Molecular recognition of modified adenine nucleotides by the P2Y(1)-receptor. 1. A synthetic, biochemical, and NMR approach

The remarkably high potencies of 2-thioether-adenine nucleotides regarding the activation of the P2Y(1)-receptor (P2Y(1)-R) in turkey erythrocyte memb ranes represent some of the largest substitution-promoted increases in pote ncies over that of a natural receptor ligand.(9,10) This paper describes th e investigation regarding the origin of the high potency of these P2Y(1)-R ligands over that of ATP. For this study, an integrated approach was employ ed combining the synthesis of new ATP analogues, their biochemical evaluati on, and their SAR analysis involving NMR experiments and theoretical calcul ations. These experiments and calculations were performed to elucidate the conformation and to evaluate the electronic nature of the investigated P2Y( 1)-R ligands. ATP analogues synthesized included derivatives where C2 or C8 positions were substituted with electron-donating groups such as ethers, t hioethers, or amines. The compounds were tested for their potency to induce P2Y(1)-R-mediated activation of phospholipase C in turkey erythrocytes and Ca2+ response in rat astrocytes. 8-Substituted ATP and AMP derivatives had little or no effect on phospholipase C or on calcium levels, whereas the c orresponding 2-substituted ATP analogues potently increased the levels of i nositol phosphates and [Ca2+](i). AMP analogues were ineffective except for 2-butylthio-AMP which induced a small Ca2+ response. P2Y(1)-R activity of these compounds was demonstrated by testing these ligands also on NG108-15 neuroblastoma x glioma hybrid cells. NMR data together with theoretical cal culations imply that steric, rather than electronic, effects play a major r ole in ligand binding to the P2Y(1)-R. Hydrophobic interactions and H-bonds of the C2 substituent appear to be important determinants of a P2Y(1)-R li gand affinity.