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Vol. 62, Issue 5, 1249-1257, November 2002
Department of Pharmacology, University of North Carolina School of
Medicine, Chapel Hill, North Carolina (G.L.W., J.C., J.L.B., T.K.H.);
Molecular Recognition Section, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
Maryland (G.R., H.S.K., X.J., K.A.J.); and Receptor Ligand Biology,
PerkinElmer Life Sciences, Boston, Massachusetts (J.L.)
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
min1) and dissociation (0.72 min1) 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.
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