Induction of Novel Agonist Selectivity for the ADP-Activated P2Y₁Receptor Versus the ADP-Activated P2Y₁₂and P2Y₁₃Receptors by Conformational Constraint of an ADP Analog

Abstract

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 non-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, G␣ 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 EC 50 values in the sub to low nanomolar range. In contrast, whereas (N)-methanocarba-2MeSADP was an extremely potent (EC 50 ϭ 1.2 Ϯ 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.The G protein-coupled P2Y receptor family is comprised of at least eight different human receptors that are activated by nucleoside diphosphates, nucleoside triphosphates, or nucleotide sugars to regulate a broad range of physiological responses including neurotransmission, muscle contraction, ion secretion, and platelet aggregation (Dubyak and ElMoatassim, 1993;Harden et al., 1998;Ralevic and Burnstock, 1998). A complex set of ectoenzymes that metabolize extracellular nucleotides has complicated the study of these receptors Zimmermann, 2000). This difficulty is exacerbated by the lack of selective agonists or antagonists for most of the P2Y receptors.

P2Y Purinergic Regulation of the Glycine Neurotransmitter Transporters

“…Fig. 1, B and C, shows that time and dose dependence of GLYT2 inhibition by 2MeSADP was also consistent with the reported P2YR features (45,46). Inhibition was maximal at the shortest assayed time of 1 min and was lost within 10 min.…”

Agonists and Antagonists for P2 Receptors

“…At the P2Y 1 receptor the (S)-methanocarba analogue had similar potency to ATP. Applying the (N)-methanocarbo modification to 2-methylthio-ADP, itself a potent agonist of P2Y 1 , P2Y 12 and P2Y 13 receptors, resulted in MRS2365 (Chhatriwala et al 2004). MRS2365 proved to be the most potent agonist reported for the P2Y 1 receptor (EC 50 0.4 nM) with high selectivity (>10 000-fold) in comparison to the P2Y 12 and P2Y 13 receptors.…”

Caged agonist of P2Y1 and P2Y12 receptors for light-directed facilitation of platelet aggregation

“…The activity at the P2Y 12 receptor was tested using a bioassay consisting of stimulation of calcium mobilization in stably transfected 1321N1 astrocytoma cells. Although in a previous study the 1321N1 cells were cotransfected with a chimeric G protein, Gαq/i, to confer a capacity of the Gi-linked P2Y 12 receptor to directly activate phospholipase C [26], in the current study only the endogenous G proteins were present. The coupling of the P2Y 12 receptor to a calcium response in the 1321N1 cells was likely as reported previously [17] through G-protein β,γ subunits.…”

“…We have not evaluated the light-directed activation of the P2Y 13 receptor by MRS2703. However, such activation is expected because the parent nucleotide and photoproduct, 2-MeSADP, is a potent agonist of the P2Y 13 receptor [1,26].…”

“…Thus, use of an o-nitrobenzyl-type phosphodiester would allow that monophosphate moiety to be regenerated upon irradiation. Alternately, the P2Y 1 selective agonist MRS2365, an (N)-methanocarba derivative of 2MeSADP [26], could be derivatized in a manner analogous to MRS2703 for the selective light-directed activation of P2Y 1 receptors. Furthermore, caged P2Y receptor ligands, such as MRS2703 and caged MRS2500 may have potential for hemodynamic control in the clinic, in a manner that is more spatially selective than with use of anti-thrombotic drugs.…”