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

Abstract

ADP is the cognate agonist of the P2Y1, P2Y12, and P2Y13 receptors. With the goal of identifying a high potency agonist that selectively activates the P2Y1 receptor, we examined the pharmacological selectivity of the conformationally constrained non-nucleotide 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 Gi-linked P2Y12 and P2Y13 receptors, a chimeric G protein, Galphaq/i, was coexpressed to confer a capacity of these Gi-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 P2Y1 receptor, this non-nucleotide analog exhibited no agonist activity at the P2Y12 receptor and very low activity at the P2Y13 receptor. (N)-Methanocarba-2MeSADP also failed to block the action of 2MeSADP at the P2Y12 and P2Y13 receptors, indicating that the (N)-methanocarba analog is not an antagonist at these receptors. The P2Y1 receptor selectivity of (N)-methanocarba-2MeSADP was confirmed in human platelets where it induced the shape change promoted by P2Y1 receptor activation without inducing the sustained platelet aggregation that requires simultaneous activation of the P2Y12 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.

Fig. 1

Structure of 2MeSADP and (N)-methanocarba-2MeSADP.

Fig. 2

Agonist activities of 2MeSADP and (N)-methanocarba-2MeSADP at the human P2Y₁, P2Y₁₂, and P2Y₁₃ receptors. COS-7 cells transfected with mammalian expression vectors for the human P2Y₁, P2Y₁₂, or P2Y₁₃ receptors were preloaded with [³H]inositol, and agonist-promoted inositol lipid hydrolysis was quantified as described under Materials and Methods. Cells transfected with P2Y₁₂ or P2Y₁₃ receptor expression vectors also were cotransfected with an expression vector for Gα_q/i, which confers capacity of these two G_i-coupled receptors to activate phospholipase C. Inositol phosphate responses were measured to the indicated concentrations of 2MeSADP (▲) or (N)-methanocarba-2MeSADP (●) in P2Y₁ receptor expressing cells (A), P2Y₁₂ receptor expressing cells (B), and P2Y₁₃ receptor expressing cells (C). The data are presented as counts per minute (mean ± S.E.M.; n = 3) of [³H]inositol phosphate accumulation, and the results are representative of results obtained in at least three full concentration-effect curves generated for both agonists with each receptor.

Fig. 3

Comparative response of P2Y₁, P2Y₁₂, and P2Y₁₃ receptors to (N)-methanocarba-2MeSADP. COS-7 cells expressing the P2Y₁ receptor, the P2Y₁₂ receptor + Gα_q/i, or the P2Y₁₃ receptor + Gα_q/i were challenged with the indicated concentrations of (N)-methanocarba-2MeSADP. The response in each case is compared with that of a maximally effective concentration (100 nM) of 2MeSADP, which was assigned a value of 100%. The results are the mean ± S.E.M. of four independent experiments carried out in triplicate.

Fig. 4

Induction of shape change of human platelets by (N)-methanocarba-2MeSADP. Washed human platelets were prepared, and drug-induced shape change (decrease in light transmission) and aggregation (increase in light transmission) were quantified in an aggregometer as described under Materials and Methods. Whereas addition of 30 nM 2MeSADP resulted in a transient shape change followed by platelet aggregation, addition of 1 or 10 μM (N)-methanocarba-2MeSADP only resulted in a rapidly occurring but sustained shape change. Platelets incubated with 1 μM (N)-methanocarba-2MeSADP for 3 min still responded with an aggregation response to 2MeSADP. These results are representative of results obtained in three different experiments.

Fig. 5

Concentration-dependent induction of platelet shape change by (N)-methanocarba-2MeSADP. Washed human platelets were prepared, and drug-induced shape change (decrease in light transmission) in response to the indicated concentrations of (N)-methanocarba-2MeSADP was quantified in an aggregometer as described under Materials and Methods. The results are representative of those obtained in three separate experiments.

Fig. 6

Antagonism of (N)-methanocarba-2MeSADP-induced platelet shape change by the P2Y₁ receptor antagonist MRS2179. Washed human platelets were prepared, and drug-induced shape change (decrease in light transmission) and the capacity of the P2Y₁ receptor antagonist MRS2179 to antagonize the shape change response to the indicated concentrations of (N)-methanocarba-2MeSADP was quantified in an aggregometer as described under Materials and Methods. In each case, 10 μM MRS2179 was added for 2 min prior to the addition of (N)-methanocarba-2MeSADP. The results are representative of those from three separate experiments.

Fig. 7

Concentration-effect curve for (N)-methanocarba-2MeSADP in the absence or presence of MRS2179. The platelet shape change response was measured exactly as described in Fig. 5 for various concentrations of (N)-methanocarba-2MeSADP alone and in Fig. 6 for various concentrations of (N)-methanocarba-2MeSADP in the presence of 10 μM MRS2179. The resulting shape change tracings were transformed into concentration-effect curves by quantifying the relative maximal shape change occurring 1 min after agonist addition in each case. The results are presented as the average mean values (S.E.M. varied by less than 15% for each value) from three experiments.