2-Chloro-N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2279) was developed previously as a selective high-affinity, non-nucleotide P2Y1 receptor (P2Y1-R) antagonist (J Med Chem 43:829–842, 2002; Br J Pharmacol 135:2004–2010, 2002). We have taken advantage of the N6-methyl substitution in the adenine base to incorporate [3H]methylamine into the synthesis of [3H]MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [3H]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 P2Y2 or P2Y12 receptors. Equilibrium binding of [3H]MRS2279 to P2Y1-R expressed in Sf9 membranes was with a high affinity (Kd = 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 Kd calculated from independent determinations of the rate constants of association (7.15 × 107 M−1 min−1) and dissociation (0.72 min−1) of [3H]MRS2279 also was in good agreement with the Kd derived from equilibrium binding studies. Competition binding assays with [3H]MRS2279 and P2Y1-R expressing Sf9 cell membranes revealed Ki values for the P2Y1-R antagonists MRS2279 (Ki = 13 nM), N6-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179; Ki = 84 nM), adenosine-3′, 5′-bisphosphate (Ki = 900 nM), and pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (Ki = 6 µM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [3H]MRS2279 also bound with high affinity (Kd = 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. [3H]MRS2279 bound with high affinity (Kd = 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 [3H]MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor
