Novel modified adenosine 5′-triphosphate analogues pharmacologically characterized in human embryonic kidney 293 cells highly expressing rat brain P2Y₁ receptor: Biotinylated analogue potentially suitable for specific P2Y₁ receptor isolation

Rat brain P2Y₁ (rP2Y₁) receptor-transfected human embryonic kidney cells (HEK 293) were recently shown to have enhanced reactivity to both ATP and ADP (Vöhringer C, Schäfer R, Reiser G. Biochem Pharmacol 2000;59:791-800). Here, we demonstrated the usefulness of this cell line as a system for further studying novel adenine nucleotide analogues (Halbfinger et al. J Med Chem 1999;42:5325-37) and for the biochemical characterization of the P2Y₁ receptor. By measurement of intracellular Ca²⁺ release, for 2-butylthio-, 2-butylamino-, and 2-butyloxy-ATP (2-BuS-, 2-BuNH-, 2-BuO-ATP), EC₅₀ values of 1.3, 5, and 60 nM were determined, markedly lower than the value for ATP (130 nM). The EC₅₀ for 2-BuSADP was 1.1 nM. The corresponding 8-substituted ATP analogues showed a substantially lower potency than ATP (ATP > 8-BuSATP > 8-BuNHATP ≈ 8-BuOATP). AMP induced intracellular Ca²⁺ release with a very low potency; 2- and 8-substitutions on AMP caused no significant potency shift, except for 2-BuSAMP (EC₅₀ = 180 nM). Another new P2Y receptor probe, 2-[(6-biotinylamido)-hexylthio]ATP, was 22-fold more potent than ATP (EC₅₀ = 6 nM), revealing that even more bulky substituents linked to the C-2 position bind with high affinity at the P2Y₁ receptor. This biotinylated probe was successfully used for the enrichment of the P2Y₁ receptor tagged with green fluorescent protein from a crude membrane fraction. This one-step enrichment provides a substantial advance for P2Y₁ receptor purification. Thus, human embryonic kidney 293 cells stably transfected with the rP2Y₁ receptor represent a powerful model system for pharmacological characterization of the P2Y₁ receptor, circumventing problems associated with natural systems. They provide a means for the development of P2Y₁ ligands of high potency and a good source for obtaining purified P2Y₁ receptor.