Identification of Novel d-Aspartate Oxidase Inhibitors by in Silico

Article pubs.acs.org/jmc

Identification of Novel D‑Aspartate Oxidase Inhibitors by in Silico Screening and Their Functional and Structural Characterization in Vitro Masumi Katane,† Shota Yamada,† Go Kawaguchi,† Mana Chinen,† Maya Matsumura,† Takemi Ando,† Issei Doi,‡ Kazuki Nakayama,† Yuusuke Kaneko,† Satsuki Matsuda,† Yasuaki Saitoh,† Tetsuya Miyamoto,† Masae Sekine,† Noriyuki Yamaotsu,‡ Shuichi Hirono,‡ and Hiroshi Homma*,† Downloaded by UNIV OF PRINCE EDWARD ISLAND on September 6, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jmedchem.5b00871

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Laboratory of Biomolecular Science, Graduate School of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan ‡ Laboratory of Physical Chemistry for Drug Design, Graduate School of Pharmaceutical and Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan S Supporting Information *

ABSTRACT: D-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for acidic D-amino acids, including D-aspartate, a potential agonist of the N-methyl-D-aspartate (NMDA) receptor. Dysfunction of NMDA receptor-mediated neurotransmission has been implicated in the onset of various mental disorders, such as schizophrenia. Hence, a DDO inhibitor that increases the brain levels of D-aspartate and thereby activates NMDA receptor function is expected to be a useful compound. To search for potent DDO inhibitor(s), a large number of compounds were screened in silico, and several compounds were identified as candidates. They were then characterized and evaluated as novel DDO inhibitors in vitro (e.g., the inhibitor constant value of 5-aminonicotinic acid for human DDO was 3.80 μM). The present results indicate that some of these compounds may serve as lead compounds for the development of a clinically useful DDO inhibitor and as active site probes to elucidate the structure−function relationships of DDO.

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INTRODUCTION Among the free D-amino acids found in mammals, D-serine (DSer) and D-aspartate (D-Asp) have been studied most extensively. D-Ser persists at high concentrations throughout the life of an animal and is concentrated predominantly in the mammalian forebrain. D-Ser binds to the glycine-binding site of the N-methyl-D-Asp (NMDA) receptor, a subtype of the Lglutamate (L-Glu) receptor family, and potentiates glutamatergic neurotransmission in the central nervous system.1,2 Astroglia- and/or neuron-derived D-Ser regulates NMDA receptor-dependent long-term potentiation and/or depression in hypothalamic and hippocampal excitatory synapses.3−5 These lines of evidence suggest that D-Ser plays an important role in the regulation of brain function by acting as a coagonist for the NMDA receptor. Indeed, perturbation of D-Ser levels in the nervous system was recently implicated in the pathophysiology of various neuropsychiatric disorders, including schizophrenia,6−9 Alzheimer’s disease,10,11 and amyotrophic lateral sclerosis.12,13 Unlike the tissue-specific expression of D-Ser, substantial amounts of free D-Asp are present in a wide variety of mammalian tissues and cells, particularly those of the central © XXXX American Chemical Society

nervous, neuroendocrine, and endocrine systems. Several lines of evidence suggest that D-Asp plays an important role in regulating developmental processes, hormone secretion, and steroidogenesis.14−16 D-Asp levels in human seminal plasma and spermatozoa are significantly lower in oligoasthenoteratospermic and azoospermic donors than in normospermic donors.17 In female patients undergoing in vitro fertilization, the D-Asp content of preovulatory follicular fluid is lower in older patients than in younger patients; this decrease in D-Asp content appears to reflect a reduction in oocyte quality and fertilization competence.18 Overall, current evidence suggests that decreased D -Asp levels may play a role in the pathophysiology of infertility. D-Asp stimulates the NMDA receptor by acting as an agonist that binds to the L-Glu-binding site of the receptor.19,20 Recent studies suggested that, similar to D-Ser, D-Asp acts as a signaling molecule in nervous and neuroendocrine systems, at least in part, by binding to the NMDA receptor, and plays an important role in the regulation of brain functions.15,16,21 This was supported by a recent report Received: May 1, 2015

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DOI: 10.1021/acs.jmedchem.5b00871 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Downloaded by UNIV OF PRINCE EDWARD ISLAND on September 6, 2015 | http://pubs.acs.org Publication Date (Web): September 4, 2015 | doi: 10.1021/acs.jmedchem.5b00871

Table 1. IC50 Values of Candidate Compounds against Recombinant Human DDO and DAO

a

Data are shown as the mean ± standard deviation of three to four independent assays. bData are from Katane et al. (2015).38

that showed that D-Asp levels in the prefrontal cortex and striatum of post-mortem brains of schizophrenic patients are

significantly lower than those of nonpsychiatrically ill individuals.22 B




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their potential value as active site probes to elucidate the structure−function relationships of DDO was demonstrated.

In mammalian tissues, two types of degradative enzymes that are stereospecific for D-amino acids have been identified, namely, D-amino acid oxidase (DAO, also abbreviated as DAAO; EC 1.4.3.3) and D-Asp oxidase (DDO, also abbreviated as DASPO; EC 1.4.3.1). DAO and DDO are flavin adenine dinucleotide (FAD)-containing flavoproteins that catalyze the oxidative deamination of D-amino acids to generate 2-oxo acids along with hydrogen peroxide and ammonia.23 DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids, including D-Ser. On the other hand, DDO is highly specific for acidic D-amino acids, such as D-Asp and DGlu, none of which are substrates of DAO.24 DAO and DDO have both been identified in various organisms, and their physiological roles in vivo are being investigated extensively. In mammals, DAO and DDO activities are highest in the kidney, followed by the liver and brain, and are low in other peripheral tissues. DAO and DDO localize to the peroxisome,25−27 where catalase degrades toxic hydrogen peroxide, one of the enzymatic reaction products. The mammalian DAO and DDO are presumed to regulate the levels of several endogenous and exogenous D-amino acids, including D-Ser and D-Asp, in various organs;28,29 however, their physiological roles in vivo remain to be fully clarified. Reduced levels of D-Asp in the nervous system and the resulting dysfunction of NMDA receptor-mediated neurotransmission are thought to occur during the onset of various mental disorders, including schizophrenia;15,16,21,22 hence, a substance capable of increasing D-Asp levels and activating NMDA receptor function may provide a novel foundation for the development of antipsychotic drugs. One way to increase the level of D-Asp is to prevent its metabolic degradation by DDO. Indeed, DDO-deficient mice have elevated concentrations of D-Asp in several brain regions and exhibit specific behaviors suggestive of potential antidepressant and antischizophrenic activities.30,31 In addition, DDO mRNA expression is increased in the prefrontal cortex of post-mortem brains of schizophrenic patients compared with that of nonpsychiatrically ill individuals,32 which is consistent with the decreased D-Asp levels in this tissue, as described above.22 These findings support the concept that DDO inhibitors that activate NMDA receptor function by augmenting the levels of D-Asp in the brain would be new and useful lead compounds for drug discovery for the treatment of NMDA receptor-related diseases. DDO inhibitors may also be lead compounds for the development of new drugs to treat infertility, since D-Asp is thought to be involved in the quality control of germ cells.17,18 Overall, human DDO is an attractive therapeutic target, and several compounds that inhibit the activity of mammalian DDO in vitro have been identified to date; however, the inhibitory potency of these compounds is limited.33−38 The main purpose of the present study was to identify novel, potential DDO inhibitor(s). Previously, we identified a number of novel DAO inhibitors based on in silico screening of many compounds listed in the chemical structure database.39 In the present study, a similar approach was used to search for novel DDO inhibitor(s). Namely, a computer ligand-docking method was used to screen a large number of compounds in silico for their ability to inhibit human DDO, and several compounds were identified as candidates. Characterization and in vitro evaluation of these compounds as novel DDO inhibitors indicated that some of them could be lead compounds for the development of a clinically useful DDO inhibitor. In addition,

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RESULTS AND DISCUSSION In Silico Screening for Compounds That Inhibit the Activity of Human DDO. Approximately 4 million compounds available commercially were screened in silico for their binding to the active site of human DDO (the procedural details of the screening are described in the Experimental Section). As a result of the screening, ten compounds (1−10) were identified as candidate inhibitors. Chemical structures and chemical information on these compounds are listed in Table 1 and Supporting Information Table S1, respectively. Subsequently, these compounds were characterized in great detail and evaluated as novel DDO inhibitors, as described in the following sections. Inhibitory Activities of the Candidate Compounds. The dose-dependent effects of the ten candidate compounds on the enzymatic activities of recombinant human DDO and DAO were examined. Several compounds inhibited the activity of DDO against D-Asp in a dose-dependent manner (Figure 1), and the values for the 50% inhibitory concentration (IC50) of three compounds (5-aminonicotinic acid [compound 2], 2amino-5-methylthiophene-3-carboxylic acid [compound 8], and 7-hydroxy-4-hydro-1,2,4-triazolo[4,3-a]pyrimidine-6-carboxylic acid [compound 9]) were determined (Table 1 and Supporting Information Table S2). Compounds 2 and 9 inhibited the activity of DDO more efficiently than malonic acid (compound 11), which reportedly inhibits mammalian DDO by competing with its substrates.34,36 On the other hand, 2 and 9 failed to inhibit the activity of DAO against D-Ala, while benzoic acid 12, which reportedly inhibits mammalian DAO by competing with its substrates,39,40 efficiently inhibited the activity of DAO in a dose-dependent manner, as expected. In contrast to 2 and 9, 8 also inhibited DAO activity; however, the inhibitory activity of 8 was relatively weaker than that of 12 (Table 1 and Supporting Information Table S2). Thus, these results indicated that 2 and 9 are efficient inhibitors of DDO activity and that these compounds are selective for DDO but not for DAO. Inhibitory Compounds 2 and 9. Compounds 2 and 9 were selected for further study because of their relatively high inhibitory activity against DDO, but not DAO (Table 1 and Supporting Information Table S2). The effects of these two compounds on the flavin absorbance spectrum of recombinant human DDO were examined. The effects of 11 and 12 were also tested as positive and negative control inhibitors, respectively. The addition of 11 perturbed the absorption spectrum of the enzyme (data not shown), and these spectral changes were also observed when the enzyme was mixed with 2 and 9 (Figure 2A), confirming the direct binding of these compounds to the enzyme. By contrast, the addition of 12 had no effect (data not shown). The types of inhibition of 2 and 9 against DDO were examined, and 11 was tested as a control inhibitor. Specifically, 1/v versus 1/[D-Asp] plots, which show the apparent kinetic parameters (Michaelis constant [Km] and maximal velocity [Vmax]) for D-Asp, were generated in the absence or presence of these compounds.41 Compound 11 showed a competitive type of inhibition (data not shown), similar to that observed for 2 and 9 (Figure 2B and C). Taken together, these results indicated that 2 and 9 are novel, active site-directed DDO inhibitors. C




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Figure 2. Types of inhibition of DDO by compounds 2 and 9. (A) Absorption spectra of human DDO in the absence or presence of the compounds. Wavelength scans were carried out at room temperature with recombinant human DDO (1.5 μg/μL) in 10 mM sodium pyrophosphate buffer (pH 8.3) containing 2 mM EDTA, 5 mM 2mercaptoethanol, and 10% (v/v) glycerol, before (black line) and after the addition of compounds to a final concentration of 1000 μM (2; red line) or 200 μM (9; blue line). The scan range was 300 to 600 nm in the absence of compound, 365 to 600 nm in the presence of 2, and 330 to 600 nm in the presence of 9. (B and C) 1/v versus 1/[S] plots of the enzymatic activity of human DDO in the absence or presence of the compounds. Enzymatic activities on D-Asp were assayed using recombinant human DDO in the absence or presence of 2 (B) and 9 (C). The compounds were used at the indicated final concentrations. The Vmax and Km values for D-Asp were determined, and the lines in the figures were drawn by fitting these kinetic parameters to the Lineweaver−Burk equation.41 Data are representative of three independent experiments, and similar results were obtained in all experiments.

Figure 1. Dose-dependent effects of several compounds on the enzymatic activity of human DDO. Enzymatic activities against D-Asp were assayed using recombinant human DDO in the absence or presence of compounds 2 (A), 8 (B), and 9 (C). The activities of the enzyme in the presence of compounds are presented as a percentage of its activities in the absence of compounds. Data are shown as the mean ± standard deviation of three independent assays. Where not shown, the error bars are smaller than the symbols used.

Binding Affinity of Compounds 2 and 9 for Human and Rodent DDOs. Preclinical studies using experimental rodents, such as rats and mice, are effective methods of evaluating the in vivo effects of DDO inhibitors; however, species-related differences in the properties of human and rodent DDOs may affect these studies and have a significant impact on the development of effective inhibitors. In our recent report,38 predicted three-dimensional (3D) structural models of human, rat, and mouse DDOs were generated and compared, which showed that the differences between these proteins occur in regions involved in the migration of the substrate/product into and out of the active site. These structural differences may be reflected by the distinct inhibitor binding property of human DDO compared with its rat and mouse homologues. Indeed, the inhibitor constant (Ki) values of 11 for recombinant human, rat, and mouse DDOs differed significantly from one another (P < 0.05 for rat versus mouse, and P < 0.001 for human versus rat and human versus mouse, based on a Tukey− Kramer multiple comparison test) (Table 2).38

Table 2. Ki Values of Several Inhibitors against Recombinant Human, Rat, and Mouse DDO Ki (μM)b Inhibitor a

11 2 9

Human DDO

Rat DDO

Mouse DDO

153 ± 26 3.80 ± 0.96 15.1 ± 2.8

1562 ± 120 5.89 ± 0.61 65.9 ± 8.0

1220 ± 133 8.69 ± 0.72 32.6 ± 2.9

Data are from Katane et al. (2015).38 bData are shown as the mean ± standard deviation of three to four independent assays. a

D




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The differences in the binding to 2 and 9 between human, rat, and mouse DDOs were examined. Compounds 2 and 9 both inhibited the enzymatic activities of recombinant rat and mouse DDOs on D-Asp in a dose-dependent manner, and the IC50 values of these compounds were determined (data not shown). Using the IC50 values and the previously determined Km values toward D-Asp,38 the Ki values of these compounds for human, rat, and mouse DDOs were calculated (Table 2).42 The Ki values of 2 and 9 for all the enzymes tested were markedly lower than those previously reported for 11, confirming relatively high levels of inhibition of DDO by 2 and 9. The lowest Ki value of 2 was obtained for human DDO, followed by rat DDO and then mouse DDO. The differences between these Ki values were significant (P < 0.05 for human versus rat and rat versus mouse, and P < 0.001 for human versus mouse, Tukey− Kramer test). On the other hand, the lowest Ki value of 9 was obtained for human DDO, followed by mouse DDO and then rat DDO. The differences between these Ki values were also significant (P < 0.05 for human versus mouse, and P < 0.001 for human versus rat and rat versus mouse, Tukey−Kramer test). These results suggested that there are species-related differences in the binding to 2 and 9, as well as in the binding to 11, between human and rodent DDOs. From a pharmacological point of view, the distinct inhibitor binding properties of human DDO in relation to those of its rodent counterparts may need to be considered when the in vivo effects of these compounds are evaluated using rats or mice. Recently, it was reported that human DAO also differs from the rat counterpart in the affinity for the binding to several DAO inhibitors.43 Inhibitory Activities of Compounds 2 and 9 against Human DDO Expressed in Cultured Mammalian Cells. To assess whether 2 and 9 inhibit the enzymatic activity of human DDO expressed in mammalian cells, human cervical adenocarcinoma HeLa cells were stably transfected with the Nterminally HA-tagged human DDO expression plasmid. The DDO-overexpressing mammalian cell line HeLa.HA-hDDO-16 was established. The HeLa/NEO cell line was also established and used as a control cell line. This cell line was derived from HeLa cells after stable transfection with the parental plasmid and was not forced to express DDO. Our recent report demonstrated that intracellular biosynthesis of D-Asp occurs in HeLa cells.44 Indeed, during the culture of the HeLa/NEO cells, substantial amounts of D-Asp were detected in this cell line (Figure 3A). D-Asp was also detected in the HeLa.HAhDDO-16 cells, but the D-Asp levels in this cell line were remarkably lower than those in the HeLa/NEO cells. These results suggested that D-Asp synthesized in the HeLa.HAhDDO-16 cells is efficiently degraded by DDO expressed in this cell line. The effects of 11, 2, and 9 on D-Asp levels in the HeLa/NEO and HeLa.HA-hDDO-16 cells were examined. Specifically, the cells were treated with 11, 2, or 9 for 24 h, and the concentrations of D- and L-Asp in the cells were examined. On the basis of preliminary experiments, 11, 2, and 9 were used at concentrations of 1000, 250, and 100 μM, respectively, to exclude direct cytotoxicity. Treatment of HeLa/NEO cells with 11 and 9 had no significant effect on D-Asp levels in this cell line (Figure 3A). On the other hand, the D-Asp content of HeLa/NEO cells treated with 2 was higher than that of vehicletreated cells, but this difference was not significant. The tendency for the increase in D-Asp content in HeLa/NEO cells treated with 2 was presumably due to the inhibitory effect of 2 on the enzymatic activity of endogenously expressed DDO,

Figure 3. Effects of the DDO inhibitors on intracellular D-Asp levels in cultured mammalian cells. HeLa/NEO and HeLa.HA-hDDO-16 cells (1 × 106 cells) were seeded in 6-well plates and cultured in appropriate media. On the next day, the media were replaced with fresh media containing the respective DDO inhibitors (compound 11, 2, or 9) or vehicle, and the cells were cultured for a further 24 h. Amino acids were then extracted from the cells. The concentrations of D-Asp (A) and L-Asp (B) in the cells were determined by HPLC. Data are shown as the mean ± standard deviation of three to four independent assays. The triple asterisk indicates a significant difference (P < 0.001) compared to the vehicle-treated cells, based on the Dunnett’s multiple comparison test.

since detectable amounts of DDO mRNA were observed in the parental HeLa cells via real-time polymerase chain reaction in our previous study.44 Treatment of HeLa.HA-hDDO-16 cells with 11 and 9 had no significant effect on D-Asp levels (Figure 3A), whereas treatment with 2 significantly increased D-Asp levels in this cell line. The L-Asp levels in the HeLa/NEO and HeLa.HA-hDDO-16 cells were essentially unchanged by treatment with any compound (Figure 3B). These results suggested that 2 crosses the cell membrane and reaches DDO in the peroxisome, resulting in the effective inhibition of the enzymatic activity of DDO expressed in the cells. It should be noted that the L-Asp levels in the HeLa.HAhDDO-16 cells were relatively lower than those in the HeLa/ NEO cells (Figure 3B). The molecular mechanism underlying the decrease in the L-Asp content by the overexpression of DDO is currently unclear. However, the biosynthesis of D-Asp was observed in HeLa cells,44 as described above, although the enzyme responsible for the synthesis of D-Asp in human cells remains to be identified. A likely candidate for this enzyme is Asp-specific amino acid racemase, which converts L-Asp to DAsp; in fact, Asp racemase (EC 5.1.1.13) of animal origin has been identified in the bivalve Scapharca broughtonii and the sea slug Aplysia californica.45−47 In HeLa.HA-hDDO-16 cells, where intracellular D-Asp is actively degraded by DDO expressed in the cells, the L-Asp substrate may be used as a source for D-Asp biosynthesis to regulate D-Asp homeostasis in the cells, resulting in a decrease in the L-Asp content. Identification of the enzyme(s) involved in the synthesis of D-Asp in mammals will enhance our understanding of the mechanism that regulates the D-Asp content in cells. E



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Structural Insights into the Binding of Compound 2 to DDO. To investigate the interaction of 2 with DDO, a structural model of human DDO complexed with 2 was generated (the procedural details of the computational modeling are described in the Experimental Section) (Figure 4A). In this model, the carboxy and amino groups of 2 interact


is consistent with the results of the kinetic analysis described above, where 2 was shown to be an active site-directed DDO inhibitor that competes with the substrate for binding to the active site (Figure 2B). To assess the validity of the proposed model, the inhibitory activities of derivatives of 2 against human DDO and DAO were examined. The derivative (13) lacks the carboxy group contained in 2, and the three other derivatives (14−16) have positionally altered carboxy groups. All four compounds failed to inhibit the activity of DDO against D-Asp or that of DAO against D-Ala (Table 3 and Supporting Information Table S2). The results suggest that the carboxy group of 2 is important for its interaction with the active site of DDO. Subsequently, the inhibitory activities of other derivatives of 2 against human DDO and DAO were examined to determine whether the amino group of 2 is also involved in its interaction with the active site of DDO. The derivative (17) lacks the amino group contained in 2, and the other three derivatives (18−20) have positionally altered amino groups. Compounds 17 and 20 inhibited DDO activity, whereas 18 and 19 failed to inhibit DDO; however, the inhibitory activities of 17 and 20 were significantly weaker than that of 2 (Table 4 and Supporting Information Table S2). Compound 17 also inhibited DAO activity, while 18, 19, and 20 failed to inhibit DAO. The inhibition of human DAO by 17 is consistent with previous reports showing the inhibition of pig and sheep DAO activities by this compound.48,49 The inhibitory activities of 13 derivatives of 2 (21−33), in which the amino group of 2 is substituted by other groups, against human DDO and DAO were also examined. All the compounds, except for 21, 23, and 24, failed to inhibit DDO (Table 5 and Supporting Information Table S2). Compounds 21, 23, and 24 inhibited the activity of DDO, although their inhibitory activities were significantly weaker than that of 2. Compound 24 also inhibited DAO, whereas 21 and 23 failed to inhibit DAO activity. Collectively, these results suggest that, in addition to the carboxy group, the amino group of 2 is an important determinant of its interaction with the active site of DDO. In the model of DDO complexed with 2 (Figure 4B), no hydrogen bonding interaction was observed between the pyridine nitrogen of 2 and any amino acid residue of DDO. To investigate whether the pyridine nitrogen of 2 is involved in its binding to the active site of DDO, the inhibitory activities of four derivatives of 2 (34−37) were also examined. The derivative (34) lacks the pyridine nitrogen contained in 2, and the three other derivatives (35−37) have positionally altered pyridine nitrogens. Unexpectedly, all the compounds failed to inhibit the activity of DDO (Table 6 and Supporting Information Table S2). Compounds 35, 36, and 37 also failed to inhibit DAO, while 34 inhibited DAO activity. The inhibition of human DAO by 34 is consistent with a previous report showing the inhibition of pig DAO activity by this compound.50 These results suggest that, in addition to the carboxy and amino groups, the pyridine nitrogen of 2 is an important determinant of its interaction with the active site of DDO. The structure−activity relationships of 2 with respect to the pyridine nitrogen (Table 6) are incompatible with the aforementioned findings of the structural model. In the model of DDO complexed with 2 (Figure 4B), however, a space exists between the pyridine nitrogen of this compound and the side-chain guanidino group of Arg-216. Therefore, a water molecule may be located in this space and interact with

Figure 4. Structural model of human DDO complexed with compound 2. In the model with the lowest energy conformation, 2 is situated in the binding pocket of the active site of DDO (A). In this model, the carboxy and amino groups of 2 interact with the side-chain guanidino group of Arg-278 and the backbone carbonyl of Ser-308, respectively (B). The carbon atoms in FAD, 2, and side chains of amino acid residues are colored yellow, cyan, and green, respectively. Other atoms are colored as follows: nitrogen, blue; oxygen, red; and sulfur, brown. Black dotted lines denote possible hydrogen bonds.

with the side-chain guanidino group of Arg-278 and the backbone carbonyl of Ser-308, respectively (Figure 4B). This model predicted that 2 binds to the active site of DDO through the above-mentioned interactions and thereby interferes with the proper orientation of the substrate in the active site, resulting in the inhibition of enzymatic activity. This prediction F



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Table 3. Effect of Alterations of the Carboxy Group of Compound 2 on Its Inhibitory Activity against Human DDO and DAO

a

Data are shown as the mean ± standard deviation of three independent assays.

hydroxyquinolin-2(1H)-one against DDO is relatively stronger than that of 2. Determination of the crystal structure of DDO complexed with 3-hydroxyquinolin-2(1H)-one will provide a clue to improve the inhibitory activity of 2 against DDO.

the pyridine nitrogen of 2 and with the side-chain guanidino group of Arg-216 to form the hydrogen bond. The pyridine nitrogen of 2 presumably contributes to binding to the active site of DDO through indirect interaction with the side-chain guanidino group of Arg-216 via the water molecule. Alternatively, since the side chain of the Arg residue is highly flexible, it is possible that the side-chain guanidino group of Arg-216 is in fact located in the vicinity of 2 bound to the active site of DDO and interacts directly with the pyridine nitrogen of this compound to form the hydrogen bond. Molecular dynamics simulations should help evaluate whether the sidechain guanidino group of Arg-216 can interact directly with the pyridine nitrogen of 2. Furthermore, determination of the crystal structure of DDO complexed with 2 may be necessary for a full understanding of the mechanics of binding of this compound to DDO. Several compounds have been recently reported as novel DAO inhibitors, including 3-hydroxyquinolin-2(1H)-one.51 Notably, 3-hydroxyquinolin-2(1H)-one also inhibited the enzymatic activity of human DDO, and the IC50 value of this compound against human DDO was reported to be 0.855 μM under conditions where the final concentration of the substrate 51 D-Asp in the assay is 0.25 mM. Using the IC50 value and the previously determined Km value toward D-Asp,38 the Ki value of this compound for human DDO was calculated to be 0.764 μM.42 Since the Ki value of 2 for human DDO was 3.80 ± 0.96 μM (Table 2), it appears that the inhibitory activity of 3-

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CONCLUSION Several compounds were identified in the present study as novel DDO inhibitors based on in silico screening. Among them, 2 and 9 have the potential to serve as active site probes to elucidate the structure−function relationships of DDO. These compounds could also be useful lead compounds for drug discovery because of their relatively low molecular weight. The chemical modification of specific moieties in these compounds to fill the space between the compounds and the enzyme, and to raise their affinity for the active site of the enzyme, would be an effective approach for improving the inhibitory activity of these compounds against DDO.

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EXPERIMENTAL SECTION

Chemicals. D-Asp, L-Asp, D-Ala, bovine serum albumin, catalase from Aspergillus niger, and G418 were purchased from Sigma-Aldrich (St. Louis, MO, USA). FAD, malonic acid (11), benzoic acid (12), and o-phthalaldehyde (OPA) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from Nacalai Tesque (Kyoto, Japan). Fetal bovine serum and Boc-L-cysteine were purchased from Gibco-BRL (Gaithersburg, MD, USA) and Novabiochem (Läufelfingen, Switzerland), respectively. The comG



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Table 4. Effect of Alterations of the Amino Group of Compound 2 on Its Inhibitory Activity against Human DDO and DAO

a

Data are shown as the mean ± standard deviation of three independent assays.

pounds selected by in silico screening and the derivatives of 2 were purchased from 14 vendors: 1, 3, 16, 18, 19, 20, and 34 were from Wako Pure Chemical Industries, Ltd.; 2, 4, 5, 6, 13, 23, 26, 27, and 29 were from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); 7 was from Labotest (Niederschöna, Germany); 8, 35, and 37 were from Vitas-M Laboratory, Ltd. (Moscow, Russia); 9 was from Matrix Scientific (Columbia, SC, USA); 10 was from Enamin Ltd. (Kiev, Ukraine); 14 and 32 were from Apollo Scientific, Ltd. (Cheshire, UK); 15 was from Ark Pharm, Inc. (Libertyville, IL, USA); 17 was from Nacalai Tesque; 21 and 22 were from Life Chemicals Inc. (Burlington, Canada); 24, 25, 28, and 31 were from Sigma-Aldrich; 30 was from Supelco (Bellefonte, PA, USA); 33 was from J&W PharmLab, LLC. (Levittown, PA, USA); and 36 was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). All these compounds were filtered for Pan Assay Interference Compounds (PAINS)52 and passed the filter. The purity of all eight experimentally validated hits (2, 8, 9, 17, 20, 21, 23, and 24) was greater than 95% as verified using high-performance liquid chromatography (HPLC), titration, nuclear magnetic resonance (NMR), or gas chromatography by the vendors or in our hands (1H NMR spectra of all these compounds are depicted in Supporting Information Figure S1). All other chemicals used were of the highest grade available and were purchased from commercial sources. In Silico Screening of Compounds Inhibiting DDO Activity. Since the 3D structure of DDO has not been determined, structural models of human DDO were first generated using the homology modeling method with the Prime v21211 (Schrödinger Suite 2009; Schrödinger, LLC, New York, NY, USA). The 3D X-ray crystallographic structures of human DAO complexed with its respective ligands (PDB ID: 2DU8, 2E49, 2E4A, 3CUK, and 3G3E)51,53−55 were used as the template structures. The structural models obtained were

then subjected to 3−6 ns of molecular dynamics simulations at 310 K in the TIP3P box water using the Sander module of AMBER9.56 These simulations allowed the identification of five representative structures using the average-linkage clustering algorithm in the PTRAJ module of AMBER9.56 All the representative structures were prepared for docking simulation analysis using the Protein Preparation Wizard tool (Schrödinger Suite 2009; Schrödinger, LLC.). Docking grids were generated around the Arg-216, Tyr-223, Arg-237, Arg-278, and Ser308 residues, which were previously identified as important for substrate binding and the catalytic activity of DDO,35,37,57 using the Glide Grid Generation v55212 (Schrödinger Suite 2009; Schrödinger, LLC.). The commercially available 4 million compounds listed in the chemical structure database of Namiki Shoji Co. (http://www.namikis.co.jp/english/) (Namiki HTS integrated database in SDFile format that was obtained through the user registration process [e-mail: info@ namiki-s.co.jp]) were screened in silico for their chemicostructural similarities to three compounds (malonic acid [11], benzoic acid [12], and 3-hydroxyquinolin-2(1H)-one). These three compounds were reported to inhibit the enzymatic activity of mammalian DDO and/or DAO, although their relative inhibitory activities significantly differ from one another.15,34−36,38,39,51 The Topomer Search module in the SYBYL 8.1 (Tripos, Inc., St. Louis, MO, USA) was used for the screen, and 2499 compounds that exhibited good similarity scores were selected as first-hit compounds. Subsequently, the energy minimized 3D molecular structures of these first-hit compounds were generated using the LigPrep v23212 (Schrödinger Suite 2009; Schrödinger, LLC.). Docking of the resulting 3D structures of the compounds to the target human DDO proteins (prepared as described above) was achieved using the Standard Precision (SP) Glide v55212 H



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(Schrödinger Suite 2009; Schrödinger, LLC). The best-docked pose with the lowest energy conformation was selected for each compound, and 1050 compounds whose docking scores were equal to or less than −7.0 were selected as second-hit compounds. Of these second-hit compounds, 192 that passed the filter to remove the compounds having reactive functional groups, such as aldehyde and acyl halide groups, using the Ligfilter (Schrödinger Suite 2009; Schrödinger, LLC) and were presumed to form the hydrogen bonds with the sidechain guanidino group of Arg-278 and the backbone carbonyl of Ser308 residues of DDO were selected as third-hit compounds. These third-hit compounds were then subjected to cluster analysis using Canvas v12212 (Schrödinger Suite 2009; Schrödinger, LLC), and 38 compounds were selected further as fourth-hit compounds. Finally, ten compounds (1−10) were selected among these fourth-hit compounds by means of visual inspection (selection of the compounds presumed to form two hydrogen bonds with the side-chain guanidino group of Arg-278 residue of DDO) and/or availability and were used to test the inhibitory activity against DDO in this study. Construction of Recombinant Protein Expression Plasmids. The construction of expression plasmids containing N-terminally Histagged human DDO, rat DDO, mouse DDO, and human DAO (pRSET-His-hDDO, pRSET-His-rDDO, pRSET-His-mDASPO, and pRSET-His-hDAO, respectively) has been described previously.35,36,38,58 The construction of the plasmid pT7-hDDO, in which the cDNA fragment corresponding to the entire human DDO-coding sequence was cloned into pT7Blue (Novagen, Madison, WI, USA), has been described previously.36,58 Using pT7-hDDO as a template, the human DDO cDNA was amplified by a polymerase chain reaction. The

Table 5. Effect of Replacement of the Amino Group of Compound 2 by Other Groups on Its Inhibitory Activity against Human DDO and DAO


IC50 (μM)a Compound

R

Human DDO

Human DAO

2 21 22 23 24 25 26 27 28 29 30 31 32 33

NH2 NH(CH3) N(CH3)2 OH CH3 OCH3 F Cl Br COOH NO2 CF3 C6H5 SO3H

21.9 ± 5.6 3541 ± 217 >1000 3351 ± 293 5472 ± 977 >10 000 >10 000 >10 000 >10 000 >10 000 >10 000 >10 000 >5000 >10 000

>2500 >10 000 NDb >10 000 1499 ± 319 ND ND ND ND ND ND ND ND ND

a Data are shown as the mean ± standard deviation of three independent assays. bNot determined.

Table 6. Effect of Alterations in the Pyridine Nitrogen of Compound 2 on Its Inhibitory Activity against Human DDO and DAO

a

Data are shown as the mean ± standard deviation of three to four independent assays. I




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com). The Ki values of several compounds were determined using the following formula described by Cheng and Prusoff (1973):42 Ki = IC50/(1 + [S]/Km), where [S] is the substrate concentration. These Ki values determined were confirmed by replots of the slope of the Lineweaver−Burk plot versus inhibitor concentration.41 Absorbance Spectrum of DDO. A plate reader (PowerWave XS, Bio-Tek Instruments) was used to measure the absorption spectra of human DDO. Wavelength scans were carried out at room temperature with purified recombinant human DDO (1.5 μg/μL) in 10 mM sodium pyrophosphate buffer (pH 8.3) containing 2 mM EDTA, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, before and after the addition of compounds to a final concentration of 1000 μM (2, 11, and 12) or 200 μM (9). The spectra were recorded soon after compound addition. The scan range was 365−600 nm in the presence of 2, 330−600 nm in the presence of 9, and 300−600 nm in the presence of 11 and 12. In these ranges, the absorption spectrum of each compound did not interfere with the measurement of spectral changes of DDO. Cell Lines. Human cervical adenocarcinoma HeLa cells were cultured in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C in 5% CO2/95% air. For preparation of the DDO-overexpressing mammalian cell line, pcDNA-HA-hDDO was transfected into HeLa cells using the Agilent Mammalian Transfection Kit (Agilent Technologies, Santa Clara, CA, USA), according to the manufacturer’s instructions. The cells were selected for resistance to 900 μg/mL G418, and several drug-resistant cell clones were isolated. Lysates were prepared from the individual cell clones by sonication in 10 mM phosphate-buffered saline (pH 7.4) containing protease inhibitors (Nacalai Tesque) using a model 250 Sonifier (Branson Ultrasonics Co., Danbury, Connecticut) and centrifugation at 10 000 × g for 10 min at 4 °C to remove insoluble material. The cell lysates were then subjected to SDSpolyacrylamide gel electrophoresis and Western blot analysis using a mouse anti-HA antibody (Anti-HA [mouse IgG1-κ], Monoclonal [HA124]; Nacalai Tesque) (1:2500 dilution) as the primary antibody, and horseradish peroxidase-conjugated antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) (1:5000 dilution) as the secondary antibody. Because the most intensely positive band was detected in the sample prepared from clone 16, this cell line (HeLa.HA-hDDO-16) was used for further experiments. For preparation of a cell line resistant to G418 that does not overexpress DDO, HeLa cells were transfected with the parental plasmid pcDNA3.1(+) and selected for resistance to G418. Drugresistant cells were mixed as a stably transfected cell pool (HeLa/ NEO) and used as the control cell line in this study. HeLa.HA-hDDO16 and HeLa/NEO cells were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, 100 mg/ mL streptomycin, and 600 μg/mL G418 at 37 °C in 5% CO2/95% air. Determination of Amino Acid Contents in Cells. HeLa.HAhDDO-16 and HeLa/NEO cells (1 × 106 cells) were seeded in 6-well plates and cultured in 2 mL of medium. On the next day, the medium was replaced with 1.2 mL of fresh medium containing each compound (1000 μM 11, 250 μM 2, or 100 μM 9) or the vehicle (5% [v/v] dimethyl sulfoxide), and the cells were cultured for a further 24 h before extraction of amino acids. The amino acids in the cells were extracted using methanol, as described previously39,44,60 with some modifications. Specifically, after the culture media was removed, plated cells were washed twice with ice-cold 10 mM phosphate-buffered saline (pH 7.4), collected with a cell scraper, and then transferred into a 1.5 mL microtube. Subsequently, the cells were mixed with 600 μL of 100% (v/v) methanol, and the mixture was sonicated in a water bath for 10 min, followed by incubation at −80 °C for 1 h to extract the amino acids. The methanol extracts from the cells were centrifuged at 10 000 × g for 10 min at 4 °C to remove the precipitated proteins. The supernatant (500 μL) was then evaporated to dryness, and the residue was dissolved in 50 μL of 400 mM borate buffer (pH 9.0) and filtered through a 0.45 μm Millex-LH filter (Millipore, Bedford, MA, USA). The filtered solution was appropriately diluted with the same buffer and stored at −80 °C until use.

primers used were as follows: 5′-AAG CTT GAG ACA GGC CAT GTA CCC ATA CGA TGT TCC AGA TTA CGC TGA CAC AGC ACG GAT TGC AG-3′ (forward primer) and 5′-AAG CTT GAG ACA GGC CAT GTA CCC ATA CGA TGT TCC AGA TTA CGC TGA CAC AGC ACG GAT TGC AG-3′ (reverse primer). These primers were designed to contain additional HindIII and EcoRI sites at the 5′-ends of the forward and reverse primers, respectively. The forward primer was also designed to contain an HA-tag-coding sequence. The reaction product was cloned into pT7Blue, and the sequence was confirmed (pT7-HA-hDDO). Subsequently, the 1.1 kb HindIII−EcoRI fragment of pT7-HA-hDDO containing the entire Nterminally HA-tagged human DDO-coding sequence was subcloned into pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA) to generate the N-terminally HA-tagged human DDO expression plasmid (pcDNAHA-hDDO). Expression and Purification of Recombinant Proteins. Escherichia coli BL21(DE3)pLysS cells were transformed with expression plasmids and cultured at 37 °C with shaking in Luria− Bertani medium containing ampicillin (100 μg/mL). Crude extracts were prepared from cells transformed with pRSET-His-hDDO, pRSET-His-rDDO, pRSET-His-mDASPO, and pRSET-His-hDAO, as described previously.38,57,59 All recombinant proteins were purified by affinity chromatography using a chelating column. Specifically, crude extracts were applied to a His GraviTrap column (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) equilibrated with 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl and 10 mM imidazole. The column was then washed with the same buffer, and bound proteins were eluted using a stepwise gradient of 50−500 mM imidazole. Each fraction (2 mL) containing recombinant protein was dialyzed at 4 °C for 1 d against 1 L of 10 mM sodium pyrophosphate buffer (pH 8.3) containing 2 mM EDTA, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol. The buffer was changed once during dialysis. The dialyzed fractions were recovered and centrifuged at 10 000 × g for 10 min at 4 °C to pellet the proteins denatured during dialysis. The supernatants were recovered as purified enzyme and used immediately for enzyme assays or stored at −80 °C until use. All recombinant proteins were determined to be purified to nearhomogeneity when examined by SDS-polyacrylamide gel electrophoresis. The protein concentrations of the purified enzyme preparations were determined using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA), with bovine serum albumin as a standard. Enzymatic Activity Assays. The activity of DDO and DAO was determined using a colorimetric assay for 2-oxo acid production, as described previously.60 Briefly, appropriate amounts (0.095−1.5 μg) of the purified enzymes were added to a reaction mixture of air-saturated 40 mM sodium pyrophosphate buffer (pH 8.3), 33 ng/μL A. niger catalase, 60 μM FAD, and 10 mM amino acids (D-Asp and D-Ala for the determination of the DDO and DAO activities, respectively) in a final volume of 150 μL. The reaction mixture was incubated at 37 °C for 10 min, and then 10 μL of 100% (w/v) trichloroacetic acid was added to stop the reaction. The 2-oxo acid products were reacted with 2,4-dinitrophenylhydrazine and quantified by measuring the A445 against a blank mixture lacking amino acids. To test the inhibitory activity of candidate compounds, each compound was added to the reaction mixture individually, and its relative inhibitory activity was determined by considering the activity of the enzyme in the absence of compound as 100%. The IC50 values of the compounds tested were determined using the following formula, as described previously:39 IC50 = 10(log[A/B] × [50 − C]/[D − C] + log B), where A and B are the highest and lowest concentrations closest to the middle of the curve, and C and D are the inhibition percentages at B and A, respectively. For determination of the type of inhibition of DDO by several compounds, different final concentrations (1−20 mM) of D-Asp were used as the substrate. The data obtained were fitted to the Michaelis−Menten equation, and the Vmax and Km values in the absence or presence of the compounds tested were estimated using the nonlinear least-squares fitting algorithm in pro Fit 6.1 software (Quantum soft, Zürich, Switzerland; http://www.quansoft. J




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(3) Henneberger, C.; Papouin, T.; Oliet, S. H. R.; Rusakov, D. A. Long-term potentiation depends on release of D-serine from astrocytes. Nature 2010, 463, 232−236. (4) Panatier, A.; Theodosis, D. T.; Mothet, J.-P.; Touquet, B.; Pollegioni, L.; Poulain, D. A.; Oliet, S. H. R. Glia-derived D-serine controls NMDA receptor activity and synaptic memory. Cell 2006, 125, 775−784. (5) Rosenberg, D.; Artoul, S.; Segal, A. C.; Kolodney, G.; Radzishevsky, I.; Dikopoltsev, E.; Foltyn, V. N.; Inoue, R.; Mori, H.; Billard, J.-M.; Wolosker, H. Neuronal D-serine and glycine release via the Asc-1 transporter regulates NMDA receptor-dependent synaptic activity. J. Neurosci. 2013, 33, 3533−3544. (6) Bendikov, I.; Nadri, C.; Amar, S.; Panizzutti, R.; De Miranda, J.; Wolosker, H. A CSF and postmortem brain study of D-serine metabolic parameters in schizophrenia. Schizophr. Res. 2007, 90, 41− 51. (7) Hashimoto, K.; Fukushima, T.; Shimizu, E.; Komatsu, N.; Watanabe, H.; Shinoda, N.; Nakazato, M.; Kumakiri, C.; Okada, S.; Hasegawa, H.; Imai, K.; Iyo, M. Decreased serum levels of D-serine in patients with schizophrenia: evidence in support of the N-methyl-Daspartate receptor hypofunction hypothesis of schizophrenia. Arch. Gen. Psychiatry 2003, 60, 572−576. (8) Hashimoto, K.; Engberg, G.; Shimizu, E.; Nordin, C.; Lindström, L. H.; Iyo, M. Reduced D-serine to total serine ratio in the ̈ schizophrenic patients. Prog. Neurocerebrospinal fluid of drug naive Psychopharmacol. Biol. Psychiatry 2005, 29, 767−769. (9) Yamada, K.; Ohnishi, T.; Hashimoto, K.; Ohba, H.; IwayamaShigeno, Y.; Nakamura, K.; Shimizu, E.; Itokawa, M.; Mori, N.; Iyo, M.; Yoshikawa, T. Identification of multiple serine racemase (SRR) mRNA isoforms and genetic analysis of SRR and DAO in schizophrenia and D-serine levels. Biol. Psychiatry 2005, 57, 1493− 1503. (10) Inoue, R.; Hashimoto, K.; Harai, T.; Mori, H. NMDA- and βamyloid1−42-induced neurotoxicity is attenuated in serine racemase knock-out mice. J. Neurosci. 2008, 28, 14486−14491. (11) Wu, S.-Z.; Bodles, A. M.; Porter, M. M.; Griffin, W. S. T.; Basile, A. S.; Barger, S. W. Induction of serine racemase expression and Dserine release from microglia by amyloid β-peptide. J. Neuroinflammation 2004, 1, 2. (12) Sasabe, J.; Chiba, T.; Yamada, M.; Okamoto, K.; Nishimoto, I.; Matsuoka, M.; Aiso, S. D-Serine is a key determinant of glutamate toxicity in amyotrophic lateral sclerosis. EMBO J. 2007, 26, 4149− 4159. (13) Sasabe, J.; Miyoshi, Y.; Suzuki, M.; Mita, M.; Konno, R.; Matsuoka, M.; Hamase, K.; Aiso, S. D-Amino acid oxidase controls motoneuron degeneration through D-serine. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 627−632. (14) Di Fiore, M. M.; Santillo, A.; Baccari, G. C. Current knowledge of D-aspartate in glandular tissues. Amino Acids 2014, 46, 1805−1818. (15) Katane, M.; Homma, H. D-Aspartatean important bioactive substance in mammals: a review from an analytical and biological point of view. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2011, 879, 3108−3121. (16) Ota, N.; Shi, T.; Sweedler, J. V. D-Aspartate acts as a signaling molecule in nervous and neuroendocrine systems. Amino Acids 2012, 43, 1873−1886. (17) D’Aniello, G.; Ronsini, S.; Guida, F.; Spinelli, P.; D’Aniello, A. Occurrence of D-aspartic acid in human seminal plasma and spermatozoa: possible role in reproduction. Fertil. Steril. 2005, 84, 1444−1449. (18) D'Aniello, G.; Grieco, N.; Di Filippo, M. A.; Cappiello, F.; Topo, E.; D'Aniello, E.; Ronsini, S. Reproductive implication of D-aspartic acid in human pre-ovulatory follicular fluid. Hum. Reprod. 2007, 22, 3178−3183. (19) Fagg, G. E.; Matus, A. Selective association of N-methyl aspartate and quisqualate types of L-glutamate receptor with brain postsynaptic densities. Proc. Natl. Acad. Sci. U. S. A. 1984, 81, 6876− 6880.

The concentrations of D-Asp and L-Asp in the cells were determined by HPLC using the OPA precolumn derivatization technique, as described previously.44 Specifically, a 10 μL aliquot of the sample was prepared as described above, and then 30 μL of 400 mM borate buffer (pH 9.0) and 20 μL of OPA/Boc-L-cysteine reagent (prepared by mixing 10 mg of OPA with 10 mg of Boc-L-cysteine in 1 mL of 100% [v/v] methanol) were added to fluorescently derivatize the amino acids in the sample.62 After incubation at room temperature for 2 min, a 10 μL aliquot of the sample was injected into a Jasco chromatographic system, which consisted of a model PU-2089 pump, a model FP-2025 fluorescence detector, and a model 807-IT integrator (Jasco Corp., Tokyo, Japan). The amino acids were separated on an octadecylsilyl silica gel column (Mightysil RP-18GP, 150 mm × 4.6 mm internal diameter; Kanto Chemical Co., Tokyo, Japan) at a flow rate of 1 mL/min. The column was first equilibrated with a 9:1 ratio of solvent A (200 mM sodium acetate buffer [pH 6.2]) to solvent B (100% [v/v] acetonitrile). After injection, the sample was eluted for 20 min with a linear gradient (10−14%) of solvent B. The fluorescence was detected at an excitation wavelength of 344 nm and an emission wavelength of 443 nm. The levels of D-Asp and L-Asp were determined based on the peak areas in the chromatograms.

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ASSOCIATED CONTENT

S Supporting Information *

. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00871. Chemical information on the compounds selected by in silico screening, molecular formula strings of the compounds tested in this study, and 1H NMR spectra for experimentally validated hits (PDF) IC50 values (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-3-5791-6229. Fax: +81-3-5791-6381. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Ms. Noriko Sato and Dr. Kenichiro Nagai (Graduate School of Pharmaceutical Sciences, Kitasato University) for performing the NMR analyses. This work was supported in part by a Grants-in-Aid for Scientific Research (24590090) from the Japan Society for the Promotion of Science and a Kitasato University Research Grant for Young Researchers (to M.K.).

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ABBREVIATIONS USED DAO, D-amino acid oxidase; DDO, D-aspartate oxidase; DMEM, Dulbecco’s modified Eagle’s medium; FAD, flavin adenine dinucleotide; HPLC, high-performance liquid chromatography; IC50, 50% inhibitory concentration; NMDA, Nmethyl-D-aspartate; NMR, nuclear magnetic resonance; OPA, o-phthalaldehyde

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REFERENCES

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Identification of Novel d-Aspartate Oxidase Inhibitors by in Silico

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