Abstract
The P2Y12 receptor plays a crucial role in platelet aggregation. In the present study, we analyzed the properties of non-nucleotide antagonists at the recombinant human P2Y12 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 P2Y12 receptor or mutant constructs. 2-Methylthio-ADP inhibited the CRE-dependent luciferase expression with an IC50 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 (pA2 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 pA2 value of 9.8. In cells expressing the R256A-mutant receptor, the potencies of both reactive blue 2 (apparent pKB, 5.9) and PSB-0739 (apparent pKB, 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 pKB 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 P2Y12 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 P2Y12 receptor.
- AZD6140, (1S,2S,3R,5S)-3-[7-[(1R,2S)-2-(3,4-difluorophenyl)cyclopropylamino]-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol
- ANOVA, analysis of variance
- CHO cells, Chinese hamster ovary cells
- CRE, cAMP response element
- HBSS, Hank′s balanced salt solution
- 2-MeSADP, 2-methylthio-ADP, 2-methylthioadenosine 5′-diphosphate
- BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid
- RB-2, reactive blue 2, amino-4-[[4-[[4-chloro-6-[[3 (or 4)-sulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-3-sulfophenyl]amino]-9,10-dihydro-9,10-dioxo-2-anthracenesulfonic acid
- PSB-0739, 1-amino-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate
- PSB-0826, 1-amino-4-[4-anilino-phenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonate, lacking a sulfonic acid residue at ring D
- PSB-0801, 1-amino-2-methyl-4-[4-phenylamino-3-sulfophenylamino]-9,10-dioxo-9,10-dihydroanthracene monosodium salt
- Bay u 9421, 2,2′-(6-chloro-1,3,5-triazine-2,4-diyl)-bis-(azanediyl)-bis-(N-(8-hydroxy-3,6-disulfonaphthalen-1-yl)benzamide tetrasodium salt
- MG 38-1, (1-amino-4-[4-amino-3-sulfonatophenylamino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfonic acid disodium salt.
Footnotes
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This work was supported by the German Research Foundation [Grant GRK804] (to Y.B., C.E.M.) and by a scholarship from the German Academic Exchange Service (to Y.B.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.109.156687
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ABBREVIATIONS:
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↵1 Current affiliation: Elsevier Pharma Biotech Group, Frankfurt, Germany.
- Received May 26, 2009.
- Accepted August 17, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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