Quantitation of the P2Y1 receptor 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 [H-3] methylamine into the synthesis of [H-3] MRS2279 to high (89 Ci/mmol) specific radioactivity and have used this molecule as a radioligand for the P2Y1-R. [H-3] 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 [H-3] 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 x 10(7) M-1 min(-1)) and dissociation (0.72 min(-1))of [H-3] MRS2279 also was in good agreement with the K-d derived from equilibrium binding studies. Competition binding assays with [H-3] 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 muM) that were in good agreement with antagonist activities of these molecules previously determined at the P2Y1-R in intact tissues. Moreover, [H-3] 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. [H-3] 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 [H-3] MRS2279 is the first broadly applicable antagonist radioligand for a P2Y receptor.