Molecular Modeling of the Human P2Y₂ Receptor and Design of a Selective Agonist, 2‘-Amino-2‘-deoxy-2-thiouridine 5‘-Triphosphate

A rhodopsin-based homology model of the nucleotide-activated human P2Y₂ receptor, including loops, termini, and phospholipids, was optimized with the Monte Carlo multiple minimum conformational search routine. Docked uridine 5‘-triphosphate (UTP) formed a nucleobase π−π complex with conserved Phe3.32. Selectivity-enhancing 2‘-amino-2‘-deoxy substitution interacted through π-hydrogen-bonding with aromatic Phe6.51 and Tyr3.33. A “sequential ligand composition” approach for docking the flexible dinucleotide agonist Up₄U demonstrated a shift of conserved cationic Arg3.29 from the UTP γ position to the δ position of Up₄U and Up₄ ribose. Synthesized nucleotides were tested as agonists at human P2Y receptors expressed in 1321N1 astrocytoma cells. 2‘-Amino and 2-thio modifications were synergized to enhance potency and selectivity; compound 8 (EC₅₀ = 8 nM) was 300-fold P2Y₂-selective versus P2Y₄. 2‘-Amine acetylation reduced potency, and trifluoroacetylation produced intermediate potency. 5-Amino nucleobase substitution did not enhance P2Y₂ potency through a predicted hydrophilic interaction possibly because of destabilization of the receptor-favored Northern conformation of ribose. This detailed view of P2Y₂ receptor recognition suggests mutations for model validation.