Abstract 1313: Evidence for Distinct Ligand Binding Sites on Recombinant Human P2Y12 Receptors
Background: The P2Y12 receptor is a Gi-coupled GPCR activated by ADP that is expressed on platelets and plays an integral role in platelet aggregation. AZ11931285 is an investigational triazolo-pyrimidine compound that binds to the recombinant human P2Y12 receptor (Kd = 2nM), antagonizes ADP-mediated GTPγS activation (pKi = 8.3), and inhibits ADP-induced aggregation in washed platelets (pIC50 = 8.2). When examining ADP-mediated GTPγS binding in the presence of increasing concentrations of AZ11931285 using CHO-K1 cell membranes stably expressing rh-P2Y12 receptors, we observed only a small shift in ADP potency but a significantly decreased efficacy. In contrast, AZ11931285 caused only a potency shift when 2Me-S-ADP was used as agonist, suggesting that the relationship between the two agonists and AZ11931285 is different.
Methods: To further investigate these observations, we used 125I-AZ11931285, 3H-ADP, and 33P-2Me-S-ADP in competition binding experiments using the rh-P2Y12 receptor.
Results: These experiments showed that ADP was able to displace itself (IC50 = 90 nM) and 33P-2Me-S-ADP (IC50 = 454 nM), but was unable to displace 125I-AZ11931285. In contrast, AZ11931285 was able to displace itself (IC50 = 87 nM) and 33P-2Me-S-ADP (IC50 ± 86 nM), but not 3H-ADP. Consistent with these observations, 2Me-S-ADP was able to displace itself, 3H-ADP, and 125I-AZ11931285. Additionally, ATP, ATPβS, and ADPβS all competed with 3H-ADP and 33P-2Me-S-ADP, whilst not affecting 125I-AZ11931285 binding. In contrast, 2Me-S-AMP targets 125I-AZ11931285 and 33P-2Me-S-ADP, but has no effect on 3H-ADP binding.
Conclusions: Our data demonstrate that ADP and AZ11931285 bind independently to the recombinant human P2Y12 receptor, whilst the binding of 2Me-S-ADP overlaps with both. Preliminary data using AZD6140, a cyclopentyl-triazolo- pyrimidine, suggest that it binds to the rh-P2Y12 receptor in a similar way to AZ11931285, but further experiments will investigate whether the endogenous receptor expressed on platelet membranes demonstrates similar properties.