Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5′‐O‐(1‐boranotriphosphate) derivatives

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Abstract
P2Y‐nucleotide receptors represent important targets for drug development. The lack of stable and receptor specific agonists, however, has prevented successful therapeutic applications. A novel series of P‐boronated ATP derivatives (ATP‐α‐B) were synthesized by substitution of a nonbridging O at P with a BH3 group. This introduces a chiral center, thus resulting in diastereoisomers. In addition, at C2 of the adenine ring a further substitution was made (Cl‐ or methylthio‐). The pairs of diastereoisomers were denoted here as A and B isomers. Here, we tested the receptor subtype specificity of these analogs on HEK 293 cells stably expressing rat P2Y1 and rat P2Y2 receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y1‐GFP, rP2Y2‐GFP). We investigated agonist‐induced receptor endocytosis, [Ca2+]i rise and arachidonic acid (AA) release. Agonist‐induced endocytosis of rP2Y1‐GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP‐α‐B analogs. Both 2‐MeS‐substituted diastereoisomers induced a greater degree of agonist‐induced receptor endocytosis as compared to the 2‐Cl‐substituted derivatives. Endocytosis results are in accordance with the potency to induce Ca2+ release by these compounds in HEK 293 cells stably transfected with rP2Y1. In case of rP2Y2‐GFP, the borano‐nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca2+ release, AA release and in inducing receptor endocytosis. The different ATP‐α‐B derivatives and also the diastereoisomers were equally ineffective. Thus, the new agonists may be considered as potent and highly specific agonist drug candidates for P2Y1 receptors. The difference in activity of the ATP analogs at P2Y receptors could be used as a tool to investigate structural differences between P2Y receptor subtypes. British Journal of Pharmacology (2004) 142, 869–878. doi:10.1038/sj.bjp.0705859