title: Interaction of new, very potent non-nucleotide antagonists with Arg256 of the human platelet P2Y12 receptor.
abstract: The P2Y(12) receptor plays a crucial role in platelet aggregation. In the present study, we analyzed the properties of non-nucleotide antagonists at the recombinant human P2Y(12) receptor and searched for amino acids involved in the molecular interaction. Receptor function was assessed by measuring the cAMP response element (CRE)-directed luciferase expression in Chinese hamster ovary cells. The cellular cAMP production was accelerated by forskolin; 2-methylthio-ADP was used to activate the wild-type P2Y(12) receptor or mutant constructs. 2-Methylthio-ADP inhibited the CRE-dependent luciferase expression with an IC(50) value of approximately 1 nM. The anthraquinone derivative reactive blue 2 used at increasing concentrations shifted the concentration-response curve of 2-methylthio-ADP to the right in a manner compatible with competitive antagonism (pA(2) value, 7.4). Its analog, 1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate (PSB-0739), showed a markedly higher antagonistic potency with a pA(2) value of 9.8. In cells expressing the R256A-mutant receptor, the potencies of both reactive blue 2 (apparent pK(B), 5.9) and PSB-0739 (apparent pK(B), 9.1) were decreased. The same was true for the pure reactive blue 2 meta- and para-isomers and for the ortho-isomer cibacron blue 3GA. In contrast, the analog, 1-amino-4-[4-anilino-phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, lacking a sulfonic acid residue at ring D (PSB-0826), showed similar pK(B) values at wild-type (8.4) and R256A-mutant receptors (8.3). In summary, the results demonstrate that PSB-0739 is the most potent competitive non-nucleotide antagonist at the human P2Y(12) receptor described so far. The results also indicate that the sulfonic acid residue at ring D is involved in the interaction of antagonists derived from reactive blue 2 with the residue Arg256 of the human P2Y(12) receptor.
authors.affiliation: Department of Pharmacology and Toxicology, University of Bonn, Bonn, Germany. kugelgen@uni-bonn.de
authors.forename: Kristina
authors.initials: K
authors.lastname: Hoffmann
authors.forename: Younis
authors.initials: Y
authors.lastname: Baqi
authors.forename: María Sol
authors.initials: MS
authors.lastname: Morena
authors.forename: Markus
authors.initials: M
authors.lastname: Glänzel
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
authors.forename: Ivar
authors.initials: I
authors.lastname: von Kügelgen
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D001120:Arginine / Q000187:drug effects*; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D051786:Cyclic AMP Response Element Modulator / Q000494:pharmacology; D006801:Humans; D007536:Isomerism; D008156:Luciferases / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2 / Q000737:chemistry / Q000235:genetics; D058925:Receptors, Purinergic P2Y12; D013451:Sulfonic Acids / Q000737:chemistry; D013498:Suramin / Q000494:pharmacology; D013873:Thionucleotides / Q000737:chemistry / Q000494:pharmacology; D014227:Triazines / Q000737:chemistry / Q000494:pharmacology*
chemical_list: C548576:P2RY12 protein, human; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D013451:Sulfonic Acids; D013873:Thionucleotides; D014227:Triazines; D051786:Cyclic AMP Response Element Modulator; D005576:Colforsin; C002480:methylthio-ADP; C012842:Cibacron Blue F 3GA; D013498:Suramin; D000244:Adenosine Diphosphate; D001120:Arginine; D000242:Cyclic AMP; D008156:Luciferases