Competitive mode and site of interaction of ticagrelor at the human platelet P2Y12‐receptor

Abstract

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.

Inverse agonism at the P2Y12 receptor and ENT1 transporter blockade contribute to platelet inhibition by ticagrelor

“…5 A study assessing P2Y 12 R function in CHO cells indicated that ticagrelor acted in a competitive manner based on functional readouts although only low concentrations of ticagrelor (,30 nM) were included in the analysis. 28 In conflicting studies, ticagrelor has been shown to display characteristics of a noncompetitive antagonist, decreasing the maximum response (E max ) as well as right-shifting the ADP concentration-response curve. 4 Ticagrelor did not displace [ 3 H]ADP from the receptor (K i .10 mM) but displaced 2MeS-ADP from membranes prepared from human washed platelets.…”

“…Our Schild analysis therefore suggests that ticagrelor and AR-C 66096 are acting as noncompetitive and competitive antagonists, respectively, with pA 2 values in broad agreement to those reported. 28 Upon closer examination of forskolin-stimulated adenylyl cyclase activity, a significant reduction was noted in P2Y 12 R-expressing cells vs vector control cells ( Figure 7D), indicating constitutive (agonistindependent) activity of the P2Y 12 R. This attenuation of forskolin responsiveness because of P2Y 12 R expression was reversed by ticagrelor (10 mM; 30 minutes) but not with the other P2Y 12 R antagonists ( Figure 7E). The ability of ticagrelor to modulate this agonist-independent P2Y 12 R activity was concentration dependent For personal use only.…”

Ocular Purine Receptors as Drug Targets in the Eye

“…Two of them (thienopyridines Clopidogrel 80 and Prasugrel 81) are prodrugs that must be first activated enzymatically in vivo; the other 2 are nucleotide Cangrelor 77 and nucleoside Ticagrelor 78, both of which are competitive at the receptor. 87 Applications of P2YR agonists in the eye and elsewhere are envisioned, 3 and consequently, selective agonists for P2Y 1 R, P2Y 2 R, P2Y 4 R, P2Y 6 R, P2Y 11 R, and P2Y 14 R have been reported.…”

Antiplatelet drugs: which targets for which treatments?

“…In the latter case, this allowed visualization of an adjacent binding site, close to the agonist site. However, AZD1283 was found to behave as a competitive antagonist [33][34][35]. In addition, these reports confirmed the role of the cysteine at position 97 as a key moiety for the action of the active metabolites of thienopyridine compounds [28,29].…”