Identification of a Potent Inverse Agonist at a Constitutively Active Mutant of Human P2Y₁₂ Receptor

Abstract

Human platelets express two P2Y receptors: G q -coupled P2Y 1 , and G i -coupled P2Y 12 . Both P2Y 1 and P2Y 12 are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y 12 receptor with its C terminus replaced by the corresponding part of human P2Y 1 receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y 12 mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y 12 mutant was dramatically inhibited [(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y 12 receptor and the identification of potent inverse agonist.

Identification of Determinants Required for Agonistic and Inverse Agonistic Ligand Properties at the ADP Receptor P2Y12

“…P2Y 12 expressed in yeast showed a similar EC 50 value for MeS-ADP (EC 50 value, 6 nM; Fig. 1) as when expressed in mammalian cells ranging from low nanomolar to 25-80 nM concentrations (Zhang et al, 2001;Simon et al, 2002;Bodor et al, 2003;Zhong et al, 2004;Ding et al, 2006;Hoffmann et al, 2008). ADP was approximately 500-fold less potent to MeS-ADP, which is consistent with previous findings showing an approximately 30-fold to 1000-fold lower potency in mammalian cells (Zhang et al, 2001;Simon et al, 2002;Bodor et al, 2003).…”

“…For example, many b-blockers and atropine are inverse agonists at b1-adrenoceptors and muscarinic acetylcholine receptors, respectively (Thor et al, 2009;Baker et al, 2011). Therapeutically used P2Y 12 ligands are high-affinity antagonists, but inverse activity was described only for the experimental P2Y 12 blocker AR-C78511 (Vasiljev et al, 2003;Ding et al, 2006). AR-C78511 is a 2-alkylthio-substituted ATP analog but, in contrast to mant-dATP, has no modification at the 29 or 39 OH residues of the ribose.…”

“…In previous experiments, we and others demonstrated that the human P2Y 12 is functionally expressed in the yeast system (Schulz and Schoneberg, 2003;Pausch et al, 2004), which lacks such problems. Numerous constitutively activating mutations have been described for GPCRs in natural or recombinant systems, but only a few have been reported for P2Y receptors (Ding et al, 2006). From more than 1000 single point mutations, we identified 28 constitutively active P2Y 12 mutants.…”

“…Therefore, inverse agonists may be therapeutically beneficial compared with antagonists. Because only a few inverse agonists of P2Y 12 have been described (Ding et al, 2006), we therefore screened for compounds that reduce the basal activity of constitutively active P2Y 12 mutants.…”

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

“…55 A follow-up study also revealed that AR-C78511 had superior antiplatelet activity compared with a neutral antagonist (cangrelor) in transgenic mice expressing the same chimeric receptor. 56 Interestingly, AR-C78511, like ticagrelor, could not be docked to the 2MeSADP-bound conformation of the P2Y 12 R, seemingly because of its own bulky N6 substituents.…”