights into structural and conformational requirements for ligands at P2Y nucleotide receptors and at adenosine receptors (ARs). Methanocarba derivatives (containing a rigid bicyclic ring system in place of ribose) of adenosine, ATP, ADP, UTP, UDP, and other receptor agonist analogs were synthesized. Biological evaluation led to the conclusion that in general the Northern (N)-conformation was favored over the Southern (S)-conformation of the pseudoribose moiety at A₁ and A₃ ARs and at P2Y₁, P2Y₂, P2Y₄, or P2Y₁₁ receptors, but not P2Y₆ receptors. At the hA₃ AR a new full agonist, MRS1898, the (N)-methanocarba equivalent of CI-IB-MECA (2-chloro-N ⁶-(3-iodobenzyl)-5'-N-methylcarbamoyladenosine), had a K_i value of 1.9 nM in binding to the hA₃ AR expressed in CHO cells. Functional assays confirmed the selectivity of MRS1898, although CI-IB-MECA was even more functionally selective for human A₃ vs. hA₁ and hA_2A ARs. Thirty microM MRS1898 did not induce apoptosis in HL-60 cells, suggesting that some of the proapoptotic effects of CI-IB-MECA may be nonreceptor-mediated. Manipulation of the sequence of A₃ ARs through site-directed mutagenesis has led to pharmacologically unique constructs: constitutively active receptors and "neoceptors." Such engineered receptors may later prove to have potential for cardioprotection through gene transfer. Effects of single amino acid replacement were interpreted using a rhodopsin-based model of ligand-A₃ receptor interactions, leading to the proposal that a movement of the conserved W243 in TM6 may be involved in AR activation.