title: Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12 -receptor.
abstract: BACKGROUND
  The G-protein-coupled P2Y12 -receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y12 -receptor antagonist.
  
  
  OBJECTIVE
  The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y12 -receptors.
  
  
  METHODS
  Recombinant wild-type or mutant human P2Y12 -receptors were stably expressed in Chinese hamster ovary Flp-In cells. Receptor function was assessed by quantification of ADP- and 2-methylthio-ADP-mediated inhibition of forskolin-induced cellular cAMP production either using a [(3) H]cAMP-radioaffinity assay or a cAMP response element-driven luciferase reporter gene assay.
  
  
  RESULTS
  The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y12 -receptor with a lower potency (EC50 209 nm) than the synthetic agonist 2-methylthio-ADP (EC50 1.0 nm). Ticagrelor shifted the concentration-response curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA2 values of 8.85 for ticagrelor against ADP, and 8.69 against 2-methylthio-ADP, and slopes of the regression lines not different from unity. In cells expressing a recombinant C194A(5.43) -mutant P2Y12 -receptor construct, ticagrelor lost antagonistic potency when tested against ADP or 2-methylthio-ADP.
  
  
  CONCLUSIONS
  The experiments reveal a surmountable and competitive mode of antagonism of ticagrelor at P2Y12 -receptors activated by either the natural agonist ADP or the synthetic agonist 2-methylthio-ADP. Cys194(5.43) is likely to be involved in the interaction of ticagrelor with ADP and 2-methylthio-ADP. The data give new insights into the site and mode of action of ticagrelor at the human P2Y12 -receptor.
authors.affiliation: Pharma Center Bonn, Department of Pharmacology and Toxicology, University of Bonn, Bonn, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Hoffmann
authors.forename: D A
authors.initials: DA
authors.lastname: Lutz
authors.forename: J
authors.initials: J
authors.lastname: Straßburger
authors.forename: Y
authors.initials: Y
authors.lastname: Baqi
authors.forename: C E
authors.initials: CE
authors.lastname: Müller
authors.forename: I
authors.initials: I
authors.lastname: von Kügelgen
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D058912:Purinergic P2Y Receptor Agonists / Q000494:pharmacology; D058921:Purinergic P2Y Receptor Antagonists / Q000378:metabolism / Q000494:pharmacology*; D058925:Receptors, Purinergic P2Y12 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D020218:Response Elements; D013873:Thionucleotides / Q000494:pharmacology; D000077486:Ticagrelor; D014162:Transfection
chemical_list: C548576:P2RY12 protein, human; D010975:Platelet Aggregation Inhibitors; D058912:Purinergic P2Y Receptor Agonists; D058921:Purinergic P2Y Receptor Antagonists; D058925:Receptors, Purinergic P2Y12; D013873:Thionucleotides; D005576:Colforsin; C002480:methylthio-ADP; D000244:Adenosine Diphosphate; D000242:Cyclic AMP; D008156:Luciferases; D000077486:Ticagrelor; D000241:Adenosine
keywords: binding site; mode of action; platelets; receptors, purinergic P2Y12