Summary The G protein-coupled P2Y12 receptor (P2Y12R) is an important antithrombotic target and of great interest for pharmaceutical discovery. Its recently solved, highly divergent crystallographic structures in complex either with nucleotides (full or partial agonist) or with a nonnucleotide antagonist raise the question of which structure is more useful to understand ligand recognition. Therefore, we performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Supervised molecular dynamics (SuMD) of ticagrelor binding indicated interactions with the extracellular regions of P2Y12R, defining possible meta-binding sites. Ureas, sulfonylureas, sulfonamides, anthraquinones and glutamic acid piperazines docked readily to the antagonist-bound P2Y12R. Docking dinucleotides at both agonist- and antagonist-bound structures suggested interactions with two P2Y12R pockets. Thus, our structure-based approach consistently rationalized the main structure-activity relationships within each ligand class, giving useful information for designing improved ligands.

Summary. Background: The G-protein-coupled P2Y 12 -receptor plays a crucial role in platelet aggregation. Recently, ticagrelor was licensed as the first perorally active and reversible P2Y 12 -receptor antagonist. Objective: The present study investigated the site and the antagonistic mode of action of ticagrelor at wild-type or mutant human P2Y 12 -receptors. Methods: Recombinant wild-type or mutant human P2Y 12 -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 forskolininduced cellular cAMP production either using a [ 3 H] cAMP-radioaffinity assay or a cAMP response elementdriven luciferase reporter gene assay. Results: The natural agonist ADP inhibited forskolin-induced cAMP formation at the wild-type P2Y 12 -receptor with a lower potency (EC 50 209 nM) than the synthetic agonist 2-methylthio-ADP (EC 50 1.0 nM). Ticagrelor shifted the concentrationresponse curves of both agonists in a parallel and surmountable manner to the right. Increasing concentrations of ticagrelor caused increasing shifts. Schild-plot analysis revealed pA 2 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 P2Y 12 -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 P2Y 12 -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 P2Y 12 -receptor.

Ticagrelor is a perorally available P2Y12 receptor antagonist used for the prevention of cardiovascular events. In the present study, we analyzed the mode of action of ticagrelor at the recombinant human P2Y12 receptor and mutant constructs stably expressed in CHO Flp‐In cells. ADP and its analogue 2‐methylthio‐ADP (2‐MeSADP) decreased forskolin‐induced cAMP‐accumulation in a concentration dependent manner with 2‐MeSADP being more potent than ADP. Ticagrelor (3 to 10 nM) itself did not change basal and forskolin‐induced cAMP‐levels. Addition of ticagrelor at increasing concentrations led to increasing rightward shifts of the concentration‐response curves of ADP and 2‐MeSADP. An analysis according to Schild revealed pA2‐values of 8.7 with slopes of the linear regression lines not different from unity. These data are in agreement with a competitive mode of antagonism of ticagrelor against both agonists. The antagonistic potency of ticagrelor was maintained in cells expressing S101A‐, K173A/K174A‐, C248A‐, R256A‐ and K280A‐mutant constructs. In contrast, there were no rightward shifts of the concentration‐response curves of 2‐MeSADP and ADP by ticagrelor in cells expressing the C194A‐mutant P2Y12 receptor construct. Interestingly, antagonistic potencies of reactive blue 2 and analogues including PSB‐0739 (1‐amino‐4‐[4‐phenylamino‐3‐sulfophenylamino]‐9,10‐dioxo‐9,10‐dihydroanthracene‐2‐sulfonate) were increased in cells expressing the C194A‐mutant construct. In summary, our results suggest a competitive mode of antagonism of ticagrelor at the human P2Y12 receptor. The residue Cys194 (located in transmembrane region 5) appears to belong to the binding site of ticagrelor within the P2Y12 receptor protein. Grant Funding Source: Supported by the University of Bonn