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Published in final edited form as: Drug Dev Res. 2000 Jun 12;49(4):253–259. doi: 10.1002/1098-2299(200004)49:4<253::AID-DDR4>3.0.CO;2-1

Activity of Novel Adenine Nucleotide Derivatives as Agonists and Antagonists at Recombinant Rat P2X Receptors

Sean G Brown 1, Brian F King 1, Yong-Chul Kim 2, Soo Yeon Jang 2, Geoffrey Burnstock 1, Kenneth A Jacobson 2,*
PMCID: PMC3393598  NIHMSID: NIHMS385207  PMID: 22791931

Abstract

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The effects of structural modifications of adenine nucleotides previously shown to enhance either agonist (2-thioether groups) or antagonist (additional phosphate moieties at the 3′- or 2′-position) properties at P2Y1 receptors were examined at recombinant rat P2X1, P2X2, P2X3, and P2X4 receptors expressed in Xenopus oocytes. The potency of P2Y1 agonists HT-AMP (2-(hexylthio)adenosine-5′-monophosphate) and PAPET (2-[2-(4-aminophenyl)ethylthio]adenosine-5′-triphosphate) was examined at P2X receptors. Both nucleotides showed a preference for the Group I (α,β-meATP-sensitive, fast-inactivating) P2X sub-units. HT-AMP was 5-fold more potent than ATP at P2X3 receptors and a partial agonist at all except P2X2 receptors, at which it was a full agonist. The efficacy of HT-AMP was as low as 23% at P2X4 receptors. PAPET was a weak partial agonist at rat P2X4 receptors and a nearly full agonist at the other subtypes. At rat P2X3 receptors, PAPET was more potent than any other known agonist (EC50 = 17 ± 3 nM). MRS 2179 (N6-methyl-2′-deoxyadenosine 3′, 5-bisphosphate, a potent P2Y1 receptor antagonist) inhibited ATP-evoked responses at rat P2X1 receptors with an IC50 value of 1.15 ± 0.21 μM. MRS 2179 was a weak antagonist at rat P2X3 receptors, with an IC50 value of 12.9 ± 0.1 μM, and was inactive at rat P2X2 and P2X4 receptors. Thus, MRS 2179 was 11-fold and 130-fold selective for P2Y1 receptors vs. P2X1 and P2X3 receptors, respectively. MRS 2209, the corresponding 3′-deoxy-2′-phosphate isomer, was inactive at rat P2X1 receptors, thus demonstrating its greater selectivity as a P2Y1 receptor antagonist. Various adenine bisphosphates in the family of MRS 2179 containing modifications of either the adenine (P2Y1 antagonists with 2- and 6-substitutions), the phosphate (a 3′,5′-cyclic diphosphate, inactive at P2Y1 receptors), or the ribose moieties (antagonist carbocyclic analogue), were inactive at both rat P2X1 and P2X3 receptors. An anhydrohexitol derivative (MRS 2269) and an acyclic derivative (MRS 2286), proved to be selective antagonists at P2Y1 receptors, since they were inactive as agonist or antagonist at P2X1 and P2X3 receptors.

Keywords: ion channel, oocytes, purines, ATP derivatives, bisphosphates, deoxyadenosine derivatives

INTRODUCTION

Extracellular ATP and other adenine nucleotides play an important physiological role in the central and peripheral nervous systems and in the immune, cardiovascular, renal, and musculo-skeletal systems through the activation of P2 receptors [North and Barnard, 1997; Burnstock, 1996]. Two families of P2 receptors have been defined [Abbracchio and Burnstock, 1994]: ligand-gated cation channels (P2X subtype), activated by adenine nucleotides, and G-protein coupled receptors (P2Y subtype), most of which are activated by both adenine and uracil nucleotides. Several of the P2Y subtypes are activated exclusively by uracil nucleotides [Communi and Boeynaems, 1997]. P2X1–7 and P2Y1,2,4,6,11 designations have been unambiguously assigned to mammalian nucleotide receptors [Burnstock and King, 1996; Fredholm et al., 1997; Communi et al., 1997], although there is still uncertainty about the correspondence of these cloned sequences to the pharmacological phenotypes of native P2 receptors. The P2X3 receptors found in dorsal root ganglion neurons are a therapeutic target for pain control [Burgard et al., 1999].

Synthetic ligands which display high potency and/or selectivity at various subtypes of P2 receptors are currently being designed [Lambrecht et al., 1996; Williams and Bhagwat, 1996; Jacobson et al., 1997; Fischer, 1999]. One of the first advances in the design of highly potent P2 receptor agonists was the introduction of 2-thioether groups, i.e., analogs of 2-MeSATP. For example, PAPET (2-[2-(4-aminophenyl)ethylthio]adenosine-5′-triphosphate), 1a, and 2-cyanohexylthioATP, 1b (Fig. 1) were shown to activate turkey erythrocyte P2Y1 receptors with EC50 values of 2.5 and 1.0 nM, respectively [Fischer et al., 1993]. The linkage of ATP with bulky 2-thioether groups not only enhanced affinity, but also decreased susceptibility to degradation by nucleotidases [Zimmet et al., 1993]. In the series of 5′-monophosphates, certain 2-thioether groups enhanced potency as P2 receptor agonists. Thus, Boyer et al. [1996] found that HT-AMP (2-(hexylthio)adenosine-5′-monophosphate), 2 (Fig. 1) was 72-fold more potent than ATP in activating turkey P2Y1 receptors, with an EC50 of 59 nM. Compound 3 (2-(5-hexenyl)thioadenosine-5′-triphosphate, MRS 2055), is a moderately potent agonist at the adenylate cyclase-coupled P2Y receptor [P2YAC; Boyer et al., 1996] and inactive at P2Y1 receptors.

Fig. 1.

Fig. 1

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Structure of adenosine-5′-triphosphate and monophosphate 2-thioether derivatives previously found to be potent P2 receptor agonists.

Selective antagonists have been designed for P2Y1 receptors [Camaioni et al., 1998; Boyer et al., 1998; Nandanan et al., 1999] and the yet uncloned P2T receptors [Ingall et al., 1999]. MRS 2179 (N6-methyl-2′deoxyadenosine 3′,5′-bisphosphate), 4, a bisphosphate derivative (Fig. 1), was found to be a pure, competitive antagonist at turkey and human P2Y1 receptors, with a KB value of 100 nM [Boyer et al., 1998]. Before such ligands can be used as definitive pharmacological probes, for example, in platelets, in which P2X1, P2Y1, and P2YAC receptors coexist [Jin et al, 1998; Hechler et al., 1998; Fagura et al., 1998], it is necessary to explore their activity at the full range of P2 receptors [Bianchi et al, 1999]. This is the first study of the activity of these novel agents at recombinant P2X receptors.

MATERIALS AND METHODS

Adenosine nucleotide derivatives were synthesized as reported [Fischer et al., 1993; Boyer et al., 1996; Nandanan et al., 1999; Kim et al., 2000].

Antagonist Activity at Recombinant P2X Receptors

Xenopus oocytes were harvested and prepared as previously described [King et al., 1997]. Defolliculated oocytes were injected cytosolically with 40 nl of a solution of cRNA of rat P2X1, P2X3, or P2X4 receptors (1 μg/ml) or rat P2X2 receptors (0.002 μg/ml) incubated for 24 h at 18°C in Barth’s solution and kept for up to 12 days at 4°C until used in electrophysiological experiments.

ATP-activated membrane currents (Vh = −90 mV) were recorded from cRNA-injected oocytes using the twin-electrode voltage-clamp technique (Axoclamp 2B amplifier). Voltage recording (1–2 MΩ tip resistance) and current-recording microelectrodes (5 MΩ tip resistance) were filled with 3.0 M KCl. Oocytes were held in an electrophysiological chamber and superfused with Ringer’s solution (5 ml/min, at 18°C) containing (in mM): NaCl, 110; KCl, 2.5; HEPES, 5; BaCl2, 1.8, adjusted to pH 7.5.

ATP was superfused over oocytes for 120 sec then washed out for a period of 20 min. The agonist concentration (1 μM for P2X1, 30 μM for P2X2, 3 μM for P2X3, or 10 μM for P2X4) was approximately equal to the EC70 value at each subtype. For inhibition curves, data were normalized to the current evoked by ATP at pH 7.5. Test substances were added for 5 min prior to ATP exposure; all compounds were tested for reversibility of their effects. The concentration required to inhibit the ATP-response by 50% (IC50) was taken from Hill plots constructed using the formula log(I/Imax − I), where I was the current evoked by ATP in the presence of an antagonist. Data are presented as mean ± SEM (n = 4) for data from different batches of oocytes.

RESULTS

ATP agonists were tested in functional assays of recombinant rat P2X1,2,3,4 receptors expressed in Xenopus oocytes (Fig. 2). Values for potency (EC50) in activating membrane currents and maximum efficacy (as percent) are reported (Table 1). HT-AMP was 5-fold more potent than ATP at P2X3 receptors and a partial agonist at all except P2X2 receptors, at which it was a full agonist. The efficacy of HT-AMP was as low as 23% at P2X4 receptors. PAPET was a weak partial agonist at rat P2X4 receptors and a nearly full agonist at the other subtypes. At rat P2X3 receptors, PAPET was more potent than any other known agonist (EC50 = 17 ± 3 nM).

Fig. 2.

Fig. 2

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Effects of various analogs (n = 4) as indicated on current induced at recombinant rat P2X receptors, expressed in Xenopus oocytes. The twin electrode voltage clamping technique was used. The medium consisted of Ba2+ Ringer’s buffer at pH 7.50.

Table 1.

Agonist Potency and Efficacy at Rat P2X 1,2,3,4 Receptors

Receptor ATP PAPET HT-AMP
EC50 Values (μM)
P2X1 0.30 ± 0.01 0.098 ± 0.010 0.84 ± 0.11
P2X2 11.0 ± 3.2 10.2 ± 1.0 180 ± 60a
P2X3 1.85 ± 0.37 0.017 ± 0.003 0.35 ± 0.22
P2X4 4.14 ± 1.34 15.3 ± 5.3 20.4 ± 0.31
Efficacy (maximum) receptor
P2X1 100% 91 ± 5% 49 ± 2%
P2X2 100% 91 ± 2% ≤100%b
P2X3 100% 83 ± 2% 50 ± 2%
P2X4 100% 27 ± 2% 23 ± 1%
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a

Estimated: the log concentration–response curve for HT-AMP at P2X2 receptors did not reach a definable maximum count and accordingly, the stated EC50 value is an estimate.

b

The maximum was not determined over the concentration range studied.

Various adenine bisphosphates (Fig. 3) in the family of MRS 2179 (a potent P2Y1 receptor antagonist) containing modifications of either the adenine (2- and 6-substitutions), the phosphate (a 3′,5′-cyclic diphosphate), or the ribose moieties (carbocyclic and anhydrohexitol analogs), were examined for the ability to interact with P2X receptors. MRS 2179 (Fig. 1), itself, inhibited ATP-evoked responses at rat P2X1 receptors with an IC50 value of 1.15 ± 0.21 μM (Fig. 4a). MRS 2179 was a weak antagonist at rat P2X3 receptors, with ~40% inhibition occurring at 10 μM, and was inactive at rat P2X2 and P2X4 receptors (Fig. 4b). The IC50 value of MRS 2179 at rat P2X3 receptors was determined to be 12.9 ± 0.1 μM. At rat P2X1,2,3,4 receptors, MRS 2179 displayed no agonist properties in the absence of ATP.

Figure 3.

Figure 3

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Structures of deoxyadenosine bisphosphate derivatives previously tested as P2Y1 receptor agonists/antagonists [Camaioni et al., 1998; Nandanan et al., 1999; Kim et al., 2000]. IC50 values (μM) for inhibition of phospholipase C elicited by 10 nM 2-MeSADP at the turkey erythrocyte P2Y1 receptors were found to be: 5a, 0.206; 5b, 0.362; 5c, 1.85; 6, 0.324; 7, 2.53; 8, >50; 9, 1.64; 10, no effect; 11, 0.84.

Fig. 4.

Fig. 4

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Effects of MRS 2179 on current induced by activation of recombinant rat P2X1 and P2X3 receptors (concentration–response curve, a) and rat P2X1–4 receptors (at 10 μM, b), expressed in Xenopus oocytes (n = 8). The IC50 at P2X1 receptors was found to be 1.15 ± 0.21 μM. The twin electrode voltage clamping technique was used. The medium consisted of Ba2+ Ringer’s buffer at pH 7.50.

Figure 3 shows the structures of a variety of bisphosphate derivatives that have been tested at turkey erythrocyte P2Y1 receptors as antagonists of the effects of 2-MeSADP. We have currently tested these compounds at recombinant rat P2X1 and P2X3 receptors. All of the bisphosphate analogs shown in Figure 3, i.e., compounds 5–10, were demonstrated to be inactive at P2X1 and P2X3 receptors, either as agonists (in the absence of ATP) or antagonists (at 1 or 10 μM for rat P2X1 and at 1 μM for rat P2X3 receptors, vs. either 1 or 3 μM ATP, respectively).

Compounds 5a–5c, which are 2-substituted analogs of MRS 2179, and MRS 2209, 6, the 3′-deoxy-2′-phosphate isomer of MRS 2179, were inactive at rat P2X1 and P2X3 receptors, thus demonstrating their selectivity as P2Y1 receptor antagonists. Similarly, other ribose-modified bisphosphate antagonist of P2Y1 receptors, in which the ribose ring was replaced with either cyclopentane (7) or anhydrohexitol (9), were found to be inactive at both rat P2X1 and P2X3 receptors. Compounds 7, a cyclic diphosphate corresponding to an anhydride of MRS 2179, and 10, a six-membered ring morpholine derivative having an ethyl phosphonate on a ring nitrogen, were inactive at P2Y1 receptors as either agonists or antagonists and were found to be similarly inactive at P2X1 and P2X3 receptors. The acyclic nucleotide analog MRS 2286 [Kim et al., 2000], 11, is an antagonist at turkey P2Y1 receptors, but is inactive at both rat P2X1 and P2X3 receptors.

DISCUSSION

Since agonist potency at P2X1 and P2X3 receptors is often linked, an aim of this study has been to distinguish these two subtypes with selective ligands. At rat P2X3 receptors, PAPET, 1, was more potent than any other known agonist, with an EC50 value of 17 nM. This derivative was a full agonist at P2X1, P2X2, and P2X3 receptors and a partial agonist at P2X4 receptors. The mono-phosphate derivative HT-AMP, 2, appeared to be a partial agonist at P2X1, P2X3, and P2X4 receptors, and a weak full agonist at P2X2 receptors. Both of these 2-alkylthio nucleotide derivatives showed a preference for the Group I (α,β-meATP-sensitive, fast-inactivating) P2X subunits, i.e., P2X1 and P2X3 receptors. Thus, PAPET was 600-and 900-fold selective for P2X3 vs. P2X2 and P2X4 receptors, respectively, with nanomolar potency. HT-AMP was 510- and 58-fold selective for P2X3 vs. P2X2 and P2X4 receptors, respectively. The preference for Group I was much more pronounced for the 2-thioether derivatives, including 2-MeSATP [King, 1998], than for ATP itself. 2-MeSATP was reported to have the following EC50 values (in μM): 0.4 (P2X1), 7.1 (P2X2), 0.2 (P2X3), and 74 (P2X4) [King, 1998]. Thus, PAPET appears to be more potent than 2-MeSATP at recombinant P2X1 and P2X3 receptors expressed in oocytes. The utility of PAPET and HT-AMP and similar 2-thioethers derivatives as potent P2 agonists in vivo may be further enhanced due to increased resistance to nucleotidases [Zimmet et al., 1993].

Among bisphosphate derivatives tested, only MRS 2179, 4, showed any antagonist properties at P2X receptors. Thus, MRS 2179 remained a moderately selective P2Y1 receptor antagonist, with ratios of selectivity vs. P2X1 and P2X3 receptors of 11- and 130-fold, respectively, and, as reported, is inactive at P2Y2, P2Y4, and P2Y6 receptors [Boyer et al., 1998]. MRS 2179 has also been found to be inactive at P2YAC receptors in platelets [Boyer et al., 1998] and at P2Y11 receptors (B. Torres, A. Zambon, and P. Insel, unpublished observations), as well as P2X2 and P2X4 receptors. MRS 2179 (100 μM) is also inactive as either substrate or inhibitor of recombinant rat ecto-ATPase and ecto-apyrase expressed in CHO cells (C. Hoffmann, K. Jacobson, and H. Zimmermann, unpublished observations).

Nucleotides that distinguish between P2X1 and P2Y1 receptors may be useful pharmacological probes to study aggregation effects in platelets. Among the most important findings of the present study are that compounds 5–7, 9, and 11, which bind to P2Y1 receptors in the micromolar range, appear to be very selective for that subtype. The substitution of MRS 2179 at the 2-position with a chloro-, methylthio-, or amino- group, 5a–5c, abolished activity at P2X1–4 receptors, and similarly, the 2′, 5′-isomer of MRS 2179, 6, was inactive at P2X receptors. The carbocyclic derivative, 7, was a moderately potent P2Y1 receptor antagonist (IC50 2.53 μM) and inactive at P2X1,3 receptors. Thus, compounds 5a–5c, 6, and 7, which are relatively potent as antagonists at P2Y1 receptors (IC50 < 3 μM), were highly selective at that subtype.

The anhydrohexitol derivative 9, an antagonist at P2Y1 receptors, appeared to be a selective antagonist of P2Y1 receptors, i.e., to the extent that it has no effect at P2X1,3 receptors. The corresponding 6-NH2 derivative (MRS 2255), a pure agonist at P2Y1 receptors with an EC50 value of 2.99 μM [Nandanan et al., 1999], for stimulation of phospholipase C in turkey erythrocytes, should also be evaluated at P2X receptors and may prove to be a selective P2Y1 agonist. A selective P2Y1 receptor agonist may be useful as a hypotensive agent or in the treatment of diabetes, while a selective P2Y1 receptor antagonist may be useful as an antithrombotic agent.

Acknowledgments

Grant sponsor: Gilead Sciences.

We thank Gilead Sciences (Foster City, CA) for financial support to S.B and S.Y.J. We thank Dr. Lewis Pannell and Wesley White for determination of HRMS and NMR.

Abbreviations

ATP

adenosine 5′-triphosphate

EC50

50% effective concentration

EFS

electrical field stimulation

HT-AMP

2-hexylthioadenosine-5′-monophosphate

IC50

50% inhibitory concentration

KB

Schild constant

me-ATP

adenosine-5′-methylenetriphosphate, (α,β) or (β,γ) isomers

2-MeSATP

2-methylthioadenosine-5′-triphosphate

MRS 2055

2-(5-hexenyl)thioadenosine-5′-triphosphate

MRS 2179

N6-methyl-2′-deoxyadenosine 3′, 5-bisphosphate

MRS 2209

N6-methyl-3′-deoxyadenosine 2′, 5-bisphosphate

PAPET

2-[2-(p-aminophenyl)ethylthio]adenosine-5′-triphosphate

PPADS

pyridoxal-5′-phosphate-6-phenylazo-2,4-disulfonate

SAR

structure–activity relationship

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