Quantitation of the P2Y1Receptor with a High Affinity Radiolabeled Antagonist

2-Chloro- N 6 -methyl-( N )-methanocarba-2′-deoxyadenosine-3′,5′- bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y 1 receptor (P2Y1-R) antagonist ( J Med Chem 43: 829–842, 2002; Br J Pharmacol 135: 2004–2010, 2002). We have taken advantage of the N 6 -methyl substitution in the adenine base to incorporate [ 3 H]methylamine into the synthesis of [ 3 H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [ 3 H]MRS2279 bound to membranes from Sf9 insect cells expressing recombinant human P2Y1-R but not to membranes from wild-type Sf9 cells or Sf9 cells expressing high levels of recombinant P2Y 2 or P2Y 12 receptors. Equilibrium binding of [ 3 H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity ( K d = 8 nM) essentially identical to the apparent affinity of MRS2279 determined previously in studies of P2Y1-R–promoted inositol phosphate accumulation or platelet aggregation. A kinetically derived K d calculated from independent determinations of the rate constants of association (7.15 × 10 7 M −1 min −1 ) and dissociation (0.72 min −1 ) of [ 3 H]MRS2279 also was in good agreement with the K d derived from equilibrium binding studies. Competition binding assays with [ 3 H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed K i values for the P2Y1-R antagonists MRS2279 ( K i = 13 nM), N 6 -methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179; K i = 84 nM), adenosine-3′, 5′-bisphosphate ( K i =900 nM), and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid ( K i = 6 μM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [ 3 H]MRS2279 also bound with high affinity ( K d = 4–8 nM) to Chinese hamster ovary (CHO) or 1321N1 human astrocytoma cells stably expressing the human P2Y1-R, but specific binding was not observed in wild-type CHO or 1321N1 cells. [ 3 H]MRS2279 bound with high affinity ( K d = 16 nM) to a binding site on out-dated human platelets (5–35 receptors/platelet) and rat brain membranes (210 fmol/mg protein) that fit the expected drug selectivity of a P2Y1-R. Taken together, these results indicate that [ 3 H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.