Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate
Abstract
The antagonist activity of N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate (N6MABP) has been examined at the phospholipase C-coupled P2Y1 receptor of turkey erythrocyte membranes. N6MABP antagonized 2MeSATP-stimulated inositol phosphate hydrolysis with a potency approximately 20 fold greater than the previously studied parent molecule, adenosine 3′,5′-bisphosphate. The P2Y1 receptor antagonism observed with N6MABP was competitive as revealed by Schild analysis (pKB=6.99±0.13). Whereas N6MABP was an antagonist at the human P2Y1 receptor, no antagonist effect of N6MABP was observed at the human P2Y2, human P2Y4 or rat P2Y6 receptors.
Introduction
Pharmacological classification of natively expressed G protein-coupled P2Y receptors and unambiguous definition of their physiological roles have been problematic due to lack of selective receptor antagonists. The available antagonists interact with non-receptor ATP-binding proteins and antagonize both P2Y and P2X receptors (Ziganshin et al., 1993; Bultmann & Starke, 1994; Harden et al., 1995; Charlton et al., 1996).
The turkey erythrocyte has been utilized as a model cell-free assay system for pharmacological and biochemical characterization of the P2Y1 receptor and its associated signalling proteins (Boyer et al., 1996b). Using this test system we recently identified a group of molecules, the adenosine bisphosphates, that exhibit partial agonist/antagonist activities at the P2Y1 receptor (Boyer et al., 1996a). To identify antagonist molecules that interact with higher affinity with the P2Y1 receptor, a series of adenosine bisphosphate derivatives were synthesized with various adenine base- or ribose-substitutions (Camaioni et al., 1998). In this paper we describe the P2Y1 receptor activity and P2Y receptor subtype specificity of one of the most promising of these molecules, N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate (N6MABP or MRS2179).
Methods
Assay of P2Y receptor-promoted inositol lipid hydrolysis Turkey erythrocytes were labelled with [3H]-inositol and P2Y1 receptor-promoted phospholipase C activity was determined in membranes prepared from these cells as previously described (Boyer et al., 1996a). Inositol phosphate accumulation in 1321N1 human astrocytoma cells expressing the human P2Y1 receptor, the human P2Y2 receptor, the human P2Y4 receptor or the rat P2Y6 receptor was determined as previously described (Boyer et al., 1997).
Chemical synthesis N6MABP was prepared as previously described (Camaioni et al., 1998). The chemical structure of the phosphorylated nucleoside was verified by use of 1H-n.m.r. and 31P-n.m.r. techniques, as well as by high resolution fast atomic bombardment mass spectroscopy.
Results
A series of second generation molecules was synthesized (Camaioni et al., 1998) based on our observation that adenosine bisphosphates are P2Y1 receptor antagonists (Boyer et al., 1996a). Absence of a hydroxyl in the 2’ position of these molecules resulted in analogues that, in contrast to the originally studied adenosine bisphosphates, do not interact with A1-adenosine receptors (data not shown). N6MABP (insert in Figure 1a) was one of the most promising of these molecules in preliminary studies, since no partial agonist activity was observed at the turkey erythrocyte membrane P2Y1 receptor and the effect of the full agonist 2MeSATP was inhibited with an IC50 approximately 20 fold lower than that of the parent compound 2′-deoxyadenosine 3′,5′-bisphosphate (data not shown). The nature of the antagonism was determined by generating a series of concentration-effect curves for 2MeSATP for stimulation of phospholipase C in the presence of different concentrations of N6MABP. N6MABP caused a parallel shift to the right of the concentration-response curve for 2MeSATP (Figure 1a). In contrast to the effects of reactive blue-2, suramin and PPADS in the same assay system (Boyer et al., 1994), the effects of high concentrations of N6MABP were completely surmountable by high concentrations of 2MeSATP (Figure 1a). Schild analysis (Figure 1b) confirmed that the antagonism produced by N6MABP was competitive with a pKb of 6.99 ± 0.13 (n= 3). Identical antagonist properties were observed with the positional isomer of N6MABP, N6-methyl 3′-deoxyadenosine 2′,5′-bisphosphate (data not shown).
Competitive inhibition of 2MeSATP-promoted activation of P2Y1 receptors by N6MABP. (a) [3H]-inositol-labelled turkey erythrocyte membranes were incubated with the indicated concentrations of 2MeSATP in the presence or absence of the indicated concentrations of N6MABP (inset). (b) Schild regression analysis of data shown in (a). The data shown are the results from a representative experiment repeated three times to yield a mean pKB of 6.99 ± 0.13 and Schild slope of 0.93 ± 0.05.
The selectivity of N6MABP was tested by examining its effects at the human P2Y1, human P2Y2, human P2Y4 and rat P2Y6 receptors (Figure 2). N6MABP completely blocked P2Y1 receptor-promoted stimulation of phospholipase C activity induced by a near maximal concentration (100 nM) of 2MeSATP (Figure 2). This inhibition also was competitive (data not shown). In contrast, no significant inhibition was observed of the response to 100 nM UTP at the human P2Y2 and P2Y4 receptors, or to 100 nM UDP at the P2Y6 receptor (Figure 2).
Effects of N6MABP on mammalian P2Y receptors stably expressed in 1321N1 human astrocytoma cells. [3H]-inositol-labelled 1321N1 human astrocytoma cells stably expressing the human P2Y1, human P2Y2, human P2Y4 or rat P2Y6 receptors were incubated with 100 nM 2MeSATP (P2Y1), 100 nM UTP (P2Y2 and P2Y4), or 100 nM UDP (P2Y6) in the absence or in the presence of 30 μM N6MABP. The data are from a representative experiment repeated three times.
Discussion
The data presented here illustrate that N6MABP is a high affinity competitive antagonist at the P2Y1 receptor. This antagonist also is remarkably selective since it apparently does not block the three other cloned mammalian P2Y receptors. The high affinity and receptor selectivity of N6MABP make it unique among the molecules now available for investigation of P2Y receptors. Although useful as P2 receptor antagonists, reactive blue 2 and suramin are well known to interact with many nucleotide binding proteins in addition to the P2 receptors, and they bind to multiple of the known P2 receptors (Harden et al., 1995). Lambrecht and coworkers have provided an important advance by introducing PPADS (Lambrecht et al., 1992), which apparently exhibits selectivity for P2 receptors over other types of proteins. However, PPADS also antagonizes both P2X and P2Y receptors, although it does exhibit selectivity for the P2Y1 receptor over other P2Y receptor subtypes (Ziganshin et al., 1993; Boyer et al., 1994; Bultmann & Starke, 1994; Charlton et al., 1996). A notable exception to the relatively non-selective P2 receptor antagonists is the molecule, 2-propylthio-D-β,γ-dichloromethylene ATP (ARL 67085), which is a selective antagonist of very high affinity at the Gi/adenylyl cyclase-linked P2Y receptor of platelets (Humphries et al., 1995). This molecule does not block P2Y1 receptors, which prominently distinguishes it from adenosine 3′,5′-bisphosphate which competitively blocks the P2Y1 receptor but does not interact with the Gi/adenylyl cyclase-linked receptor of C6 rat glioma cells (Boyer et al., 1996a).
A major advantage of N6MABP is that it is an adenine nucleotide derivative in a group of second generation molecules that have followed from the observation that phosphate-substitution in the 2′- or 3′-position results in loss of agonist activity at P2Y receptors without a coincident loss of receptor affinity. Discovery of antagonist activity in an adenine nucleotide derivative places us in position to take further advantage of the wide ranging structure activity studies that have preceded on adenine nucleotide derivatives at the P2Y1 receptor. Moreover, the pharmacological selectivity and apparent affinity of the adenosine bisphosphate derivative demonstrated here should prove useful for pharmacological resolution of the P2Y1 receptor in various mammalian tissues.
