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CELLULAR AND MOLECULAR

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

Department of Pharmacology, University of North Carolina, School of Medicine, Chapel Hill, North Carolina (M.C., J.L.B., T.K.H.); National Institute of Diabetes & Digestive & Kidney Diseases, National Institutes of Health, Bethesda, Maryland (R.G.R., K.A.J.); and Inspire Pharmaceuticals, Inc., Durham, North Carolina (R.I.P., J.L.B.)

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 G_i-linked P2Y₁₂ and P2Y₁₃ 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 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.

Address correspondence to: T. Kendall Harden, Kenan Professor, University of North Carolina, School of Medicine, Department of Pharmacology, CB #7365, Chapel Hill, NC 27599-7365. E-mail: tkh{at}med.unc.edu

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