Adenosine 5′-(α,β-methylene)diphosphate (α,β-metADP), adenosine 5′-(α,β-methylene)triphosphate (α,β-metATP), adenosine 5′-diphosphate-2′,3′-dialdehyde (dialADP), adenosine 5′-monophosphate (AMP), adenosine 5′-triphosphate (ATP), adenosine 5′-triphosphate-2′,3′-dialdehyde (dialATP), 2-methylthioadenosine 5′-diphosphate (2-MeSADP), 2-methylthioadenosine 5′-triphosphate (2-MeSATP), p-nitrophenol, p-nitrophenyl 5′-thymidine monophosphate (p-Nph-5′-TMP), reactive blue 2, suramin, and uridine 5′-triphosphate (UTP) were obtained from Sigma (Steinheim, Germany). Calcium chloride, magnesium chloride, sodium hydroxide, and zinc chloride were also from Sigma. 2-(N-Cyclohexylamino)ethanesulfonic acid (CHES) and tris-(hydroxymethyl)aminomethane (Tris) were from Applichem (Darmstadt, Germany). Disodium hydrogen phosphate was purchased from Carl Roth (Karlsruhe, Germany). 2-(6-Amino-9H-purin-8-ylthio)-N-(3,4-dimethoxyphenyl)acetamide (PZB08513136A) was synthesized and provided by the group of Prof. Dr. Piet Herdewijn as previously described (Chang et al., 2014). N-[2-[1-(6,7-Dimethoxyquinazolin-4-yl)piperidin-4-yl]ethyl]sulfuric diamide (SAR 03004) was synthesized as described in Supporting Information. [TiW₁₁CoO₄₀]⁸⁻ (PSB-POM141) (Müller et al., 2006) was provided by Dr. Holger Stephan (Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden—Rossendorf). Human recombinant soluble NPP1 (Val191—Leu591) expressed in murine myeloma NS0 cells was purchased from R&D Systems GmbH (Wiesbaden, Germany, purity > 95%, purified by using N-terminal His-tag).

p-Nitrophenyl 5′-adenosine monophosphate (p-Nph-5′-AMP) was synthesized in analogy to a described procedure (Borden and Smith, 1966; Ivanovskaya et al., 1987). Adenosine-5′-monophosphate disodium salt (17, see Figure 4, 1.0 g) was dissolved in 20 mL of deionized water. To this solution was added Dowex 50X8 proton form (2 gram) prewashed three times, each with 100 mL of deionized water. The resulting adenosine-5′-monophosphoric acid (18, see Figure 4) was separated by filtration and freeze-dried. Adenosine-5′-monophosphoric acid (18, 347 mg) was dissolved in pyridine (10 mL) containing triethylamine (1.4 mL). To this was added p-nitrophenol (1.4 g) followed by dicyclohexylcarbodiimide (2.0 g), and the mixture was stirred in the dark for 3 days at room temperature, until complete disappearance of adenosine-5′-monophosphosphoric acid as indicated by thin layer chromotagraphy (TLC, solvent system: n-butanol: acetic acid: water = 2:1:1). Pyridine was subsequently removed under reduced pressure, the residue was suspended in water (100 mL), and insoluble dicyclohexylurea was removed by filtration. The aqueous layer was washed three times with diethyl ether (3 × 50 mL) to remove excess p-nitrophenol, and the aqueous layer was freeze-dried. Then the freeze-dried crude product was subjected to C-18 reverse phase-HPLC using a gradient of acetonitrile: 50 mM aqueous NH₄HCO₃ solution from 0:100 to 25:75 for 50 min. Appropriate fractions were pooled and freeze-dried several times to obtain dry p-nitrophenyl 5′-adenosine monophosphate (19 in Figure 4) as a white solid; yield: 62%. The final product was characterized by LC-MS, ¹H, ¹³C, and ³¹P NMR spectroscopy. ¹H NMR (500 MHz, MeOD-d₄): δ 4.23–4.26 (1H, q, H4′, J = 5.70 Hz), 4.29–4.36 (2H, m, H5′and H5″CH₂, J = 4.40 Hz), 4.41–4.43 (1H, q, H3′, J = 5.05 Hz), 4.70–4.72 (1H, t, H2′, J′ = 5.03 Hz and J″ = 5.35 Hz), 6.11–6.12 (1H, d, H1′, J = 5.35 Hz), 7.38–7.40 (2H, d, Ph-CH₂, J = 9.15 Hz), 8.10–8.12 (2H, d, Ph-CH₂, J = 9.45 Hz), 8.33 (1H, s, H2), 8.56 (1H, s, H8). ¹³C NMR (125 MHz, MeOD-d₄): δ 67.05, 72.30, 76.34, 86.00, 89.85, 120.36, 121.97, 126.45, 143.36, 144.88, 147.92, 150.57, 152.41, 159.91. ³¹P NMR (202 MHz, MeOD-d₄) δ –6.47 (s). LC-ESI-MS: negative mode 467 ([M – H]⁻), positive mode 469 ([M + H]⁺).

Enzyme kinetic parameters were measured for p-Nph-5′-TMP and p-Nph-5′-AMP, both being artificial substrates of human NPP1. Solutions with different concentrations of both substrates (ranging from 1.0 to 500 μM) were prepared in 10 mM CHES buffer (in mM: 1 MgCl₂, 2 CaCl₂, 10 CHES, pH 9.0) and added in a final volume of 100 μl to 96-well-plates. The enzyme reactions were initiated by the addition of 20 ng of human NPP1 (for p-Nph-5′-TMP), or 75 ng of human NPP1 (for p-Nph-5′-AMP). The mixture was incubated at 37°C for 15 min (p-Nph-5′-TMP), or 30 min (for p-Nph-5′-AMP), respectively, and subsequently terminated by the addition of 20 μl of 1.0 N aqueous NaOH solution. The amounts of p-nitrophenolate liberated were measured at 400 nm. Each analysis was repeated twice in three separate experiments.

Concentration-response curves of NPP1 inhibition were determined for six nucleotidic inhibitors [dialADP (1), dialATP (2), α,β-metADP (3), α,β-metATP (4), 2-MeSADP (5), and 2-MeSATP (6), see Figure 2] and five non-nucleotidic inhibitors [SAR 03004 (12), reactive blue 2 (13), suramin (14), PZB08513136A (15), and [TiW₁₁CoO₄₀]⁸⁻ (16), see Figure 3], and with different substrates. For testing vs. both synthetic substrates, p-Nph-5′-TMP and p-Nph-5′-AMP, solutions containing different concentrations of each inhibitor were prepared with 10 mM CHES buffer (See Section Determination of Kinetic Parameters of Artificial Substrates) containing 400 μM p-Nph-5′-TMP, or p-Nph-5′-AMP, respectively, in a final volume of 100 μl. Incubation and operation conditions remained the same as described in Section Determination of Kinetic Parameters of Artificial Substrates, with one exception: reactive blue 2 absorbs light at 400 nm. Therefore, measurements were not performed colorimetrically, but by capillary electrophoresis (CE) with DAD detection of the formed p-nitrophenolate at 400 nm. The CE instrumentation and operation conditions were as follows: The CE instrumentation and operating conditions were as follows: P/ACE MDQ capillary electrophoresis system (Beckman Instruments, Fullerton, CA, USA) with a DAD detection system, polyacrylamide-coated capillaries of 50 cm effective length × 50 μm (id) obtained from CS Chromatographie GmbH (Langerwehe, Germany), 100 mM phosphate buffer (pH 6.5) as running buffer, electrokinetic injection (−6 kV, 60 s), separation voltage of −20 kV. The measurement was performed twice in three different experiments. Data collection and peak area analysis were performed by the 32 Karat software obtained from Beckman Coulter (Fullerton, CA, USA). The IC₅₀-values of test compounds for each substrate were calculated by plotting of three independent experiments using the program Prism 5.0 (GraphPad software, San Diego, CA, USA).

For the natural substrate ATP, the enzyme inhibition assays were performed in 10 mM CHES buffer containing 400 μM of substrate along with different inhibitor concentrations. Incubation and operation conditions were the same as described above with artificial substrates. The analysis was performed by CE and the amounts of AMP produced were quantified by their UV absorption at 260 nm. Each analysis was repeated twice in three separate experiments.

The inhibition mechanisms of the nucleotidic and non-nucleotidic inhibitors were determined using different concentrations of each substrate (from 10 to 1500 μM), and three different concentrations (0, ~0.5- and ~2-fold of IC₅₀-value) of each test compound. The instrumentations and operation conditions for the experiments were the same as those described in the Sections Determination of Kinetic Parameters of Artificial Substrates and Determination of Concentration-Dependent Inhibition Curves. Each analysis was performed in three separate experiments. The inhibition type of each inhibitor was then evaluated graphically from the Hanes-Woolf plots. For the determination of the (α)K_i-values the slope of the reciprocal lines from the Hanes-Woolf plot were plotted as a function of inhibitor concentrations using Prism 5.0.

The generated homology model of human NPP1 described in Namasivayam et al. (2017). was used for the docking procedure using AutoDock 4.2 (Morris et al., 2009). The AutoDockTools package was employed to generate the docking input files and to analyze the docking results (Sanner, 1999). The search algorithm Lamarkian genetic algorithm (LGA) and the default scoring function, a hybrid scoring function (semi-empirical and free-energy) was employed for docking calculations. Three-dimensional energy scoring grids for a box of 60 × 60 × 60 points with a spacing of 0.375 Å were computed. The grids were centered based on the co-crystallized ligand, which was transformed into the homology model. A total of 50 runs with a maximum of 250,000 energy evaluations were performed with the default parameters for the genetic algorithm (GA) and Solis-Wet local search, a method that facilitates random moving around the binding pose identified through the GA. High scoring binding poses (of lowest energy) or more populated poses were selected for the analysis on the basis of visual inspection.

Statistical data analyses of pK_i-values were performed using Prism 5.0 software. The pK_i-values [–log₁₀(α)K_i-values] were calculated from the obtained (α)K_i-values in each assay. Data were tested for statistical significance by one-way ANOVA as appropriate. When significant differences were observed, Tukey multiple comparison tests were performed. A value of p < 0.05 was considered significant.

The correlation coefficients (R²) were evaluated by comparing pK_i-values of one assay to those of another assay using Prism 5.0.

p-Nph-5′-AMP had been previously synthesized (Borden and Smith, 1966; Ivanovskaya et al., 1987), but no detailed characterization of the compound has been published. Initially we tried to prepare 19 directly from adenosine by reaction with p-nitrophenyl phosphorodichloridate. However, the reagent is toxic and difficult to handle. Moreover, tedious separation and purification procedures were required to obtain the desired product in sufficient purity, and the yield was only about 20%. Therefore, we decided to utilize commercially available AMP (17) for the preparation of 19. In a one-step reaction AMP was condensed with p-nitrophenol in the presence of N,N′-dicyclohexylcarbodiimide yielding product 19 in 62% yield after purification (see Figure 4).

In subsequent experiments, we characterized the newly synthesized alternative substrate p-Nph-5′-AMP in comparison with the common artificial substrate p-Nph-5′-TMP and the natural substrate ATP. Enzyme kinetic analysis showed the following rank order of substrate preference: ATP > > p-Nph-5′-TMP > p-Nph-5′-AMP. The k_cat/K_m-values are 674 × 10³ M⁻¹s⁻¹ (k_cat = 5.51 s⁻¹, K_m = 8.17 μM) for ATP (Namasivayam et al., 2017), 100 × 10³ M⁻¹s⁻¹ (k_cat = 22.3 s⁻¹, K_m = 222 μM) for p-Nph-5′-TMP (Namasivayam et al., 2017), and 13.4 × 10³ M⁻¹s⁻¹ (k_cat = 2.51 s⁻¹, K_m = 188 μM) for p-Nph-5′-AMP (see Figure S1 in Supporting Information). While the K_m-value of p-Nph-5′-AMP was almost identical to that of p-Nph-5′-TMP indicating similar affinities, the k_cat-value determined for p-Nph-5′-TMP was nearly 10-fold higher than that for p-Nph-5′-AMP. This means that p-Nph-5′-TMP is hydrolyzed by NPP1 much faster than p-Nph-5′-AMP. Compared to the natural substrate ATP, the new artificial substrate has a similar k_cat-value, but its K_m-value is > 20-fold higher than that for ATP. This indicates that the binding affinity of p-Nph-5′-AMP is significantly lower than that of ATP.

In order to gain insights into the molecular determinants involved in the formation of the enzyme-substrate complex, the new artificial substrate p-Nph-5′-AMP was docked into a homology model of the human NPP1 (Namasivayam et al., 2017), which was generated based on the recently solved crystal structure of the mouse NPP1 (Kato et al., 2012). The observed interactions were compared to those of ATP and p-Nph-5′-TMP. As shown in Figure 5A, the α-phosphate group of the substrate ATP is bound between the two zinc ions, and the two other phosphate groups form hydrogen bond interactions with the following amino acid residues: Lys255, Thr256, Asn277, His380, and His535. Tyr340 forms a hydrogen bond with the ribose moiety, and the adenine ring of ATP is stacked between Phe257 and Tyr340 (Namasivayam et al., 2017). Similarly, both artificial substrates form complexes with the zinc ions of the enzyme with their phosphate groups as shown in Figures 5B,C. Because the p-nitrophenylphosphate group of both artificial substrates interacts in the same way with the zinc ions in the active site of the enzyme, the ground state of binding interactions may be comparable, which explains the similar K_m-values determined for both artificial substrates. While p-Nph-5′-AMP binding is stabilized through π-π interactions of the adenine base mainly with Tyr340 of the enzyme, this interaction is expected to be weaker for the artificial substrate p-Nph-5′-TMP due to the exchange of adenine for thymine. Furthermore, a 2′-hydroxyl group at the ribose moiety is lacking in p-Nph-5′-TMP, but not in p-Nph-5′-AMP, and therefore, the interaction between that OH group and the side chain of Tyr340 is missing in p-Nph-5′-TMP. Overall, p-Nph-5′-TMP has less interactions than p-Nph-5′-AMP and therefore, it may be hydrolyzed faster via the transition state than the new artificial substrate. This may explain why p-Nph-5′-TMP showed a significantly higher k_cat-value than p-Nph-5′-AMP.

Several previous studies had indicated that the potency of enzyme inhibitors may be dependent on the substrate used for testing (Michaud et al., 1997; Hosoda et al., 1999; Schiemann et al., 2012; Ben Henda et al., 2013; Chang et al., 2014; Lee and Müller, 2014). Therefore, we decided to investigate a selection of standard NPP1 inhibitors vs. three different substrates: six nucleotidic inhibitors, dialADP (1), dialATP (2), α,β-metADP (3), α,β-metATP (4), 2-MeSADP (5) and 2-MeSATP (6), and five non-nucleotidic inhibitors, SAR 03004 (12), reactive blue 2 (13), suramin (14), PZB08513136A (15), and [TiW₁₁CoO₄₀]⁸⁻ (16) (for structures see Figures 2, 3). The concentration-inhibition curves of the test compounds vs. the natural substrate ATP and the two artificial substrates p-Nph-5′-TMP and p-Nph-5′-AMP are presented in Figure 6. Table 1 displays the (α)K_i-values and the determined inhibition types of the NPP1 inhibitors. Six of the investigated inhibitors displayed a competitive mechanism of NPP1 inhibition (also see Figure S2 in Supporting Information). As expected, this was independent of the structure of the substrate and could be confirmed for all three investigated substrates. [TiW₁₁CoO₄₀]⁸⁻, reactive blue 2 and dialADP inhibited the enzymatic activity in a non-competitive manner, while suramin and dialATP were characterized as un-competitive inhibitors vs. all investigated substrates. Differences in inhibitory potential with respect to structure of inhibitor also exist. Among the investigated competitive inhibitors, the quinazoline sulfonamide derivative SAR 03004 (12) was found to be the most potent compound vs. ATP and also vs. p-Nph-5′-AMP with K_i-values of 0.215 and 0.420 μM, respectively. To the contrary, when p-Nph-5′-TMP was employed as a substrate, the thioacetamide derivative PZB08513136A (15) was found to be the most potent competitive inhibitor with a K_i-value of 0.00500 μM. Among the investigated non-competitive inhibitors, [TiW₁₁CoO₄₀]⁸⁻ (16) was the most potent compound vs. all investigated substrates (K_i-values: 0.00146 μM vs. ATP, 0.00199 μM vs. p-Nph-5′-TMP and 0.00174 μM vs. p-Nph-5′-AMP). In the group of un-competitive inhibitors, suramin (14) was found to be the most potent compound vs. all investigated substrates (K_i-values: 0.780 μM vs. ATP, 1.07 μM vs. p-Nph-5′-TMP and 1.03 μM vs. p-Nph-5′-AMP). Among the adenosine analogs and derivatives, the two dialdehyde compounds (dialADP; non-competitive inhibitor, dialATP; un-competitive inhibitor) displayed NPP1-inhibitory activities with K_i-values around 5 μM vs. all substrates. The other investigated adenine nucleotide derivatives and analogs were mostly less active vs. the three different substrates (K_i-values: 13.0–32.8 μM vs. ATP, 1.28–4.47 μM vs. p-Nph-5′-TMP and 8.19–39.9 μM vs. p-Nph-5′-AMP).

Inhibitory potencies of non-nucleotidic and nucleotidic inhibitors vs. different substrates were compared (Figure 7). Data analysis revealed substrate-dependent inhibitory potencies of competitive inhibitors, but not of non- or uncompetitive inhibitors. When K_i-values obtained with the natural substrate ATP were compared with those obtained with the artificial substrate p-Nph-5′-TMP the competitive inhibitors were 3–3600-fold more potent against p-Nph-5′-TMP than vs. ATP [p < 0.05 for SAR 03004 (12), p < 0.01 for α,β-metATP (4) and 2-MeSATP (6), and p < 0.001 for α,β-metADP (3), 2-MeSADP (5) and PZB08513136A (15)]. Differences were also dependent on the structure of the competitive antagonists, e.g., it was particularly high for the thioacetamide derivative PZB08513136A (15), but less pronounced for the quinazoline derivative SAR 03004 (12). In contrast, results obtained vs. the new artificial substrate p-Nph-5′-AMP were similar to those obtained vs. the natural substrate ATP. As opposed to competitive inhibitors, substrate-dependent inhibition was not observed for non-competitive and un-competitive inhibitors, and very similar inhibition constants were obtained vs. all investigated substrates.

Correlation analyses of pK_i-values obtained vs. one substrate with those measured vs. another substrate were performed. Considering the competitive inhibitors, a low correlation of data obtained with p-Nph-5′-TMP as a substrate with those obtained with the natural substrate ATP was obtained [correlation coefficient (R²) = 0.5722, see Figure 8A], whereas a high correlation between the results obtained with p-Nph-5′-AMP as a substrate and those determined with ATP was observed (R² = 0.9578). Moreover, Figure 8A (left) showed that the data points were shifted to the right of the ideal correlation line [dotted line in Figure 8A (left)]. This indicates that the competitive NPP1 inhibitors were generally more potent vs. p-Nph-5′-TMP than vs. ATP as a substrate. Contrary to this, the non- and un-competitive inhibitors showed high correlations, no matter which substrates were used for comparison (R² = 0.9742 for competitive inhibitors; R² = 0.9900 for non- and un-competitive inhibitors), see Figure 8B.

The observation of significantly different potencies of competitive enzyme inhibitors when determined vs. different substrates is puzzling, and an explanation for this phenomenon is not straightforward. The different assay conditions are clearly not the reason for the observed discrepancies because the same operating conditions (e.g., same stock solutions of inhibitors, same assay buffer) were applied for the enzyme inhibition assays with different substrates. A rational explanation for the different results between assays obtained with p-Nph-5′-TMP and the natural substrate ATP could be an allosteric modulatory effect by p-Nph-5′-TMP on the enzyme, in addition to acting as a substrate (Figure 9). Such allosteric binding of the substrate has previously been reported for another nucleotide-metabolizing enzyme, bacterial UDP-N-acetylglucosamine 2-epimerase, which is allosterically modulated by its substrate UDP-N-acetylglucosamine (Velloso et al., 2008). The binding of p-Nph-5′-TMP to its allosteric binding site, which may be close or even distant from the active site, could induce a conformational change of the substrate binding site. This would modulate the interaction of competitive inhibitors with the substrate binding site, and could therefore explain the increased affinity of the investigated competitive inhibitors (Figure 9). This hypothesis is supported by the fact that the affinity increase depends on the structure of the inhibitors, e.g. some competitive inhibitors (e.g., 15) being much more strongly affected than others (see also Table 1 and Figure 9).

This hypothesis also provides a straightforward explanation for the finding that p-Nph-5′-TMP is a much better NPP1 substrate than p-Nph-5′-AMP despite the fact that—based on docking studies—the AMP derivative should have stronger interactions with the substrate binding site. p-Nph-5′-TMP may additionally bind to an allosteric site and thereby act as a positive allosteric modulator which increases its binding affinity to the substrate binding site and accelerates its hydrolysis.

Further, investigations to corroborate this hypothesis of allosteric modulation of the active site by p-Nph-5′-TMP are warranted.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.