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Research ArticleCELLULAR AND MOLECULAR

NF546 [4,4′-(Carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-α,α′-diphosphonic Acid) Tetrasodium Salt] Is a Non-Nucleotide P2Y11 Agonist and Stimulates Release of Interleukin-8 from Human Monocyte-Derived Dendritic Cells

Sabine Meis, Alexandra Hamacher, Darunee Hongwiset, Claudia Marzian, Michael Wiese, Niels Eckstein, Hans-Dieter Royer, Didier Communi, Jean-Marie Boeynaems, Ralf Hausmann, Günther Schmalzing and Matthias U. Kassack
Journal of Pharmacology and Experimental Therapeutics January 2010, 332 (1) 238-247; DOI: https://doi.org/10.1124/jpet.109.157750

Abstract

The G protein-coupled P2Y11 receptor is involved in immune system modulation. In-depth physiological evaluation is hampered, however, by a lack of selective and potent ligands. By screening a library of sulfonic and phosphonic acid derivatives at P2Y11 receptors recombinantly expressed in human 1321N1 astrocytoma cells (calcium and cAMP assays), the selective non-nucleotide P2Y11 agonist NF546 [4,4′-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-α,α′-diphosphonic acid) tetrasodium salt] was identified. NF546 had a pEC50 of 6.27 and is relatively selective for P2Y11 over P2Y1, P2Y2, P2Y4, P2Y6, P2Y12, P2X1, P2X2, and P2X2-X3. Adenosine-5′-O-(3-thio)triphosphate (ATPγS), a nonhydrolyzable analog of the physiological P2Y11 agonist ATP, and NF546 use a common binding site as suggested by molecular modeling studies and their competitive behavior toward the nanomolar potency antagonist NF340 [4,4′-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt] in Schild analysis. The pA2 of NF340 was 8.02 against ATPγS and 8.04 against NF546 (calcium assays). NF546 was further tested for P2Y11-mediated effects in monocyte-derived dendritic cells. Similarly to ATPγS, NF546 led to thrombospondin-1 secretion and inhibition of lipopolysaccharide-stimulated interleukin-12 release, whereas NF340 inhibited these effects. Further, for the first time, it was shown that ATPγS or NF546 stimulation promotes interleukin 8 (IL-8) release from dendritic cells, which could be inhibited by NF340. In conclusion, we have described the first selective, non-nucleotide agonist NF546 for P2Y11 receptors in both recombinant and physiological expression systems and could show a P2Y11-stimulated IL-8 release, further supporting the immunomodulatory role of P2Y11 receptors.

P2 receptors are divided into ionotropic (ligand-gated ion channel), P2X, and metabotropic (G protein-coupled) P2Y receptors. Up to now, eight subtypes of human P2Y receptors have been cloned and characterized (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11–14) (Abbracchio et al., 2006). P2 receptors play important roles in diverse (patho)-physiological processes; for example, in the immune system (Di Virgilio et al., 2001; Marteau et al., 2005), platelet aggregation (Gachet, 2008), neurotransmission (Franke et al., 2006), oncology (White and Burnstock, 2006), and inflammation and pain (Burnstock, 2004). P2Y receptors are activated by nucleoside triphosphates, nucleoside diphosphates, and nucleotide sugars (ATP-, UTP-, ADP-, UTP-, and UDP-glucose). P2Y1, P2Y2, P2Y4, and P2Y6 receptors couple to Gq, whereas P2Y12, P2Y13, and P2Y14 receptors couple to Gi. Only P2Y11 receptors are coupled to both the cAMP and the phosphoinositide pathway (Gq and Gs) (Qi et al., 2001a). Since the cloning and characterization of the P2Y11 receptor (Communi et al., 1997), a lot of knowledge has been gathered on multiple pharmacological actions of P2Y11 receptors. Carriers of the Ala-87-Thr polymorphism of P2Y11 receptors have an increased risk of acute myocardial infarction (Amisten et al., 2007). P2Y11 receptors (among other P2 receptors) are involved in smooth muscle relaxations (King and Townsend-Nicholson, 2008). Swennen et al. (2008) reported that ATP inhibited tumor necrosis factor-α release via activation of P2Y11 receptors. A few studies were performed on the role of P2Y11 receptors in human monocyte-derived dendritic cells. P2Y11 receptors mediate ATP-induced semimaturation of monocyte-derived dendritic cells (Wilkin et al., 2001). However, ATP effects on monocyte-derived dendritic cells are mediated by multiple P2Y receptors and also include effects of ADP as the ATP degradation product (Marteau et al., 2004). Marteau et al. (2005) reported on thrombospondin-1 (TSP-1) release from monocyte-derived dendritic cells upon ATP stimulation most likely mediated by P2Y11 receptors, thus making P2Y11 receptors potential targets for dendritic cell-based immunotherapy. P2Y11 receptors furthermore play a role in the ATP-mediated inhibition of neutrophil apoptosis. Thus, specific targeting of P2Y11 receptors could reduce neutrophil-mediated inflammatory processes (Vaughan et al., 2007).

ATP is the endogenous ligand at P2Y11 receptors. To study in depth the contribution of P2Y11 receptors, selective and potent ligands are required. So far, non-nucleotide and selective agonists and antagonists are lacking for P2Y11 receptors, with the exception of NF157 and marine sponge-derived iantherans (Ullmann et al., 2005; Greve et al., 2007). NF157 was recently introduced by our group as an antagonist at P2Y11 receptors, selective over P2Y1 and P2Y2, but not P2X1. The iantherans are of limited availability because of their marine sponge origin. Naphthalene sulfonic acid urea derivatives have been an excellent source of selective and potent P2 receptor ligands (Damer et al., 1998; Braun et al., 2001; Kassack et al., 2004; Ullmann et al., 2005; Hausmann et al., 2006). We have thus performed a systematic screening of a compound library of naphthalene sulfonic and phosphonic acid urea derivatives for P2Y11 receptor activation or inhibition by use of a fluorescence-based calcium assay (Kassack et al., 2002). This screen has resulted in the identification of NF340, a 4-fold more potent antagonist than NF157 and the first non-nucleotide P2Y11 agonist NF546 (Fig. 1). This article describes the characterization of the non-nucleotide agonist NF546 at P2Y11 receptors recombinantly expressed in human 1321N1 astrocytoma cells and the modulation of cytokine release by NF340 and NF546 from human monocyte-derived dendritic cells. Now-available selective P2Y11 ligands (NF546 and NF340) allow in-depth physiological evaluation of the role of the P2Y11 receptors.

Fig. 1.
Fig. 1.

Structural formulas of the P2Y11 antagonist NF340 and the P2Y11 agonist NF546.

Materials and Methods

Materials.

All nucleotides [ATP, adenosine 5′-O-(3-thiotriphosphate) (ATPγS), adenosine 5′-O-(2-thiodiphosphate) (ADPβS), 2′-3′-O-(4-benzoylbenzoyl)-ATP (BzATP), UTP, UDP, ADP, 2-(methylthio)adenosine-5′-triphosphate (2-MeSATP)], lipopolysaccharide (LPS), forskolin, and other reagents were obtained from Sigma-Aldrich (Taufkirchen, Germany), unless otherwise stated. NF340 and NF546 were synthesized according to published methods (Kassack et al., 2004; Ullmann et al., 2005). Structures of NF340 and NF546 (Fig. 1) were confirmed by 1H and 13C NMR. Purity was checked by elemental analysis (carbon, hydrogen, nitrogen) and high-performance liquid chromatography and was >95% (Kassack and Nickel, 1996). Synthesis will be published elsewhere. Molecular masses are as follows: NF340, 986.83 g/mol, and NF546, 1180.75 g/mol.

Cell Culture and Stable Transfection of Cells.

Human 1321N1 astrocytoma cells were stably transfected with pcDNA3.1(+) vector (Invitrogen, Karlsruhe, Germany) containing the coding sequences of P2Y1 (GenBank accession no. AY136752), P2Y2 (GenBank accession no. AY136753), P2Y4 (GenBank accession no. NM_002565), P2Y6 (GenBank accession no. AF498920), P2Y11 (MAANVSGAK-, GenBank accession no. AY449733 or MDRGAK-, GenBank accession no. AF030335), or P2Y12 (GenBank accession no. NM_176876), respectively. All plasmids were from the Missouri S&T cDNA Resource Center (www.cdna.org) with the exception for P2Y11-MDRGAK, which was from Communi et al. (1997). Unless otherwise stated, P2Y11 indicates the P2Y11-MAANVSGAK clone. Cloned cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) with sodium pyruvate, glucose (4500 mg/liter), and pyridoxine supplemented with 5 mM l-glutamine, 100 U/ml penicillin G, 100 μg/ml streptomycin, 10% fetal bovine serum (Sigma-Aldrich), and 400 μg/ml G418 (Calbiochem, San Diego, CA). Cells were incubated at 37°C in a humidified atmosphere under 5% CO2.

Measurements of Intracellular Calcium.

Ca2+ fluorescence was measured as described previously by use of a fluorescence microplate reader with a pipettor system (NOVOstar; BMG LabTech, Offenburg, Germany) (Kassack et al., 2002; Ullmann et al., 2005). The following (standard) agonists were used to stimulate the respective receptors: P2Y1, 2-MeSADP (pEC50, 8.46 ± 0.18); P2Y2, UTP (pEC50, 6.86 ± 0.05); P2Y4, UTP (pEC50, 7.69 ± 0.15); P2Y6, UDP (pEC50, 6.89 ± 0.12); and P2Y11, ATPγS (pEC50, 7.26 ± 0.03). Concentration-inhibition curves of antagonists were obtained by preincubation of the cells with test compounds for 30 min at 37°C and a subsequent injection of agonist [31.6 nM 2-MeSADP (P2Y1), 1 μM UTP (P2Y2,4), 1 μM UDP (P2Y6), or 1 μM ATPγS (P2Y11), respectively].

Analysis of cAMP Levels.

cAMP levels were estimated by use of a reporter-gene assay as described previously (Hamacher et al., 2006). 1321N1-P2Y11 cells or 1321N1-P2Y12 cells were transfected with 30 μg of pCRE-luc (Stratagene, LaJolla, CA), using Polyfect (QIAGEN, Hilden, Germany). Twenty-four hours after transfection, cells were split into two white 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and incubated in complete DMEM. Seventy-two hours after transfection, medium was replaced by phenol red- and serum-free DMEM:F-12 media (1:1 mix). Cells were then stimulated with agonists or test compounds depending on their Gα-coupling. For P2Y11 receptors, agonist screening was performed by a 3-h stimulation (37°C, 5% CO2) in the absence of forskolin. Compounds were tested for antagonism by preincubation for 30 min before addition of 1 μM ATPγS and incubation for 3 h at 37°C in a 5% CO2 incubator. For P2Y12 receptors, agonist screening was performed by a 3-h stimulation (37°C, 5% CO2) in the presence of 10 μM forskolin. Compounds were tested for antagonism at P2Y12 receptors by preincubation for 30 min before addition of 100 nM 2- MeSADP (pEC50, 8.96 ± 0.14) and 10 μM forskolin and incubation for 3 h at 37°C in a 5% CO2 incubator. After incubation, cells were lysed in 100 μl of lysis buffer (8 mM Tricine, 2 mM EDTA, 1 mM dithiothreitol, 5% Triton, pH 7.8) and incubated for 20 min at 4°C in the dark. Luciferase activity was determined after adding 100 μl of luciferase assay reagent (30 mM Tricine, 10 mM MgSO4, 0.5 mM EDTA, 10 mM dithiothreitol, 0.5 mM ATP, 0.5 mM coenzyme A, 0.5 mM d-luciferin) in a LUMIstar microplate reader (BMG LabTech).

Electrophysiological Evaluation at Recombinant P2X Receptors.

The inhibitory or activating potency of NF340 and NF546 at P2X receptors was evaluated on Xenopus laevis oocytes recombinantly expressing various rat P2X subtypes (rP2X1, rPX2, rP2X2-X3) with use of previously described protocols (Ullmann et al., 2005; Hausmann et al., 2006).

Preparation of Human Monocyte-Derived Dendritic Cells.

Immature human dendritic cells (DCs) were generated from adherent peripheral blood monocytes obtained from buffy coats of healthy volunteer donors as described previously (Romani et al., 1994; Wilkin et al., 2001). After 5 or 6 days of culture in the presence of 800 U/ml granulocyte-macrophage colony-stimulating factor (Invitrogen) and 500 U/ml of interleukin-4 (IL-4) (Invitrogen), cells were replated at 106 cells/ml in 24 multiwells in complete medium with granulocyte-macrophage colony-stimulating factor and IL-4.

Stimulation of Monocyte-Derived Dendritic Cells for ELISA Measurements.

DCs were treated with NF546, ATPγS, or NF340 in the absence or in the presence of LPS (100 ng/ml) for 24 h. Supernatants of treated DCs were collected for measurements of cytokine profile and enzyme-linked immunosorbent assay (ELISA) measurements of TSP-1, and interleukins 8 and 12 (IL-8 and IL-12p70).

ELISA.

Human TSP-1 was measured by use of a commercially available ELISA kit from Millipore Bioscience Research Reagents (Temecula, CA). Human IL-8 and IL-12p70 were measured by use of commercially available kits from Pierce (Rockford, IL).

Measurement of Cytokine and Chemokine Release.

Human cytokines in cell culture supernatants of dendritic cells were measured by use of the proteome profiler Human Cytokine Array Kit, Panel A (R&D Systems, Wiesbaden, Germany) according to the manufacturer's instructions. The kit consists of a nitrocellulose membrane containing 36 different anticytokine antibodies spotted in duplicate. In brief, membranes were blocked with blocking buffer at room temperature for 1 h. One milliliter of DC supernatants was mixed with a biotinylated detection antibody cocktail at room temperature and then incubated with membranes overnight at 4°C. Arrays were then washed three times for 10 min and subsequently incubated with streptavidin-horseradish peroxidase for 30 min at room temperature and washed again. Next, arrays were exposed to peroxidase substrate (ECL Plus Western Blotting detection reagent; GE Healthcare, Freiburg, Germany) for 1 min before imaging with a LAS 3000 chemiluminescence reader (Fujifilm Corp., Kleve, Germany). Time of exposure was between 1 and 30 min.

Data Analysis.

Effects of single doses of agonists were expressed as the percentage of the standard agonist control responses: 31.6 nM 2-MeSADP (P2Y1), 1 μM UTP (P2Y2,4), 1 μM UDP (P2Y6), or 1 μM ATPγS (P2Y11), 100 nM 2-MeSADP (P2Y12), respectively. Effects of single doses of antagonists were expressed as percentage inhibition (= standard agonist response − standard agonist response in the presence of antagonist). Apparent functional Ki values were calculated according to the equation of Cheng and Prusoff (1973): Ki = IC50/(1 + L/EC50), where IC50 is the 50% inhibitory concentration of the antagonist, EC50 is the 50% effective concentration of the agonist used, and L is the molar concentration of the agonist used. IC50 values for antagonists and EC50 values for agonists were derived from −log concentration–effect (inhibition) curves where pooled normalized data were fitted to the nonlinear four-parameter logistic equation (Prism 4.00; GraphPad Software, San Diego, CA). All experiments were performed in triplicate assays and repeated at least three times.

Receptor Modeling.

The crystallographic structure of bovine rhodopsin was used as a template to construct a homology model of the hP2Y11 receptor with the MOE software package (Chemical Computing Group, Montreal, QC, Canada). Because the modeling algorithm tends to prefer compact packing, the resulting binding pocket is quite narrow beneath the long second extracellular loop. Thus, the best model obtained was refined by a molecular dynamics simulation in a 1-palmitoyl-2-oleoyl-phosphatidylcholine membrane within physiological sodium chloride solution under periodic boundary conditions. All molecular dynamics calculations were performed using the GROMACS software (http://www.gromacs.org). After extensive minimization with steepest descent and conjugated derivatives algorithm the model was subjected to molecular dynamics with restraints on the receptor coordinates to first equilibrate the membrane environment. The molecular dynamics simulation of the relaxed receptor model was carried out for a simulation time of 5 ns, monitoring the overall system energy to ensure stability of the system. After this simulation of the unoccupied receptor, NF340 was docked into the resulting binding pocket by use of GOLD (Cambridge Crystallographic Database, Cambridge, UK), and the complex was subjected again to a short molecular dynamics simulation (500 ps). NF546 was docked into the putative binding pocket via superposition with NF340. After that, NF546 and the flexible side chains of the amino acids in the pocket and the first extracellular loop were subjected to an optimization procedure by use of the MOE Homology Modeling protocol. Molecular models shown in Fig. 7 were prepared by use of the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco.

Results

Before screening the naphthalene urea library at P2Y11 receptors, we wanted to clarify whether biological activities of agonists at both splice variants of the P2Y11 receptor described by Communi et al. (2001) were comparable. Most publications use the MDRGAK splice variant originally reported by Communi et al. (Communi et al., 1997, 1999; Patel et al., 2001; Qi et al., 2001a, 2001b; White et al., 2003). However, the amino-terminal sequence MAANVSGAK is the correct beginning of the nonchimeric P2Y11 receptor, whereas the sequence MDRGAK represents the junction between the SSF1 and P2Y11 gene products. No significant differences were observed in the pEC50 values of different ATP analogs at either receptor splice variant with use of fluorescence calcium measurements (Supplemental Table S1). The rank order of potency was the same for both splice variants and comparable with the previously published rank order of potency at P2Y11-MDRGAK receptors (ATPγS ≈ BzATP > ATP > ADPβS > 2MeSATP) (Communi et al., 1999). Based on these results, all further studies were performed with the P2Y11-MAANVSGAK receptor.

Screening of the naphthalene sulfonic and phosphonic acid urea library at P2Y11 receptors was performed by use of a fluorescence calcium assay (Kassack et al., 2002). This screen resulted in the discovery of the antagonist NF340 and the agonist NF546 (for structural formula, see Fig. 1). The inhibition by 10 μM NF340 of a response induced with standard agonists at P2Y receptors is shown in Table 1 (standard agonist concentrations and pEC50 values of agonists are given under Materials and Methods). Data for P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 are based on calcium measurements, whereas data for P2Y12 were estimated by measuring cAMP. As can be seen in Table 1, NF340 inhibited only the P2Y11 receptor. Functional characterization of NF340 at P2Y11 receptors is shown in Fig. 2 compared with the P2Y11 antagonist NF157 described recently (Ullmann et al., 2005). Apparent functional Ki values were calculated from calcium measurements as follows (Fig. 2A): NF157, 75.3 nM, and NF340, 19.2 nM. NF340 is thus ∼4-fold more potent than NF157 (calcium assay). In the cAMP assay, apparent functional Ki values were as follows (Fig. 2B): NF157, 544.7 nM, and NF340, 52.4 nM. cAMP studies leave NF340 approximately 10-fold more potent than NF157. NF340 has thus Ki values of similar range in calcium and cAMP inhibition experiments. The concentration-inhibition curves of NF340 (calcium and cAMP) displayed Hill coefficients not significantly different from unity. To further examine the mode of inhibition by NF340 of the ATPγS effect, concentration-response curves of ATPγS were monitored in the absence and presence of increasing concentrations of NF340 in the calcium assay. Figure 2C shows the rightward shift of the concentration-response curves, and Fig. 2D displays the resulting Schild analysis. The Schild plot is a straight line with a slope not significantly different from unity. We thus assume a competitive behavior of NF340 at the P2Y11 receptor. The pA2 value was estimated as 8.02 ± 0.12 (mean ± S.E.M.). The pA2 of NF340 is in a range similar to the pKi derived from the inhibition curve in Fig. 2 A (7.71 ± 0.07, mean ± S.E.M.).

View this table:
Table 1

Inhibition by 10 μM NF340 of standard agonist responses at P2Y receptors

Fig. 2.
Fig. 2.

Functional characterization of NF340. Concentration-dependent inhibition by NF157 and NF340 of a response induced by injection of 1 μM ATPγS at P2Y11 receptors in the calcium (A) and cAMP assays (B). Data shown are mean ± S.E.M. of the pooled data of n >3 experiments, each with three replicates. Hill slopes were not significantly different from unity. pIC50 (NF157-Ca2+) = 5.82 ± 0.05; pIC50 (NF340-Ca2+) = 6.43 ± 0.04; pIC50 (NF157-cAMP) = 6.12 ± 0.04; pIC50 (NF340-cAMP) = 7.14 ± 0.06. C, ATPγS concentration-response curves (calcium assay) at P2Y11 receptors in the absence and presence of increasing concentrations of NF340. Data are mean ± S.E.M., n ≥ 5. D, analysis of the functional antagonist effect of NF340 (Schild plot). Dashed line shows 95% confidence interval. The highest single fluorescence (A, C) or luminescence (B) data point obtained in any of the n experiments by injection of 1 μM ATPγS (A, B) or 316 μM ATPγS (C) is set as 100% control.

Next, the activation by NF546 of P2Y receptors was tested. Figure 3A shows concentration-effect curves of NF546 at P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors compared with ATPγS at P2Y11 in calcium assays. Table 2 lists the corresponding pEC50 values of NF546. Figure 3B shows the activation of the P2Y11 receptor by NF546 compared with ATPγS in the cAMP assay. The pEC50 values for NF546 and ATPγS in the calcium assay were 6.27 ± 0.07 and 7.26 ± 0.03, respectively. In the cAMP assay, the following pEC50 values were obtained: NF546, 5.53 ± 0.03, and ATPγS, 6.59 ± 0.04. pEC50 values determined in the calcium assay were higher than those in the cAMP assay. This is in agreement with findings published by Qi et al. (2001a, 2001b). The EC50 ratios of ATPγS and NF546, however, were similar in the calcium (9.8) and cAMP assays (11.5). The efficacy (upper plateau of the concentration-effect curve) of NF546 and ATPγS at P2Y11 is not significantly different in either assay (Fig. 3, A and B). NF546 can thus be considered a full agonist at P2Y11 with ∼10-fold less potency than ATPγS. Besides strong activation of the P2Y11 receptor, activation by NF546 of P2Y2, P2Y6, and, to a lesser extent, P2Y12 was observed (Fig. 3A, Table 2). The activation of P2Y2 by NF546 shown in Fig. 3A suggests that NF546 is a full agonist at P2Y2. However, NF546 is approximately 100-fold less potent at P2Y2 receptors (pEC50, 4.82; Table 2) than the physiological agonists UTP (pEC50, 6.86; data not shown) or ATP (pEC50, 6.74; data not shown). On the contrary, NF546 is only 2.5-fold less potent at P2Y11 (pEC50, 6.27; Table 2) than the physiological agonist ATP (pEC50, 6.67; Supplemental Table S1). These data support relative selectivity of NF546 for P2Y11.

Fig. 3.
Fig. 3.

NF546 is a novel P2Y11 agonist. Concentration-response curves of ATPγS and NF546 at P2Y receptors by use of calcium (A) and cAMP assays (B). Hill coefficients were not significantly different from unity. C shows the inhibitory effect of 10 μM NF157 on the response of 1 μM ATPγS and 10 μM NF546 (calcium assay). Data shown are mean ± S.E.M. of the pooled data of n > 3 experiments each with three replicates. The highest single fluorescence (A) or luminescence (B) data point of standard agonist controls obtained in any of the n experiments was set as 100% control. This was 100 μM ATPγS for P2Y11, 31.6 nM 2-MeSADP for P2Y1, 1 μM UTP for P2Y2 and P2Y4, 1 μM UDP for P2Y6, and 100 nM 2-MeSADP for P2Y12, respectively.

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Table 2

Potency (pEC50) of NF546 at P2Y receptors (calcium assay)

Next, the specificity of NF546 for the P2Y11 receptor was tested by addition of NF157, a recently described P2Y11 antagonist (Ullmann et al., 2005). Figure 3C shows the complete inhibition of the signals of 1 μM ATPγS and 10 μM NF546 by addition of 10 μM NF157. To further elucidate the binding behavior of NF546 compared with ATPγS and NF340, a Schild analysis using NF546 and NF340 was performed in the calcium (Fig. 4, A and B) and the cAMP assay (Fig. 4, C and D). Figure 4, A and C, shows the rightward shift of the concentration-response curves of NF546 in the presence of increasing concentrations of NF340. The corresponding Schild analyses are displayed in Fig. 4, B and D, and show straight lines with slopes not significantly different from unity. pA2 values for NF340 were estimated as 8.04 ± 0.27 (calcium) and 7.96 ± 0.25 (cAMP). Results of NF546 from calcium and cAMP studies are thus in agreement and confirmed the pA2 value of NF340 determined with ATPγS in the calcium assay (Fig. 2D).

Fig. 4.
Fig. 4.

Functional characterization of NF546. A and C show concentration-response curves of NF546 at P2Y11 receptors in the calcium (A) and cAMP assay (C) in the absence and presence of increasing concentrations of NF340. Data shown are representative for three typical experiments, each with three replicates. B and D analyze the functional antagonist effect of NF340 shown in A or C, respectively (Schild plots). Dashed lines show 95% confidence intervals. The highest single fluorescence (A) or luminescence (C) data point obtained by injection of 316 μM ATPγS (A) or 100 μM ATPγS (C) is set as 100% control.

Selectivity of NF340 and NF546 for P2Y11 over other P2Y receptors was presented earlier (Tables 1 and 2, Fig. 3A). To test the selectivity for P2Y11 over P2X receptors, NF340 and NF546 were evaluated for inhibition/activation of recombinant P2X receptors (P2X1, P2X2, and P2X2-X3) by methods described previously (Ullmann et al., 2005; Hausmann et al., 2006). Up to 3 μM NF340 or NF546 showed less than 25% inhibition and no activation of control responses (data not shown). Furthermore, no inhibition by NF340 or NF546 of soluble potato-apyrase (grade VI, 0.04 U/ml; method reported in Horner et al., 2005) was observed up to a concentration of 100 μM (data not shown).

Further experiments were undertaken to examine whether the effect of NF546 was caused by a release of ATP. The effect of NF546 on P2Y11 1321N1 astrocytoma cells was measured in the absence and the presence of soluble potato-apyrase in the calcium assay. The activity of NF546 was unaffected in the presence of soluble potato-apyrase (data not shown). This result corroborates a direct interaction of NF546 and the P2Y11 receptor and rejects a release of ATP caused by NF546.

We next tested the physiological relevance of these findings in human monocyte-derived DCs. Wilkin et al. (2001) reported that P2Y11 receptors mediate the ATP-induced semimaturation in DCs. Activation of P2Y11 receptors in DCs by ATP or ATPγS may be monitored by TSP-1 release as shown by Marteau et al. (2005). We have thus prepared DCs according to Wilkin et al. (2001) and Marteau et al. (2005), and tested NF340 and NF546 at DCs for changes in calcium signaling and TSP-1 secretion (Fig. 5). ATP, UTP, and NF546 gave a calcium signal in DCs (Fig. 5A). Because UTP gave no calcium signal at recombinant P2Y11 receptors expressed in human 1321N1 astrocytoma cells (data not shown), we assume that the UTP signal was mediated by P2Y2 (or P2Y4). Even though this is in contrast to the report from White et al. (2003), who found a UTP calcium signal at P2Y11 receptors, our result that UTP gave no P2Y11 signal fits very well to our data using the P2Y11-selective antagonist NF340; whereas NF340 had no effect on the UTP response, the ATP signal could be blocked by NF340 to the level of the UTP response. Furthermore, the NF546 signal could be completely blocked by NF340. The results from our laboratory nicely fit together: UTP is not an agonist at P2Y11, ATP is an agonist at P2Y2 and P2Y11, NF546 is a selective P2Y11 agonist, and NF340 is a selective P2Y11 antagonist. Functional effects of NF340 and NF546 in DCs were further confirmed by measuring TSP-1 secretion (Fig. 5B). TSP-1 secretion was stimulated equally by ATPγS and NF546. Furthermore, agonist-induced TSP-1 secretion was completely blocked by 10 μM NF340.

Fig. 5.
Fig. 5.

Effect of P2 ligands on calcium signaling (A) and thrombospondin-1 secretion (B) in monocyte-derived dendritic cells. ATP, ATPγS, UTP, and NF546 were used in a concentration of 100 μM. Data are mean ± S.D. of three independent experiments. ATPγS-induced TSP-1 secretion was 1977 ng/ml.

Marteau et al. (2004) have further reported modulation of cytokine release from DCs stimulated with LPS and ATP or ATP derivatives. Among others, LPS-induced release of IL-12 was found to be modulated by ATP. This prompted us to perform a cytokine profiling of DCs stimulated with ATPγS and NF546, respectively. It is interesting that ATPγS and NF546 had no effect on the release of any cytokine available in the proteome profiler Human Cytokine Array Kit, Panel A (R&D Systems), that we used, with the exception of IL-8. Figure 6A shows a detail of the cytokine array, namely effects on IL-8 and IL-12p70. The next step was to examine LPS-induced IL-12p70 secretion, to quantify IL-8 secretion, and to test for modulating effects of NF340 and NF546 on IL-12p70 and IL-8 secretion by ELISA (Fig. 6, B and C). LPS led to the expected secretion of IL-12p70 in DCs, and this effect could be inhibited by ATPγS and NF546 to control level. Furthermore, we could demonstrate that addition of NF340 to LPS and ATPγS or NF546-stimulated DCs was able to block the effect of ATPγS and NF546 back to the level of LPS alone (Fig. 6B). ATPγS and NF546-stimulated IL-8 secretion could also be inhibited to basal level by addition of 10 μM NF340.

Fig. 6.
Fig. 6.

Effect of P2Y11 ligands on cytokine release in human monocyte-derived dendritic cells. A, detail of a human proteome profiler cytokine array. Human monocyte-derived dendritic cells (DCs) were incubated with medium alone or medium containing 100 μM ATPγS or 100 μM NF546, respectively, for 24 h. B, the inhibitory effect of 100 μM ATPγS and 100 μM NF546, respectively, on the LPS-induced release of IL-12p70 (ELISA) from DCs and the reversal of the ATPγS and NF546 effect by 10 μM NF340. Control is IL-12p70 concentration in medium supernatant from untreated cells. Data are mean ± S.D. of three independent experiments. C confirms the secretion of IL-8 (ELISA) induced by 10 μM ATPγS or 100 μM NF546, respectively, which was prevented by addition of 10 μM NF340. Basal control is IL-8 concentration in medium supernatant from untreated cells. Data are mean ± S.D. of three independent experiments.

To get an insight into the molecular interaction of the agonist NF546 with the P2Y11 receptor compared with the antagonist NF340, we have constructed a homology model of the P2Y11 receptor based on the crystallographic structure of bovine rhodopsin. The nanomolar potency antagonist NF340 fits well into the derived binding pocket of the homology model in the inactive state (Fig. 7A). Arg106 and Arg307 form hydrogen bonds to the sulfonic acid in position 6 of the first naphthalene ring. Arg268 seems to stabilize the sulfonic acid in position 6 of the second naphthalene ring. These findings are in accordance to site-directed mutagenesis studies that identified these residues as essential for agonist binding (Zylberg et al., 2007). Other charged amino acids in the pocket contribute to the binding of NF340 as, for example, Lys22, Arg103, and His265, which interact with other sulfonic acid groups. Arg184 forms a hydrogen bond to the carbonyl group of the urea of NF340, and Thr110 and Thr164 also seem to stabilize the sulfonic acid in position 2 of the second naphthalene ring. The agonist NF546 also fits well into the suggested binding pocket (Fig. 7B). Nevertheless, parts of the large molecule remain in the extracellular space. Table 3 summarizes the amino acids in the putative P2Y11 receptor-binding pocket and their interaction with NF340 and NF546, respectively. Both ligands share some of the amino acid interactions (e.g., Arg103, Thr164, Arg184, and Arg268). However, NF546 shows fewer interactions and cannot occupy the region between Trp32, Arg106, and Arg307, whereas NF546 forms an additional hydrogen bond with Glu186. This latter interaction of NF546 with Glu186 is not possible for the monomeric amine precursor of NF546, which is in accordance with a complete lack of activity of the monomeric precursor at P2Y11. Likewise, the amine precursor of NF340 has no activity at P2Y11, which again is in accordance with the suggested molecular interaction displayed in Fig. 7.

Fig. 7.
Fig. 7.

Putative binding site of the P2Y11 receptor. A, NF340. B, NF546 docked into the ligand binding site. Ionic interactions and hydrogen bonds are shown as thin lines.

View this table:
Table 3

Amino acid residues in the putative P2Y11 receptor-binding pocket contributing to the binding of NF340 and NF546

Discussion

Two different splice variants for the P2Y11 receptor are described in the literature (Communi et al., 2001). Most publications use the MDRGAK splice variant representing the chimeric junction between the SSF1 and P2Y11 gene products (Communi et al., 1997, 1999; Patel et al., 2001; Qi et al., 2001a, 2001b; White et al., 2003). In comparing our data using the nonchimeric P2Y11-MAANVSGAK receptor with data from the literature using the MDRGAK splice variant, we were able to show that the differences in the amino terminus did not lead to differences in the potency of nucleotide agonists (Supplemental Table S1). This allowed us to directly compare data obtained with the different splice variants.

The purpose of this study was to characterize the non-nucleotide P2Y11 agonist NF546 identified through a screening of sulfonic and phosphonic acid derivatives. The P2Y11 receptor seems to play diverse interesting physiological roles that have not been fully explored, however, because of the lack of appropriate ligands (Wilkin et al., 2001; Marteau et al., 2004, 2005; Amisten et al., 2007; Vaughan et al., 2007; King and Townsend-Nicholson, 2008; Swennen et al., 2008). Previously, the nonselective inhibitor suramin was used as a starting point for synthetic variations of the methyl group resulting in the discovery of NF157, the first P2Y11 antagonist with an apparent Ki of 75 nM in this study (Fig. 2A) and a pA2 of 7.77 in the study by Ullmann et al. (2005). NF157 has several disadvantages. It is not fully selective among P2 receptors, it is a rather large molecule containing six sulfonic acid groups, and its potency could be improved. However, the concept of searching among sulfonic acid groups containing compounds turned out to be a fruitful approach in several P2 projects (Lambrecht et al., 2002; Kassack et al., 2004; Horner et al., 2005; Ullmann et al., 2005). We thus undertook the approach of searching in a library of sulfonic and phosphonic acid compounds for P2Y11 ligands by use of a widely applicable calcium assay (Kassack et al., 2002). This strategy was successful. We discovered the competitive P2Y11 antagonist NF340, which is selective for P2Y11 among all tested P2 receptors and 4-fold more potent than NF157 (calcium assay; Fig. 2A). Inhibition by 10 μM NF340 of standard agonist responses at other P2Y receptors was less than 10%, corresponding to Ki values were greater than 10 μM (Table 1). This leaves NF340 at least 520-fold selective for P2Y11 over P2Y1, P2Y2, P2Y4, P2Y6, and P2Y12 receptors. Further, at P2X1, P2X2, and P2X2–3 receptors, 3 μM NF340 produced no significant inhibition (data not shown), thus leaving NF340 at least 156-fold selective for P2Y11.

Most interestingly, the screening approach led to the discovery of the non-nucleotide P2Y11 agonist NF546 displaying the same efficacy as ATPγS (Fig. 3). NF546 has some structural features of NF157, the previously developed P2Y11 antagonist (Ullmann et al., 2005), with the exception of the lack of naphthalene sulfonic acid groups. Thus, the switch from antagonism to agonism lies in the exchange of naphthalene sulfonic acid groups (in NF157) to benzylic phosphonic acid groups in NF546. ATPγS is still approximately one order of magnitude more potent than NF546, but NF546 is only 2.5-fold less potent than ATP, the physiological P2Y11 ligand. At P2Y2 receptors, NF546 seems to be a full agonist, too (Fig. 3A), but remains 83-fold less potent than the physiological agonist ATP. The ratios of EC50 values for NF546 determined for activation of various P2Y receptors expressed in different stable cell lines with P2Y11 receptors differ by at least 28-fold (Table 2). Furthermore, up to 3 μM NF546 had no effect at the tested P2X1, P2X2, and P2X2–3 receptors. Thus, in contrast to ATP, which is a nonselective agonist for P2 receptors, NF546 shows relative selectivity for P2Y11 over all tested P2Y and P2X receptors.

Taken together, these data support the advantage of NF546 over ATP or derivatives thereof. NF546 is only slightly less potent than ATP at P2Y11 (2.5-fold), but much more selective for P2Y11 over other P2 receptors, and NF546 cannot be hydrolyzed by nucleotidases because of its benzylic phosphonic acid structure. Ecke et al. (2006) have recently described P2Y11-selective ATP derivatives. NF546 has an advantage over these ATP derivatives, because NF546 can be chemically modified, similarly to the antiviral drug tenofovir, to become bioavailable (Holy, 2003). This gives an additional value to this non-nucleotide agonist of P2Y11 receptors.

We have not only discovered the antagonist NF340 and agonist NF546, but have also explored their nature of interaction with the P2Y11 receptor. NF340 is a competitive antagonist as shown in Schild analysis against ATPγS (Fig. 2), and it is a competitive antagonist against NF546 (Schild analyses in Fig. 4). pA2 values of NF340 were basically identical (∼ 8) when ATPγS or NF546 are used in calcium or cAMP assays, respectively (Figs. 2D and 4, B and D). Because NF340 and NF546 are competitive and use the same binding pocket, we were interested in understanding the binding mode at the molecular level. Docking of NF340 and NF546 into the binding pocket of the P2Y11 receptor model and molecular dynamics simulations with NF340 resulted in a reasonable binding mode showing a large overlap of the NF340 and NF546 putative binding site (Fig. 7). Amino acids shown to be important for ligand binding and receptor activation form interactions, in particular, hydrogen bonds and ionic interactions, throughout the simulation time, supporting the assumption of a competitive binding mode of NF340 and NF546 or the physiological agonist ATP (Table 3). Our findings are in accordance with site-directed mutagenesis studies that identified these residues as essential for agonist binding (Zylberg et al., 2007). Of particular importance is Arg268. The mutation of Arg268Ala reduced the potency of ATP by three orders of magnitude (Zylberg et al., 2007).

Most important, NF340 and NF546 were not only active at recombinant P2Y11 receptors expressed in 1321N1 cells but also at native P2Y11 receptors in monocyte-derived dendritic cells. DCs are known to express P2Y11 receptors (Wilkin et al., 2001; Schnurr et al., 2003). NF546 and ATPγS were equi-efficacious in stimulating TSP-1 release (Fig. 5B) and inhibiting the LPS-induced release of IL-12p70 (Fig. 6B). Both release of TSP-1 and inhibition of LPS-induced release of IL-12p70 were previously reported to result from P2Y11 receptor activation (Marteau et al., 2004, 2005). Moreover, release of some cytokines by DCs upon stimulation with nucleotides has been studied by Marcet et al. (2007). To get a comprehensive picture of cytokine release from DCs stimulated with ATPγS or NF546, we used a human proteome profiler cytokine array (Fig. 6A). We discovered the release of IL-8 upon ATPγS and NF546 stimulation, which was confirmed by ELISA and could be inhibited by the P2Y11-selective antagonist NF340 (Fig. 6, A and C). We have thus reported for the first time the release of IL-8 upon P2Y11 stimulation in DCs by use of our novel P2Y11-selective ligands NF340 and NF546. These findings may have an impact on strategies to modulate immune system reactions.

In conclusion, we have introduced the P2Y11 antagonist NF340 with improved potency and selectivity compared with NF157 and the non-nucleotide P2Y11 agonist NF546. These two compounds showed functional effects in DCs and helped to discover the previously unknown IL-8 release upon P2Y11 stimulation. These ligands will be helpful in further exploration of the physiological role of P2Y11 receptors.

Footnotes

  • This work was supported by the Deutsche Forschungsgemeinschaft DFG [Graduiertenkolleg GRK677, P3-FOR748, and Schm 536/8-1]; and by the Bischöfliche Studienförderung Cusanuswerk (S.M.).

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    doi:10.1124/jpet.109.157750

  • ↵Embedded Image The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

  • ABBREVIATIONS:

    TSP-1
    thrombospondin-1
    ATPγS
    adenosine-5′-O-(3-thio)triphosphate
    ADPβS
    adenosine-5′-O-(2-thio)diphosphate
    BzATP
    2′-3′-O-(4-benzoylbenzoyl)-ATP
    DC
    human monocyte-derived dendritic cells
    IL-4
    interleukin 4
    IL-8
    interleukin 8
    IL-12
    interleukin 12
    LPS
    lipopolysaccharide
    2-MeSATP
    2-(methylthio)adenosine-5′-triphosphate
    NF340
    4,4′-(carbonylbis(imino-3,1-(4-methyl-phenylene)carbonylimino))bis(naphthalene-2,6-disulfonic acid) tetrasodium salt
    NF546
    4,4′-(carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)carbonylimino))-bis(1,3-xylene-α,α′-diphosphonic acid) tetrasodium salt
    NF157
    8,8′-[carbonylbis[imino-3,1-phenylenecarbonylimino(4-fluoro-3,1-phenylene)carbonylimino]]bis-1,3,5-naphthalene trisulfonic acid hexasodium salt
    DMEM
    Dulbecco's modified Eagle's medium
    Tricine
    N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
    ELISA
    enzyme-linked immunosorbent assay.

    • Received June 16, 2009.
    • Accepted October 8, 2009.

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Research ArticleCELLULAR AND MOLECULAR

NF546 [4,4′-(Carbonylbis(imino-3,1-phenylene-carbonylimino-3,1-(4-methyl-phenylene)-carbonylimino))-bis(1,3-xylene-α,α′-diphosphonic Acid) Tetrasodium Salt] Is a Non-Nucleotide P2Y11 Agonist and Stimulates Release of Interleukin-8 from Human Monocyte-Derived Dendritic Cells

Sabine Meis, Alexandra Hamacher, Darunee Hongwiset, Claudia Marzian, Michael Wiese, Niels Eckstein, Hans-Dieter Royer, Didier Communi, Jean-Marie Boeynaems, Ralf Hausmann, Günther Schmalzing and Matthias U. Kassack
Journal of Pharmacology and Experimental Therapeutics January 1, 2010, 332 (1) 238-247; DOI: https://doi.org/10.1124/jpet.109.157750
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