Background: The P2Y(12) receptor plays a crucial role in platelet aggregation and is the target of platelet aggregation inhibitors, including the thienopyridine compound prasugrel.
Objective: The present study analyzed the effects of R-138727 (2-[1-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid), the active metabolite of prasugrel, on recombinant wild-type and mutant human P2Y(12) receptors in order to identify the molecular site of action of R-138727.
Methods: The function of wild-type and mutant P2Y(12) receptors stably expressed in Chinese hamster ovary cells was assessed by measuring the 2-methylthio-ADP-mediated inhibition of forskolin-stimulated cellular cAMP production.
Results: In cells expressing wild-type receptors, R-138727 potently inhibited receptor function with a half-maximal concentration below 1 microm. The mode of action was irreversible. The same effect of R-138727 was observed in cells expressing Cys17Ala/Cys270Ala constructs. In contrast, in cells expressing either a Cys97Ala construct or a Cys175Ala construct, R-138727 failed to inhibit the response to the agonist. When cells expressing wild-type receptors were pretreated with the P2 receptor antagonists ATP or suramin, no effect of R-138727 was observed. Similar experiments with N-acetylcysteine 10 microm showed no interference of N-acetylcysteine with R-138727.
Conclusions: The experiments demonstrate a potent and irreversible action of R-138727 at the recombinant human P2Y(12) receptor. The data suggest that R-138727 interacts with cysteine 97 (upper portion of the predicted third transmembrane region) and cysteine 175 (second extracellular loop) of the receptor, which are likely to form a disulfide bridge in native receptors. Moreover, the data also suggest that this site of action of R-138727 is close to the ligand-binding site of the receptor.