Nonhydrolyzable ATP Analogues as Selective Inhibitors of Human

Article pubs.acs.org/jmc

Nonhydrolyzable ATP Analogues as Selective Inhibitors of Human NPP1: A Combined Computational/Experimental Study Joanna Lecka,†,‡,∥ Gal Ben-David,§,∥ Luba Simhaev,§,∥ Shay Eliahu,§ Jocelyn Oscar, Jr.,†,‡ Patrick Luyindula,†,‡ Julie Pelletier,‡ Bilha Fischer,§ Hanoch Senderowitz,§,∥ and Jean Sévigny*,†,‡,∥ †

Département de Microbiologie-Infectiologie et d′Immunologie, Faculté de Médecine, Université Laval, Québec, QC G1V 0A6, Canada ‡ Centre de Recherche du CHU de Québec, CHUL, Québec, QC G1V 4G2, Canada § Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel S Supporting Information *

ABSTRACT: Elevated nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) activity is implicated in health disorders including pathological calcification. Specific NPP1 inhibitors would therefore be valuable for studying this enzyme and as potential therapeutic agents. Here we present a combined computational/experimental study characterizing 13 nonhydrolyzable ATP analogues as selective human NPP1 inhibitors. All analogues at 100 μM inhibited (66−99%) the hydrolysis of pnp-TMP by both recombinant NPP1 and cell surface NPP1 activity of osteocarcinoma (HTB-85) cells. These analogues only slightly altered the activity of other ectonucleotidases, NPP3 and NTPDases. The Ki,app values of the seven most potent and selective inhibitors were in the range of 0.5−56 μM, all with mixed type inhibition, predominantly competitive. Those molecules were docked into a newly developed homology model of human NPP1. All adopted ATP-like binding modes, suggesting competitive inhibition with the endogenous ligand. NPP1 selectivity versus NPP3 could be explained in terms of the electrostatic potential of the two proteins that of NPP1 favoring negatively charged ligands. Inhibitor 2 that had the lowest Ki,app (0.5 μM) was also inactive toward P2Y receptors. Overall, analogue 2 is the most potent and selective NPP1 inhibitor described so far.

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INTRODUCTION Members of the ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) family are conserved eukaryotic enzymes which exist as membrane glycoproteins with an extracellular active site. Three members of this family, nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1; also known as PC-1), NPP2 (aka PD-Iα, autotaxin), and NPP3 (aka gp130RB13−6, B10, PDIα), are capable of hydrolyzing phosphodiester and pyrophosphate bonds of nucleotides. NPP1 and NPP3 are closely related, with ∼50% identity, and share 39% and 41% identity, respectively, with NPP2.1,2 However, NPP2 has a much lower ATPase activity than NPP1 and NPP3. NPP1 and NPP3 hydrolyze nucleoside triphosphates, nucleoside diphosphates, diadenosine polyphosphates, oligonucleotides, nicotinamide adenine dinucleotide (NAD+), flavin adenine dinucleotide (FAD), and uracil diphosphate (UDP) sugars.3 Hydrolysis of those nucleotide derivatives results in the formation of nucleoside monophosphates and the remaining part of the molecule. Hydrolysis of certain nucleotides prevents the activation of nucleotide receptors named P2X and P2Y receptors. Indeed, extracellular nucleotides activate G protein-coupled P2Y receptors,4−6 which are attractive pharmaceutical targets due © XXXX American Chemical Society

to their ability to modulate various functions in many tissues and organs under normal and pathophysiological conditions.7,8 Therefore the hydrolysis of ATP, uridine triphosphate (UTP), ADP, and UDP by NPPs can prevent the activation of P2 receptors.6 In this way, NPPs, especially NPP1 and NPP3, control purinergic signaling and hence may control various physiological processes.9 NPP1 was found to play a role in bone mineralization,10 signaling by insulin and nucleotides,11 differentiation, and cell motility.12,13 NPP1 has a unique role in regulating mineralization related processes. Disorders of spontaneous pathological calcification have been characterized in NPP1-deficient mice.14 A tight balance between pyrophosphate (PPi) and orthophosphate (Pi) concentrations governs physiological mineralization processes in bone, teeth, and growth plate cartilage.15,16 NPP1 is responsible for the generation of intravesicular and extracellular PPi and has been identified as the main PPigenerating enzyme of osteoblasts and chondrocytes.17−19 Matrix calcification by osteoblasts is mediated by the release of PPi-enriched matrix vesicles. These vesicles contain proteins Received: February 27, 2013

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dx.doi.org/10.1021/jm400918s | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Figure 1. Structures of the nucleotide analogues tested in this study.

a tumor marker.31,32 NPP expression has been reported to be increased in membranes of aged rat brains (NPP1) and in brain cortex of Alzheimer’s disease patients (NPP2), and NPP inhibitors have therefore been proposed as novel therapeutics for neurodegenerative diseases.33,34 Because ATP and ADP are the endogenous substrates for both nucleoside triphosphate diphosphohydrolases (NTPDases) and NPPs,35 we recently designed nucleoside di and triphosphate analogues 1−13 stable to hydrolysis by ectonucleotidases.36,37 To achieve hydrolytic resistance to NTPDases which hydrolyze the Pβ−Pγ phosphate diester bond in ATP, we substituted the bridging oxygen at Pβ−Pγ by a CX2 group (X = H, Cl or F). In addition, substitution of the nonbridging oxygen atom at Pα with a BH3 group also conferred resistance to hydrolysis by ectonucleotidases, namely NTPDase1, -2, -3, -8, NPP1, NPP3, and alkaline phosphatase. In agreement with these observations, the half-lives of these analogues in human blood serum were increased as compared to ATP.36,37 We also reported that these analogues exhibited high stability to gastric juice acidity (pH 1.4), with a half-life of 0.7−65 h. Replacement of the Pα nonbridging oxygen in analogues with a BH3 group resulted in complete resistance to hydrolysis in human blood serum over 24 h.37 Analogues 1−13

that sequester calcium and a cascade of hydrolytic enzymes including alkaline phosphatase, ATPase, and NPP1, that together control the release of Pi and PPi from ATP as well as the conversion of PPi into Pi.20 In extracellular fluids, calcium and phosphate concentrations are close to the deposition point of basic calcium phosphate such as hydroxyapatite. NPP1 activity controls this process.21−23 Interestingly, PPi not only initiates but also regulates mineralization by suppressing hydroxyapatite crystal deposition from amorphous calcium phosphate.24,25 Elevated levels of extracellular PPi often result in pathological calcification characterized by the formation of calcium pyrophosphate dehydrate crystals. A distorted balance of Pi/PPi allows crystal nucleation but ultimately leads to abnormal ossification because PPi also functions as a physiological inhibitor of this process. Importantly, a modest increase in NPP1 activity is sufficient to increase significantly extracellular PPi levels.26 Taken together, these data suggest that specific inhibitors of NPP1 would be extremely valuable as potential therapeutic agents for the treatment of calcific aortic valve disease,27 osteoarthritis,28,29 and chondrocalcinosis.30 NPP3 expression is associated with carcinogenesis and metastasis of cancer cells and has therefore been proposed as B



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Figure 2. Effect of analogues 1−13 on ectonucleotidase activities. Effect on human NPP1 (A) and on human NPP3 (B) activities. Substrate (pnpTMP) and the analogues were tested at a concentration of 100 μM each. The 100% activity tested in the absence of analogues with pnp-TMP as substrate was 42 ± 2 and 36 ± 4 [nmol p-nitrophenol·min−1·mg protein−1] for NPP1 and NPP3, respectively. Analogues 1−13 do not interfere with the hydrolysis of ATP by human NTPDase1, -2, -3, and -8 (C). Both substrate and analogues were tested at the concentration of 100 μM. The 100% activity was set with the substrate ATP alone and was: 556 ± 25, 628 ± 35, 300 ± 27, and 192 ± 11 [nmol Pi·min−1·mg protein−1] for NTPDase1, -2, -3, and -8, respectively. Data presented in A−C are the mean ± SD of 3−6 experiments carried out in triplicates. Ki,app determination (D), using Dixon and Cornish-Bowden plot, of human NPP1 by analogue 2. Pnp-TMP concentrations were 25, 50, and 100 μM, and the inhibitor concentrations were 0, 25, 50, and 100 μM. The data of one representative experiment out of three is shown. C



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potential inhibitors of ectonucleotidases activity, with a view to identify potent and selective inhibitors of NPPs. Effect of Analogues 1−13 on Human NPP1 and NPP3 Activity. All tested compounds inhibited the hydrolysis of pnitrophenyl 5′-thymidine monophosphate (pnp-TMP) by human NPP1. The compound with the weakest inhibitory properties was 1, which inhibited 66% of NPP1 activity, and the strongest inhibitors were 2 and 6−13, which blocked 90−99% of the hydrolysis of pnp-TMP (Figure 2A). Compounds 3, 4, and 5 inhibited NPP1 activity by about 80% (Figure 2A). The activity of human NPP3 was modestly affected (between 15 and 25% inhibition) by most compounds (Figure 2B). Analogues 8 and 10 had the strongest effect on NPP3 activity, inhibiting it by about 50% (Figure 2B). Effect of Analogues 1−13 on Human NTPDase Activity. Most compounds affected only partially the hydrolysis of ATP by the plasma membrane bound NTPDases (NTPDase1, -2, -3, and -8; Figure 2C). Importantly, analogues 1−5 and 10 had negligible effects on NTPDase activity, showing less than 18% inhibition, suggesting that these molecules could be used as NPP1 inhibitors. The strongest inhibition (∼50%) of ATP hydrolysis by human NTPDase1 was observed with analogues 6, 8, and 12 (Figure 2C). Fifty percent inhibition was also obtained for human NTPDase2 in the presence of 8, and NTPDase3 when incubated with analogues 6, 9, and 12 (Figure 2C). Analogues 8, 9, and 12 inhibited also 30% of human NTPDase8 activity (Figure 2C). Kinetic Parameters of Selected Human NPP1 Inhibitors. Next we evaluated the kinetic parameters for the strongest and most selective NPP1 inhibitors. These were found to have Ki,app values in the range of 0.5−56 μM, analogue 2 being the most potent (Table 1, Figure 2D). Using the

were also relatively weak agonists at the P2Y1 receptor. Replacement of the ATP nonbridging oxygen at Pα with BH3 and the Pβ,γ-bridging oxygen atom by a CH2 group as, for example, in analogue 2, resulted in a low agonist activity at this receptor.36 A nonhydrolyzable analogue that is tightly bound to NPP1 may serve as an inhibitor competing with ATP on the catalytic site. In this paper, we report on the identification of potent and selective NPP1 inhibitors exhibiting metabolic and chemical stability. Furthermore, we describe the development of ligand supported molecular models of human NPP1 and NPP3 based on the recently solved structure of mouse NPP138 and use them to explain the origin of the NPP1 inhibitory activity and selectivity of the most promising NPP1 inhibitors identified here.

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RESULTS Design of Potential NPP Inhibitors. Important characteristics of specific inhibitors of ectonucleotidases based on nucleotide scaffold are ineffectiveness toward P2 receptors as well as resistance to ectonucleotidase hydrolysis. Hence our design of potential NPP1 inhibitors focused on stabilizing the α,β- and β,γ-phosphorodiester bonds by appropriate chemical modifications. Some of these modifications may also reduce activity at P2-receptors. In addition, we explored the dependence of a nucleotide-analogue metabolic stability and affinity to NPP1 on the substitution of the adenine moiety.36 Specifically, replacing a β,γ bridging oxygen atom in ATP with a methylene group (i.e., β,γ-CH2−ATP) confers significant resistance to hydrolysis by nucleotide phosphohydrolases.36,39−42 Although β,γ-CH2−ATP was found to be a P2X1R agonist,43,44 it was a weak agonist at P2X2/3Rs,45 did not activate P2Y1R,43,44 and was a weak P2Y1R antagonist.46 We were aware of the advantage of the β,γ-methylene group as a stabilizing isostere in β,γ-CH2−ATP against ectonucleotidase-mediated hydrolysis, yet we realized that it would not protect the labile α,β-phosphodiester bond. Therefore, in addition to the β,γ-CH2 group,42 the α-phosphate was substituted by a boranophosphate moiety to stabilize the α,βphosphodiester bond of ATP against hydrolysis by NTPDases47 and NPPs. In several analogues, we also substituted the C2-position of ATP with a MeS group. The latter substitution also protects 2-MeS−ATP against hydrolysis by NTPDases.42 Under physiological pH (7.4), 91% of ATP is ionized, whereas β,γ-methylene-ATP analogues are only 9% ionized and are 91% protonated at Pγ. This may significantly reduce electrostatic interactions between the ligand and positively charged residues in ectonucleotidase catalytic sites. The effect of the methylene group could however be counteracted by a dihalogenated methylene group (e.g., CF2, CCl2) whose electronegativity lowers the pKa of phosphonate from 8.4 to 6.7−7.0, making it closer to the pKa of a terminal phosphate (i.e., 6.5).40 Thus, we explored also β,γ-dihalomethylene-ATP analogues as potential NPP1 inhibitors, assuming that the lower pKa values of their terminal phosphate may improve binding. On the basis of the above structure−activity relationship (SAR) considerations, we have previously synthesized a series of nucleotide analogues (1−13, Figure 1).36,37 We found that these analogues were stable to hydrolysis by NTPDase1, -2, -3, -8, and NPP1, -3, and additionally showed only minimal effects on P2Y1 receptor. Hence, here we selected analogues 1−13 as

Table 1. Ki,app Values of the Most Potent and Selective Inhibitors of Human NPP1a hNPP1 inhibitor 2 3 4 10 11 12 13

Ki [μM] 0.5 7 56 26 37 18 28

± ± ± ± ± ± ±

0.02 0.3 2 1 1 0.7 0.9

a

For Ki,app determination, the substrate pnp-TMP and all nucleotide analogues were used at the concentration range of 2.5 × 10−5 to 1 × 10−3 M. All experiments were performed three times in triplicate.

methods of Dixon and of Cornish-Bowden (Figure 2D and data not shown), we determined that the inhibitors presented in Table 1 showed mixed type inhibition, predominantly competitive.48,49 Effect of Analogues 2−4, 6−7, 9−11, and 13 on NPP Activity at the Surface of HTB85 Cells. Knowing the role of NPP1 in bone metabolism, we analyzed the influence of selected analogues on NPP activity at the surface of an NPP expressing cell line. We used osteocarcinoma cells (HTB85 also known as SaOS 2) as a native source of NPPs, but first we demonstrated the presence of these enzymes in these cells by immunostaining and biochemical activity. We have developed antibodies to human NPP1 and NPP3, which showed the presence of both enzymes (NPP1 and NPP3) at the surface of the HTB85 cells (Figure 3A). Although the NPP activity of D



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Figure 3. HTB85 cells express NPP1 and NPP3 at its surface which are inhibited by the analogues. (A) Immunoassays with human NPP1 antibodies hNPP1−3CI6 and human NPP3 antibodies hNPP3−4CI6 show the presence of both enzymes at the surface of COS-7 transfected cells and on the HTB85 cancer cell line. (B) NPPs activity assays on intact cells: COS-7 nontransfected, COS-7 transfected with NPP1, COS-7 transfected with NPP3, and the cancer cell line HTB85. (C) Analogues (2, 3, 4, 6, 7, 9, 10, 11, and 13) inhibit NPP activity at the surface of HTB85 cells. In (B,C) both substrate and analogues were tested at a concentration of 100 μM. The 100% was set for NPP1 with the substrate alone and was 1.2 ± 0.04 [nmol p-nitrophenol·min−1·well]. Data presented are the mean ± SD of three experiments carried out in triplicate.

HTB85 cells was much lower than in COS-7 cells transfected with NPP1 and NPP3, it was higher than the nontransfected COS-7 cells (Figure 3B). This allowed us to test inhibition strength of the designed nucleotide derivatives using human

osteoblastic HTB85 cells as a host of NPPs. The effect of the most potent and stable analogues (2, 3, 4, 6, 7, 9, 10, 11, and 13) on NPP activity at the surface of these cells was evaluated. In agreement with the results obtained for experiments with E



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Figure 4. Homology model of human NPP1. (A) Sequence alignment between human and mouse NPP1. (B) 3D model of human NPP1 with ATP docked into its catalytic site. The protein is shown as a ribbon diagram color coded according to its secondary structure, the two Zn ions are shown as orange spheres, and the ATP molecule (NPP1 site) is depicted in blue. (C) 3D model of the catalytic site of human NPP1 highlighting the interactions between ATP and binding site residues. The two Zn ions are shown as orange spheres, and the ATP molecule is colored according to atom types (nitrogen atoms are colored in blue; oxygen atoms are colored in red; carbon atoms are colored in gray; phosphate atoms are colored in purple). Hydrogen bonds and Pi interactions are shown in green and orange, respectively.

Information Figure S1). A structural analysis of the resulting model (Figure 4 and Supporting Information Table S1) reveals structural features which are consistent with the nucleotide/ nucleoside binding pattern of NPP1 and with its proposed catalytic mechanism. Thus, the catalytic binding site in the NPP1 model contains two zinc ions separated by 3.81 Å, with Zn1 coordinated by the ligand and Zn2 coordinated by the catalytic threonine (Thr256). The site is aligned by multiple residues (e.g., Asp218, Lys255, Thr256, Phe257, Asn277, Leu290, Lys295, Tyr340, Tyr371, Glu373, Asp376, His380, His424, and His535), which could form favorable interactions with the three parts of NPP1’s endogenous substrates, namely, the adenine ring, the sugar moiety, and the (mono, di, or tri) phosphate chain (Figure 4C). Docking Simulations at Human NPP1. Docking simulations with Glide successfully reproduced the crystallographic pose of AMP in mouse NPP1 with a RMSD of 0.73 Å

transfected COS-7 cell extracts, NPP activity exerted by the HTB85 cell line was inhibited by all these analogues (Figure 3C). NPP activity of HTB-85 cells was reduced by over 90% by analogues 2, 4, and 6 (Figure 3C). The other tested compounds (3, 7, 9−11, and 13) inhibited NPP activity by 41−80% (Figure 3C). Modeling of Human NPP1. To provide insight into the activities of analogues 1−13 at human NPP1 and tools for the structure based design of yet more active and selective NPP1 inhibitors, a ligand supported model of the protein was generated based on the recently solved structure of mouse NPP1 (PDB code 4GTW; sequence identity of 77.8%; Figure 4A,B).38 The model was found to have good stereochemical quality (99.2% and 0.6% of residues residing in the mostallowed and generously allowed region of the Ramachandran plot), an overall G-factor of −0.08, and a Prosa profile similar to that of the crystal structures of the template (Supporting F



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(data not shown). Furthermore, these simulations also provided a plausible binding mode for ATP, as shown in Figure 4B,C. ATP is stabilized within the binding site through an array of aromatic, hydrophobic, and H-bond interactions with binding site residues such as Tyr340 (π−π interaction with the adenine base), Phe257 (T-shape interaction with the adenine base), and Tyr340 (H-bond interactions with the sugar moiety; see Supporting Information Table S1). Furthermore, ATP is coordinated to Zn1 through its α-phosphate in accord with the NPP reaction and its triphosphate group is also stabilized through a hydrogen bond network to Asn277, Thr256, and Lys255. The results obtained for these two ligands (AMP and ATP) suggest that Glide is a suitable docking tool for this system. The seven NPP1 inhibitors presented in Table 1 were subsequently docked into the NPP1 site. To get some insight into the origin of inhibitory activity, an attempt was made to correlate the docking scores of the lowest energy poses for all analogues with their Ki,app values. This attempt however was unsuccessful (see Supporting Information Table S1 for Glide scores). Similarly, Boltzmann averaged docking scores did not correlate with analogues activity. Thus, we chose to focus our attention on the analysis of representative poses within the protein’s active site. These were obtained by first clustering the resulting poses within the binding site and then by selecting the lowest energy pose from the largest cluster. The representative poses of all seven analogues were found to adopt ATP-like conformations, suggesting that they could compete with ATP for binding site interactions (Figure 5, Supporting Information Table S1). The large catalytic site of NPP1 can in principle accommodate other poses. Indeed, a variety of binding modes were obtained for all ligands some of which clearly deviate from the ATP pattern. However, these binding modes were not representative. Similar to ATP, analogues 3 (Figure 5B) (with S chirality at Pα), 10, and 11 (with no chirality at Pα) preferentially coordinate the Zn1 through their Pα oxygen atom (although other chelation patterns were also observed). However, a similar coordination pattern for analogues 2 (Figure 5A) and 12 (with R chirality at Pα) would have forced coordination through the BH3, which is not chemically possible. Thus these two analogues coordinate Zn1 through their Pβ oxygen atom. Preference for Pβ−Zn1 coordination is also observed for analogues 4 and 13 despite their S chirality. However, these analogues also feature Zn1 coordination via Pα oxygen. Figure 5 shows the representative poses for analogues 2 (0.5 μM) and 3 (7 μM), which only differ in the chirality at Pα within the hNPP1 site. A detailed analysis of the representative poses of all analogues is provided in Supporting Information Table S1. Modeling of Human NPP3. To provide insight into analogue selectivity at human NPP3, a model of this protein was built, based once more on the crystal structure of mouse NPP3 (sequence identity of 51.7%; Figure 6A,B). As in the case of NPP1, the NPP3 model has good stereochemical quality (98.6% and 0.8% of residues residing in the most-allowed and generously allowed region of the Ramachndran plot), a good Gfactor of −0.18 and a satisfactory Prosa profile (Supporting Information Figure S1). As the data in Figure 2 clearly indicate, all analogues studied in this work inhibit NPP3 to a lesser extent than NPP1. To gain insight into the origin of this selectivity, we have aligned the sequences of the two proteins (Figure 7) and found a larger prevalence of Lys residues in the vicinity of the binding site in

Figure 5. Ligand docking into the human NPP1 model. (A) A representative pose of analogue 2 in the binding site of the human NPP1 model. The two Zn ions are shown as orange spheres, and the ligand is colored according to atom types (nitrogen atoms are colored in blue; oxygen atoms are colored in red; carbon atoms are colored in gray; phosphate atoms are colored in purple; boron atom is colored in green). Hydrogen bonds and Pi interactions are shown in green and orange, respectively. Because of the R chirality at Pα which would have pointed the nonchelating BH3 group toward Zn1, this zinc ion is chelated by the ligand’s Pβ oxygen atom. (B) A representative pose of analogue 3 in the binding site of the hNPP1 model. Color coding is as in (A). The S chirality at Pα allows for Zn1 chelation by the ligand’s Pα oxygen atom.

NPP1 relative to NPP3 (seven Lys residues, namely, Lys255, Lys278, Lys291, Lys293, Lys295, Lys338, and Lys528 in NPP1 and a single Lys residue, Lys204 in NPP3, with similar numbers of Arg, Asp, and Glu residues; see Figure 7), which is reflected in the electrostatic potential of the two proteins (Figure 8). Thus, the binding site of NPP1 is better suited to accommodate negatively charged ligands such as the ATP analogues discussed in this study. In accord with this observation, the electrostatic interactions of representative poses of analogue 2 (the most potent analogue considered in this work) are more favorable in the NPP1 binding site than in the NPP3 binding site (−335 vs −176 kcal/mol; obtained with MM-GBSA calculations performed with Maestro version 9.3). G



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Figure 6. Homology model of human NPP3. (A) Sequence alignment between human NPP3 and mouse NPP1. (B) 3D model of human NPP3. The protein is shown as a ribbon diagram color coded according to its secondary structure, and the two Zn ions are shown as orange spheres.

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was reported as an NPP1 inhibitor (Ki,app = 100 μM).54 Likewise, bis-coumarin derivatives were identified as pure noncompetitive inhibitors of snake venom and human NPP1 enzymes, with Ki,app and IC50 values of 50 and 164 μM, respectively, for human NPP1.55 Here, we examined nonhydrolyzable nucleotide analogues 1−1336,37 as potential NPP inhibitors. All tested analogues inhibited the activity of human NPP1 at the concentration of 100 μM. Among the selected, most specific analogues presented in Table 1, compound 2 showed the lowest Ki (Ki,app = 0.5 μM). Yet other tested analogues, such as 6−13, although less potent and specific than 2, are also characterized with lower Ki,app values than the few previously reported inhibitors (Table 1, Figure 2D).53−55 Analogues 1−5 and 10 did not affect the activity of NTPDases. Interestingly, analogue 10 also inhibited over 50% NPP3 activity. This ability of analogue 10 to inhibit the activity of both NPPs, which is also shared by compound 8, may reveal useful in designing either

DISCUSSION AND CONCLUSIONS As other ectonucleotidases, NPPs are expected to control P1 and P2 receptor signaling in various mammalian organs and cells. The field of NPP enzymology is still in its infancy. Therefore, NPP specific inhibitors that do not affect other ectonucleotidases, such as NTPDases, and which do not trigger nor interfere with P2 receptor activation, would be extremely valuable as novel research tools. Furthermore, potent and selective NPP inhibitors could serve as promising therapeutic agents for the treatment of calcific aortic valve disease,27 osteoarthritis,28 chondrocalcinosis,30 and/or cancer.50,51 Despite their therapeutic potential, NPP inhibitors have scarcely been reported. Suramin was reported to reduce the hydrolysis of pnp-TMP by NPP by about 36% at 250 μM.52 Yet, suramin and its derivatives antagonize most P2 receptors and also efficiently inhibit NTPDases and cannot therefore be considered as specific NPP inhibitors.53 Recently, [3-(tbutyldimethylsilyloxy)-phenyl]-1,3,3-oxadiazole-2 (3H)-thione H



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Figure 7. Sequence alignment between human NPP1 and NPP3. Binding site residues are bolded. Lys residues within 10 Å of the two binding sites are marked in red. There are seven Lys residues in the vicinity of the binding site in NPP1 and only a single one in NPP3, while the number of other charged residues in the vicinity of the binding site is similar in both proteins (one Arg residue in both NPP1 and NPP3; Asp, six and five residues in NPP1 and NPP3, respectively; Glu, nine and seven residues in NPP1 and NPP3, respectively).

more potent and selective inhibitors of NPP3, which are still not available, or in designing inhibitors of the NPP family, which would also be very valuable. The data presented in Figure 1 and Table 1 do not lend themselves to a clear interpretation in terms of SAR because all major moieties, namely BH3, methylene, or dihalomethylene bridge and a substitution at C2 of the adenine ring, are found in both active and less active analogues. Nevertheless, several trends were observed (although precise comparisons were sometimes difficult to make). Thus, NPP1 seems to favor R over S chirality at Pα (compare analogues 2 with 3 and 12 with 13) and a Pβ−Pγ methylene over a dihalomethyelene moiety (compare analogues 2 and 3 with analogues 11−13) and to disfavor substitution at C2 of the adenine ring (the three most potent analogues, 2, 3, and 12 are unsubstituted at C2, whereas three of the four less active analogues, 4, 10, and 11 are substituted at C2). Accordingly, the high potency of analogue 2 seems to result from a combination of all these features. To provide insight into the inhibitory activities of the analogues studied in this work as well as into their selectivity at NPP1, ligand supported homology models of human NPP1 and NPP3 were generated based on the recently solved structure of mouse NPP1.38 Both models were shown to have high quality stereochemical profiles and to be structurally similar to the template protein (backbone root-mean square deviation, RMSD, of 0.53 Å and 1.16 Å for the NPP1 and NPP3 models, respectively). Docking simulations using Glide successfully reproduced the crystallographic pose of AMP within the mouse NPP1 binding site (RMSD of 0.73 Å) and provided a plausible binding mode for ATP as the lowest energy structure (Figure 4 and Supporting Information Table S1). Encouraged by these results, analogues 2, 3, 4, 10, 11, 12, and 13 were docked into the binding site of the NPP1 model. The representative structures of all seven analogues adopted ATP-like binding modes, suggesting that they could effectively compete with ATP for binding site interactions. While these analogues are

structurally similar, they represent distinct chemical families (phosphates, boranophosphates, and phosphonates) which differ in their stereochemical properties. Thus, this structural similarity will not necessarily translate into similar binding modes within the NPP1 site. Indeed, Glide simulations provided multiple poses for each of the ligands studied in this work, some of which clearly differed from the ATP-like binding mode. Yet the site was able to “select” from among the docked ensemble ATP-like poses as representative ones. All analogues maintained a coordination distance to Zn1, albeit with different coordination patterns. For the set of analogues studied in this work, our docking results suggest that depending on the stereochemistry at Pα, coordination is geometrically possible through oxygen atoms of either Pα or Pβ (e.g., analogues 2 and 3). However, all attempts to correlate either the docking scores (including rescoring with multiple scoring functions; results not shown) or the interaction pattern within the binding site (see Supporting Information Table S1) for these analogues with their Ki,app values have been unsuccessful. Presumably, the accurate reproduction of binding energies for the highly charged and flexible analogues considered in this study requires the quantitative assessment of desolvation penalties and entropy effects which are not accounted for by the docking process. This is perhaps not surprising in lieu of the known deficiencies of contemporary scoring functions.70 A clear-cut selectivity for NPP1 vs NPP3 was observed for analogues 1−13 (Figure 2A vs B). These analogues inhibited NPP1 mostly by 90−99%, whereas NPP3 was inhibited in most cases by only 15−20%. This clear subtype selectivity indicates tighter binding of analogues 1−13 to NPP1. This phenomenon may be explained by the newly developed models of NPP1 and NPP3. As the data in Figures 7 and 8 clearly indicate, NPP1 features a unique arrangement of Lys residues near its nucleotide binding site, which are absent in NPP3. This large number of positively charged residues improves analogue I



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process of mineralization. We therefore conclude that analogue 2 is a plausible starting point for the rational design of yet more potent inhibitors of NPP1. Such design efforts are currently being pursued in our laboratory.

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EXPERIMENTAL SECTION

Analogues. The synthesis of compounds 1−13 (actual compound numbers are bolded and nomenclature in the previous papers are in regular characters: 1, 2; 2, 3A; 3, 3B; 4, 4B; 5, 4A;36 6, 12A; 7, 12B; 8, 10A; 9, 10B; 10, 13; 11, 8; 12, 11A; 13, 11B37) was done as reported before.36,37 The spectral data of the compounds were used for checking the consistency with the literature. The purity of the compounds was above 95% as evaluated by an analytical reverse-phase column system (Gemini 5 μ C-18 110A, 150 mm × 4.60 mm, 5 mm, Phenomenex, Torrance, CA, USA) in two solvent systems using different gradient combinations: solvent system I, (A) 100 mM triethylammonium acetate (TEAA), pH 7, (B) methanol (MeOH); solvent system II, (A) 0.01 M KH2PO4, pH 4.5, (B) MeOH.36,37 Plasmids. The plasmids encoding the human form of various ectonucleotidases used in this study have all been described in published reports: NTPDase1 (GenBank accession no. U87967),59 NTPDase2 (NM_203368),60 NTPDase3 (AF033830),61 NTPDase8 (AY330313),62 NPP1 (NM_006208),63 and NPP3 (NM_005021).64 Cell Transfection and Preparation of Membrane Fraction. Ectonucleotidases were produced by transiently transfecting COS-7 cells in 10 cm plates by use of lipofectamine (Invitrogen), as previously described.65 Briefly, COS-7 cells were transfected in 10 cm plates using lipofectamine (Invitrogen, Burlington, ON, Canada). Cells (80−90% confluent) were incubated for 5 h at 37 °C in Dulbecco’s Modified Eagle’s Medium, nutriment mix F-12 (DMEM/F-12) in the absence of fetal bovine serum (FBS) with 6 μg of plasmid DNA, and 24 μL of lipofectamine reagent. The reaction was stopped by the addition of an equal volume of DMEM/F-12 containing 20% FBS, and the cells were harvested 44−72 h later. For the preparation of protein extracts, transfected cells were washed three times with Tris-saline buffer at 4 °C, collected by scraping in the harvesting buffer (in mM, 95 NaCl, 0.1 phenylmethylsulfonyl fluoride (PMSF) and 45 Tris at pH 7.5), and washed twice by 300g centrifugation for 10 min at 4 °C.53 Cells were resuspended in the harvesting buffer containing 10 μg/mL aprotinin and sonicated. Nucleus and cellular debris were discarded by centrifugation at 300g for 10 min at 4 °C, and the supernatant (crude protein extract) was aliquoted and stored at −80 °C until used for activity assays. Protein concentration was estimated by the Bradford microplate assay using bovine serum albumin (BSA) as a standard.66 Enzymatic Assays. NPPs (EC 3.1.4.1; EC 3.6.1.9). Evaluation of the effect of analogues on human NPP1 and NPP3 activity was carried out with para-nitrophenyl thymidine 5′-monophosphate (pnp-TMP) as substrates.67 The reactions were carried out at 37 °C in 0.2 mL of the following incubation mixture, in mM, 1 CaCl2, 140 NaCl, 5 KCl, and 50 Tris, pH 8.5, with or without analogues 1−13 (100 μM) or ATP (100 μM). Human NPP1 or NPP3 extract was added to the incubation mixture and preincubated at 37 °C for 3 min. Reaction was initiated by the addition of 100 μM pnp-TMP, and the production of paranitrophenol was measured after 15 min reaction at 410 nm. For analogues, the reaction was stopped after 30 min by transferring an aliquot of 0.1 mL from the reaction mixture to 0.125 mL ice-cold 1 M perchloric acid. The samples were centrifuged for 5 min at 13000g. Supernatants were neutralized with 1 M KOH (4 °C) and centrifuged for 5 min at 13000g. An aliquot of 20 μL was separated by reversephase high-performance liquid chromatography (HPLC) to evaluate the nucleotide content of each reaction sample.36,37 The type of inhibition and Ki,app was calculated by plotting the data of three independent experiments using pnp-TMP as substrate according to Dixon and Cornish-Bowden methods.48,49 Activity assays at the surface of intact HTB-85 cells were carried out in 0.5 mL of the incubation mixture in 24-well plates. Reaction was initiated by the addition of pnp-TMP to obtain a final concentration of

Figure 8. Electrostatic potentials of human NPP1 and NPP3. Electrostatic potentials of human NPP1 (A) and NPP3 (B) binding sites calculated on their respective molecular surfaces. The enhanced positive potential (blue) at the NPP1 site is clearly visible. A representative pose of analogue 2 (stick representation) is shown at both sites.

binding to NPP1 by reducing their pKa and therefore enhancing electrostatic interactions. On the basis of its low Ki,app value (500 nM), selectivity toward human NPP1, stability in the presence of other ectonucleotidases, inactivity at P2Y1,4,6 purinoceptors,36 and ability to block NPPs activity exerted by cancer cells, analogue 2 appears as the most potent and promising NPP1 inhibitor currently known. Nevertheless, being an ATP derivative, analogue 2 (as well as the other analogues studied in this work) is not a classical “drug-like” compound. Yet related compounds such as thiazole-4-carboxamide adenine dinucleotide and Denufosol have found their way into clinical trials.56,57 Developing analogue 2 into a drug may require prodrug approaches,58 appropriate formulations, and/or administration modes other than oral (e.g., topical, inhalation; depending on the clinical indication). However, even if analogue 2 will not be eventually developed into a drug, it is still likely to serve as an important mechanistic tool for the study of the complex J



Article

100 μM. After 20 min, 0.2 mL of the reaction mixture was transferred to a 96-well plate and the production of paranitrophenol was measured at 410 nm. NTPDases (EC 3.6.1.5). Activity of human NTPDases was measured as described previously53 in 0.2 mL of the Tris-Ringer incubation medium (in mM, 120 NaCl, 5 KCl, 2.5 CaCl2, 1.2 MgSO4, 25 NaHCO3, 5 glucose, 80 Tris, pH 7.4) at 37 °C with or without analogues 1−13 (final concentration 100 μM) and with or without 100 μM ATP (or ADP) as a substrate of human NTPDases. The analogues were added without ATP or ADP when tested as potential substrate and with ATP or ADP when tested for their effect on nucleotides hydrolysis. NTPDase protein extracts were added to the incubation mixture and preincubated at 37 °C for 3 min. The reaction was initiated by the addition of the substrate and stopped after 15 min with 50 μL of malachite green reagent. The concentration of the released Pi was measured at 630 nm according to Baykov et al.68 Animals and Antibody Production. All procedures were approved by the Canadian Council on Animal Care and by the Laval University Animal Ethics Advisory Committee. For antibody production, Hartley guinea pigs were obtained from Charles River Laboratories (St-Constant, QC, Canada) and genetic immunization was carried out by direct injection of plasmids (pcDNA3.1) encoding each of the human proteins. The antibodies obtained and described in this manuscript were named guinea pig hNPP1−3CI6 to human NPP1 and guinea pig hNPP3−4CI6 to human NPP3. These antibodies can be obtained via the Internet at http://ectonucleotidases-ab.com. Immunocytochemistry. Immunocytochemistry experiments were performed as previously described.69 Briefly, COS-7 cells (105/ coverslip) were fixed with cold acetone:10%phosphate-buffered formalin (9.5:0.5). Fixed cells were incubated with the polyclonal human NPP1 and human NPP3 antiserum (hNPP1−3CI6, hNPP3− 4CI6) and the biotinylated secondary antibodies, biotin-conjugated goat antiguinea pig (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), were added before addition of Vectastain Elite ABC reagent (Vector Laboratories, Burlington, ON, Canada). The antibodies were used at a 1:500 (primary antibodies) and 1:1000 (secondary antibodies) working dilution. Endogenous peroxidase was quenched by incubating the fixed cells with 0.15% (v/v) H2O2 in phosphate buffered saline (PBS). Endogenous avidin and biotin were blocked using the Avidin/Biotin blocking kit (Vector Laboratories). The peroxidase reaction was performed in a solution containing the 3,3′-diaminobenzidine substrate (0.5 mg/mL) and 0.03% (v/v) H2O2 in PBS and stopped with extensive water rinsing. Preimmune sera were routinely included as controls. Homology Modeling of Human NPP1. A ligand supported model of human NPP1 was built based on the recently solved crystal structure of mouse NPP1 (PDB code 4GTW; sequence identity to NPP1 of 77.8%). Briefly, the sequence of the target protein (accession number P22413) was downloaded from the UniprotKB/Swiss-Prot server and aligned with the target sequence using the ClustalW70,71 method as implemented in Discovery Studio version 3.572 with default settings. Following manual refinement, the resulting alignment was used as input to the MODELER73 program as implemented in Discovery Studio, also using default settings. The highest ranked model was tested for stereochemical quality using Procheck74 and Prosa75,76 and selected for subsequent analysis. Homology Modeling of Human NPP3. A model of human NPP3 (accession code O14638) was built in a manner identical to NPP1 using the same template structure. As before, the highest ranking model was selected for further analysis. Docking. Docking simulations were performed using Glide77,78 as implemented in Maestro 9.0. Prior to docking, protein structures were prepared using the protein preparation wizard in Discovery Studio version 3.5. For docking into crystal structures, Glide’s grid box was centered on the coordinates of the ligand in the complex. For docking into the protein model, the position of the ligand within the binding site was approximated by transferring its coordinates from the crystal. A docking grid was generated within the docking box, and ligands were docked into their respective binding sites using Glide’s Extra Precision (XP) option.79

Following docking, the resulting poses were clustered in Maestro using all heavy atoms along the analogues’ “backbone” and the lowest energy poses from the largest clusters were taken as representatives.

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ASSOCIATED CONTENT

S Supporting Information *

Model validation; list of interactions between ATP and analogues 2, 3, 4, 10, 11, 12, and 13 in their representative poses with binding site residues within the catalytic site of the human NPP1 model. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 418-654-2772. Fax: 418-654-2765. E-mail: Jean. [email protected]. Address: Centre de Recherche du CHU de Québec, CHUL, 2705 Boulevard Laurier, Office T149, Québec, QC, G1V 4G2, Canada. Author Contributions ∥

J.L., G.B.-D., L.S., H.S., J.S.: These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED BSA, bovine serum albumin; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; NPP1, nucleotide pyrophosphatase/phosphodiesterase-1; NPP2, nucleotide pyrophosphatase/phosphodiesterase-2; NPP3, nucleotide pyrophosphatase/phosphodiesterase-3; PBS, phosphate buffered saline; Pi, orthophosphate; PPi, pyrophosphate; pnp-TMP, para-nitrophenyl thymidine 5′-monophosphate; SAR, structure−activity relationship

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REFERENCES

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