Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5'-O-(1-boranotriphosphate) derivatives.
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-alpha-B) were synthesized by substitution of a nonbridging O at P(alpha) with a BH(3) 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 P2Y(1) and rat P2Y(2) receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y(1)-GFP, rP2Y(2)-GFP). We investigated agonist-induced receptor endocytosis, [Ca(2+)](i) rise and arachidonic acid (AA) release. Agonist-induced endocytosis of rP2Y(1)-GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP-alpha-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 Ca(2+) release by these compounds in HEK 293 cells stably transfected with rP2Y(1). In case of rP2Y(2)-GFP, the borano-nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca(2+) release, AA release and in inducing receptor endocytosis. The different ATP-alpha-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 P2Y(1) 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.
Tulapurkar
M E
ME
Institut für Neurobiochemie, Medizinische Fakultät, Otto-von-Guericke-Universität Magdeburg, Leipziger Strasse 44, 39120 Magdeburg, Germany.
Laubinger
W
W
Nahum
V
V
Fischer
B
B
Reiser
G
G
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004705:Endocytosis / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D018613:Microscopy, Confocal; D008968:Molecular Conformation; D058910:Purinergic P2 Receptor Agonists; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D011994:Recombinant Proteins / Q000378:metabolism; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D014162:Transfection
D018076:DNA, Complementary; C546382:P2RY1 protein, human; C546385:P2RY2 protein, human; C546384:P2ry1 protein, rat; C546387:P2ry2 protein, rat; D058910:Purinergic P2 Receptor Agonists; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D016718:Arachidonic Acid; D000255:Adenosine Triphosphate; D002118:Calcium