2‐Chloro N⁶‐methyl‐(N)‐methanocarba‐2′‐deoxyadenosine‐3′,5′‐bisphosphate is a selective high affinity P2Y₁ receptor antagonist

Abstract

1 We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y 1 receptor and that modi®cation of the ribose in these analogues is tolerated in the P2Y 1 receptor binding pharmacophore. 2 Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N 6 -methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety. 3 MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pK B =7.75). High anity competitive antagonism by MRS2279 was also observed at the human P2Y 1 receptor (pK B =8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was speci®c for the P2Y 1 receptor since MRS2279 had no eect on activation of the human P2Y 2 , P2Y 4 , P2Y 6 , or P2Y 11 receptors by their cognate agonists. 4 MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. 5 In contrast, the P2Y 1 receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent anity (pK B =8.05) similar to that observed at the human P2Y 1 receptor heterologously expressed in 1321N1 cells. 6 Taken together these results illustrate selective high anity antagonism of the P2Y 1 receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.

2‐Chloro‐N6‐methyl‐(N)‐methanocarba‐2′‐deoxyadenosine‐3′,5′‐bisphosphate is a selective high affinity P2Y1 receptor antagonist: Commentary on Boyer et al.

“…The present study by Boyer et al (2002)`2-Chloro-N 6 -methyl-(N)-methanocarba-2' deoxyadenosine-3',5'-bisphosphate is a selective high a nity P2 1 receptor antagonist' introduces a highly selective and competitive antagonist for the P2Y 1 receptor, reporting K b values of 18 nM at the P2Y 1 receptor in turkey erythrocytes, 8 nM at the human P2Y 1 receptor expressed in astrocytoma 1321-N1 cells, and 9 nM at the P2Y 1 receptor in human blood platelets. This antagonist is somewhat loosely related to cyclic AMP (adenosine 3',5'-cyclic monophosphate), although it is better viewed as a highly modi®ed non-cyclic biphosphate analogue.…”

Design and synthesis of new bicyclic diketopiperazines as scaffolds for receptor probes of structurally diverse functionality

“…Exploration of selective agonists and antagonists modulators is most advanced at the P2Y 1 and P2Y 12 receptors, but at most of the P2Y receptors selective pharmacological probes are lacking. [22][23][24][25][26][27][28][29][30][31][32] Compound 20 was shown to antagonize signaling of the P2Y 1 receptor selectively and with moderate potency. At the P2Y 1 receptor, the previously described high affinity antagonists are nucleotide derivatives and therefore highly charged, which is highly limiting in pharmacological studies due to low bioavailability and stability.…”

“…Cell culture and membrane preparation-Human 1321N1 astrocytoma cells transfected individually with the hP2Y 1,2,4,6,11 receptors 26,33,40 were grown at 37 °C in a humidified incubator with 5% CO 2 -95% air in Dulbecco's modified Eagle's medium (JRH Biosciences, Inc.) supplemented with 10% fetal bovine serum (FBS), 100 Units mL −1 penicillin, 100 μg mL −1 streptomycin and 2 mM L-glutamine. The cells were grown to ca.…”

“…For example, antagonists of the P2Y 1 and P2Y 12 receptors are of interest as antithrombotic agents. [22][23][24][25][26][27][28][29][30][31][32] Agonists of the P2Y 2 receptor are of interest in the treatment of pulmonary diseases, including cystic fibrosis. The P2Y 6 receptor has recently been implicated in protection against apoptosis induced by TNFα.…”

2,2′-Pyridylisatogen tosylate antagonizes P2Y1 receptor signaling without affecting nucleotide binding

“…The assay of PLC activation was carried out as previously described [15]. 1321N1 astrocytoma cells stably expressing human P2Y receptors were harvested by trypsinization and grown in six-well plates (−10 6 cells per well; Costar, Cambridge, MA) in DMEM culture medium supplemented with 2 μCi/ml of myo-[ 3 H] inositol.…”