Induction of novel agonist selectivity for the ADP-activated P2Y1 receptor versus the ADP-activated P2Y12 and P2Y13 receptors by conformational constraint of an ADP analog

ADP is the cognate agonist of the P2Y(1), P2Y(12), and P2Y(13) receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y(1) receptor, we examined the pharmacological selectivity of the conformationally constrained nonnucleotide analog (N)-methanocarba-2MeSADP [(1'S, 2'R, 3'S, 4'R, 5'S)-4-[(6-amino-2-methylthio-9H-purin-9-yl)-1-diphosphoryloxymethyl] bicyclo[3.1.0] hexane-2,3-diol] among the three ADP-activated receptors. Each P2Y receptor was expressed transiently in COS-7 cells, and inositol lipid hydrolysis was quantified as a measure of receptor activity. In the case of the G(i)-linked P2Y(12) and P2Y(13) receptors, a chimeric G protein, Galpha(q/i), was coexpressed to confer a capacity of these G(i)-linked receptors to activate phospholipase C. 2MeSADP (2-methylthio-ADP) was a potent agonist at all three receptors exhibiting EC50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC50 = 1.2 +/- 0.2 nM) agonist at the P2Y(1) receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y(12) receptor and very low activity at the P2Y(13) receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y(12) and P2Y(13) receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y(1) receptor selectivity of (N)methanocarba- 2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y(1) receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y(12) receptor. These results provide the first demonstration of a high-affinity agonist that discriminates among the three ADP-activated P2Y receptors, and therefore, introduce a potentially important new pharmacological tool for delineation of the relative biological action of these three signaling proteins.