Identification of Novel d-Amino Acid Oxidase Inhibitors by in Silico

Feb 7, 2013 - d-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for d-amino acids, including d-serine and d-alanine, which are...
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Identification of Novel D‑Amino Acid Oxidase Inhibitors by in Silico Screening and Their Functional Characterization in Vitro Masumi Katane,† Naoko Osaka,†,∥ Satsuki Matsuda,†,∥ Kazuhiro Maeda,† Tomonori Kawata,† Yasuaki Saitoh,† Masae Sekine,† Takemitsu Furuchi,† Issei Doi,‡,§ Shuichi Hirono,‡ and Hiroshi Homma*,† †

Laboratory of Biomolecular Science, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan ‡ Laboratory of Physical Chemistry for Drug Design, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan § First Research Department, Toyama Chemical Co., Ltd., 2-4-1 Shimookui, Toyama, Toyama 930-8508, Japan S Supporting Information *

ABSTRACT: D-Amino acid oxidase (DAO) is a degradative enzyme that is stereospecific for D-amino acids, including Dserine and D-alanine, which are potential coagonists of the Nmethyl-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 DAO inhibitor that augments the brain levels of Dserine and/or D-alanine and thereby activates NMDA receptor function is expected to be an antipsychotic drug, for instance, in the treatment of schizophrenia. In the search for potent DAO inhibitor(s), a large number of compounds were screened in silico, and several compounds were estimated as candidates. These compounds were then characterized and evaluated as novel DAO inhibitors in vitro. The results reported in this study indicate that some of these compounds are possible lead compounds for the development of a clinically useful DAO inhibitor and have the potential to serve as active site probes to elucidate the structure−function relationships of DAO.



INTRODUCTION Among the free D-amino acids that are present in mammals, Daspartate (D-Asp) and D-serine (D-Ser) have been studied the most intensively. Substantial amounts of free D-Asp are present in a wide variety of mammalian tissues and cells, particularly in the central 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.1,2 Unlike the widespread expression of DAsp, D-Ser was predominantly concentrated in the mammalian forebrain, where it is synthesized by Ser racemase (EC 5.1.1.16), which is a synthetic enzyme that produces D-Ser from L-Ser.3 D-Ser persists at high concentrations throughout the life of the animal. D-Ser is now considered to be a neuromodulator that binds to the glycine-binding site of the Nmethyl-D-Asp (NMDA) receptor, a subtype of the L-glutamate (L-Glu) receptor, and potentiates glutamatergic neurotransmission in the central nervous system.4,5 In fact, astroglia-derived D-Ser has been shown to regulate NMDA receptor-dependent long-term potentiation and/or long-term depression, which are basic processes of learning and memory, in the hypothalamic and hippocampal excitatory synapses.6,7 D-Ser also is found in the cerebellum during the early postnatal period, and it was © XXXX American Chemical Society

recently reported that D-Ser derived from the Bergmann glia serves as an endogenous ligand for the δ2 Glu receptor to regulate long-term depression at synapses between parallel fibers and Purkinje cells in the immature cerebellum but not in the mature one.8 These lines of evidence suggest a physiological significance of D-Ser in the regulation of higher brain functions through L-Glu receptors. Indeed, perturbation of D-Ser levels in the nervous system has now been implicated in the pathophysiology of various neuropsychiatric disorders, including schizophrenia,9−12 Alzheimer’s disease,13,14 and amyotrophic lateral sclerosis.15,16 In addition to D-Asp and D-Ser, D-alanine (D-Ala), another Damino acid found in mammals at relatively low levels, has been investigated recently. Because D-Ala is found in the mammalian brain and peripheral tissues, and in the anterior pituitary gland and pancreas, it has been postulated to be involved in endocrine activity.17−19 Notably, D-Ala is known to stimulate the NMDA receptor by acting as a coagonist that binds to the glycine-binding site of the receptor.20,21 Received: October 12, 2012

A

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structure databases. In silico screening is a cost-effective first screening method, and results from the screening can then be used as a guide for library construction before an actual screening is performed.43−45 Therefore, this study used a computer ligand-docking method to screen a large number of compounds in silico for their ability to inhibit human DAO, and several compounds were estimated as candidates. These compounds were then characterized and evaluated in vitro as novel DAO inhibitors. This work shows that some of these compounds will be possible lead compounds for the development of a clinically useful DAO inhibitor. They also can serve as active site probes to elucidate the structure−function relationships of DAO.

In mammalian tissues, two types of degradative enzymes that are stereospecific for D-amino acids have been identified: Damino acid oxidase (DAO, also abbreviated DAAO, EC 1.4.3.3) and D-Asp oxidase (DDO, also abbreviated 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 with oxygen to generate the corresponding 2-oxo acids along with hydrogen peroxide and ammonia.22,23 DAO displays broad substrate specificity and acts on several neutral and basic D-amino acids such as D-Ser and DAla. DDO is highly specific for acidic D-amino acids, such as DAsp and D-Glu, none of which are substrates of DAO. DAO and DDO have been identified in various organisms and in mammals, where they are reported to be localized in the peroxisomes.24−27 In mammals, DAO and DDO activities are highest in the kidney, followed by the liver and brain, and are low in other peripheral tissues. The mammalian DAO and DDO are believed to regulate the levels of several endogenous and exogenous D-amino acids, including D-Asp, D-Ser, and DAla, in various organs.28,29 However, their physiological roles in vivo have yet to be fully clarified. As described above, a decrease in D-Ser levels in the central nervous system and the resultant dysfunction of NMDA receptor-mediated neurotransmission has now been postulated in the onset of various mental disorders including schizophrenia.9−12 D-Ser and D-Ala are both coagonists of the NMDA receptor4,5,20,21 and could be candidate drugs for the treatment of NMDA receptor-related diseases. Schizophrenic patients who received D-Ser administration with concomitant antipsychotic therapy have been reported to show significant improvements in their positive, negative, and cognitive symptoms.30,31 Similar clinical efficacy also was reported in the study on adjunctive administration of D-Ala to schizophrenic patients.32 However, the direct administration of these D-amino acids has some problems in their eventual therapeutic applications, including their low permeability through the blood−brain barrier and nephrotoxicity.4,33,34 Another strategy for activating NMDA receptor function is to prevent the metabolic degradation of D-Ser and/or D-Ala by DAO. In fact, a mouse mutant that lacks DAO activity (ddY/ DAO−) has significantly elevated levels of D-Ser and/or D-Ala in various brain regions, in addition to several peripheral tissues, serum, and urine.35,36 Alterations in NMDA receptor-mediated long-term synaptic plasticity have been observed in this mutant strain.37 In addition, behavioral characterizations have suggested that the mutants exhibit specific antischizophrenic responses.38,39 Considering these results, increased attention has been focused on DAO as a therapeutic target, and a DAO inhibitor(s) that augments brain D-Ser and/or D-Ala levels is now expected to be an antipsychotic drug(s) in the treatment of schizophrenia. Several compounds recently have been reported as novel DAO inhibitors with high potencies for the treatment of schizophrenia, including 6-chlorobenzo[d]isoxazol-3-ol, 4Hfuro[3,2-b]pyrrole-5-carboxylic acid and 3-hydroxyquinolin2(1H)-one.40−42 These compounds were identified by high throughput screening, which is an approach to discover novel compounds effective for a given target. However, preparation of numerous candidate compounds is prerequisite for high throughput screening, and this preparatory step is timeconsuming and costly in general.43−45 Recent advances in computational chemistry have enabled us to perform structurebased in silico screening of many compounds listed in chemical



RESULTS In Silico Screening of Compounds Inhibiting the Activity of Human DAO. As many as 4 million commercially available compounds were screened in silico for their binding capability to the active site of human DAO. Of these, 15 compounds (compounds 1−15) were estimated as candidates (Chart 1). Chemical information on these compounds is listed in Supporting Information Table S1. Subsequently, these compounds were characterized in more detail and evaluated as novel DAO inhibitors, as described in the following sections. Inhibitory Activities of the Candidate Compounds. Dose-dependent effects of the 15 candidate compounds on the enzymatic activity of recombinant human DAO were examined. The effects of benzoate, crotonate, 4H-furo[3,2-b]pyrrole-5carboxylic acid, meso-tartrate, and malonate also were tested as control inhibitors: the first three compounds as positive control inhibitors and the last two as negative control inhibitors. Benzoate, crotonate, and 4H-furo[3,2-b]pyrrole-5-carboxylic acid reportedly inhibit mammalian DAO by competing with their substrates,42,46,47 whereas meso-tartrate and malonate reportedly inhibit mammalian DDO by competing with their substrates.48−50 meso-Tartrate and malonate also reportedly inhibit mammalian Ser racemase by competing with their substrates.51,52 As expected, benzoate, crotonate, and 4Hfuro[3,2-b]pyrrole-5-carboxylic acid inhibited the activity of human DAO on D-Ser in a dose-dependent manner (Figure 1A,B and data not shown), and the values for the 50% inhibitory concentration (IC50) of these compounds were determined (Table 1). Several candidate compounds inhibited the activity of DAO on D-Ser in a dose-dependent manner (Figure 1C−G), and the IC50 values of five compounds (1, 5, 8, 11, and 14) were determined (Table 1). In contrast, mesotartrate and malonate had no effect even at a concentration of 10000 μM, as expected. Because 5, 8, 11, and 14 exhibited relatively high inhibitory activity against DAO (Table 1), structure-based molecular invention was subsequently performed in silico using the structures of these compounds as lead structures. As shown in Chart 1 and Supporting Information Table S1, four compounds (compounds 16−19) were identified as new candidates and were evaluated for their inhibitory activity against DAO. Compound 14 has a novel chemical structure in comparison with known potent inhibitors of DAO; therefore, analogues of 14 also were searched in silico, and two compounds (compounds 20 and 21) were identified as new candidates (Chart 1 and Supporting Information Table S1). Among these six new candidates (16−21), 16−20 inhibited the activity of DAO on D-Ser in a dose-dependent manner (Figure 1H−L) and the IC50 values of these compounds were determined B

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Chart 1. Chemical Structures of Compounds 1−21a

be determined because more than 50% of the activity of DAO remained (51.5 ± 1.8% and 67.1 ± 4.9% of the control activity, respectively), even at the highest concentrations tested (10000 μM). Selectivity of the Candidate Compounds. To establish whether the 10 compounds (1, 5, 8, 11, 14, 16−20) inhibit enzyme(s) other than DAO, the effects of these compounds on the enzymatic activities of recombinant human DDO and human Ser racemase were examined. Several compounds inhibited the activity of DDO on D-Asp or Ser racemase on L-Ser in a dose-dependent manner, and the IC50 values of these compounds were determined (Table 1). However, the inhibitory activities of these compounds were relatively lower than those of meso-tartrate or malonate, which inhibited the activities of DDO and Ser racemase in a dose-dependent manner (Table 1). Benzoate, crotonate, and 4H-furo[3,2b]pyrrole-5-carboxylic acid had no effect on these enzyme activities, even at the highest concentrations tested (10000 or 100 μM). Inhibitory Compounds 5, 8, and 11. Compounds 5, 8, and 11 were chosen for further study because they exhibited relatively high levels of inhibition of DAO, but not DDO or Ser racemase (Table 1). The effects of these three compounds on the flavin absorbance spectrum of recombinant human DAO were examined. The effects of benzoate, 4H-furo[3,2-b]pyrrole5-carboxylic acid, and malonate also were tested as control inhibitors. As expected, the addition of benzoate and 4Hfuro[3,2-b]pyrrole-5-carboxylic acid yielded perturbations of the absorption spectrum of the enzyme (Figure 2A,B). The spectral changes on benzoate binding were consistent with those reported for recombinant human DAO.53 The spectral changes also were observed when the enzyme was mixed with 5, 8, and 11 (Figure 2C−E), confirming the direct binding of these compounds to the enzyme. In contrast, the addition of malonate had no effect, as expected (Figure 2F). The types of inhibition of compounds 5, 8, and 11 on DAO were examined, and benzoate and 4H-furo[3,2-b]pyrrole-5carboxylic acid were tested as control inhibitors. Figure 3 shows 1/v versus 1/[S] plots in the absence or presence of the inhibitors.54 As expected, benzoate and 4H-furo[3,2-b]pyrrole5-carboxylic acid showed a competitive type of inhibition (data not shown). Similarly, 5, 8, and 11 showed a competitive type of inhibition (Figure 3). Taken together, these results indicated that 5, 8, and 11 are novel, active site-directed DAO inhibitors. The Active Site Residue Tyr-224 Is Involved in the Binding of Compounds 5 and 8 to DAO. The threedimensional (3D) X-ray crystallographic structures of porcine and human DAO have been solved.42,55−57 In the structures complexed with benzoate, the carboxyl group of benzoate interacts with the side chain guanidino group of Arg-283 and with the side chain hydroxyl group of Tyr-228 (Figure 4A).55−57 Importantly, a loop formed by residues 216−228 has been postulated to act as an “active-site lid” that opens and closes on substrate/product migration in and out of the active site.58 Tyr-224 within the lid is presumably important in the hydrophobic environment of the active site in the closed conformation,55−57 and the side chain of this residue possibly covers the bound ligand (Figure 4A). In the structures complexed with 4H-furo[3,2-b]pyrrole-5-carboxylic acid, the carboxyl group of 4H-furo[3,2-b]pyrrole-5-carboxylic acid interacts with the side chain guanidino group of Arg-283 and with the side-chain hydroxyl group of Tyr-228, whereas the heterocyclic pyrrole NH group of 4H-furo[3,2-b]pyrrole-5-

a Chemical structures of benzoate, crotonate, 4H-furo[3,2-b]pyrrole-5carboxylic acid, meso-tartrate, malonate, and 3-hydroxyquinolin-2(1H)one also are shown for comparison.

(Table 1). However, the inhibitory activity of these compounds was relatively weaker than that of the parent compounds (5, 8, 11, or 14). The 10 compounds (1, 5, 8, 11, 14, 16−20) and benzoate, crotonate, and 4H-furo[3,2-b]pyrrole-5-carboxylic acid inhibited the activity of DAO on D-Ala in a dose-dependent manner (Figure 1 and data not shown). The IC50 values of these compounds, with the exception of 1 and 16, were determined (Table 1). The IC50 values of 1 and 16 could not C

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Figure 1. Dose-dependent effects of several compounds on the enzymatic activity of human DAO. Enzymatic activities on D-Ser (filled circles) and D-Ala (open squares) were assayed using the recombinant human DAO in the absence or presence of benzoate (A), 4H-furo[3,2-b]pyrrole-5carboxylic acid (B), and compounds 1 (C), 5 (D), 8 (E), 11 (F), 14 (G), 16 (H), 17 (I), 18 (J), 19 (K), and 20 (L). 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 to four independent assays. Where not shown, the error bars are smaller than the symbols used.

aromatic ring of Tyr-224 contributes to the binding to the ligands (data not shown). To assess the validity of the proposed models, the interaction of the Tyr-224 residue of DAO with 5, 8, and 11 was tested. Two mutants of human DAO carrying Tyr-224-to-Phe or Tyr-224-to-Ala substitutions (Y224F and Y224A variants, respectively) were prepared, and kinetic properties were compared with wild-type human DAO. The apparent kinetic parameters (Michaelis constant [Km] and molecular activity [kcat] values) of the human DAO variants were determined with D-Ala as the substrate and compared with those of wild-type human DAO (Table 2). The Km and kcat values of the Y224F variant on D-Ala were comparable to those of the wild-type enzyme. In contrast, the Km value of the Y224A

carboxylic acid interacts with the backbone carbonyl of Gly-313 (Figure 4B).42 In comparison with these experimentally determined structures, structural models of human DAO complexed with 5, 8, and 11 were proposed in this study (Figure 4C−E). Docking grids were generated in silico around Leu-215, Tyr-224, Tyr-228, Arg-283, and Gly-313 residues in the active site of human DAO, and structural models complexed with the compounds were constructed using Glide software (Schrödinger Suite 2009; Schrödinger LLC, New York NY). The models with the lowest energy conformation are shown in Figure 4C−E. In all these models, the Tyr-224 residue covers the bound ligand, and computational analysis of enzyme−ligand interactions suggested that the side chain D

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benzoate against the wild-type enzyme was comparable to the reported Ki value against recombinant human DAO (7 μM).55 As expected, the Ki values of benzoate against the Y224F and Y224A variants were significantly higher than that against the wild-type enzyme. Thus, the introduction of the Y224F or Y224A mutation into human DAO decreased its ability to bind to benzoate. These results indicated that the Tyr-224 residue of DAO is involved in the interaction with benzoate and are consistent with the previously reported observations described above.55−57 The Ki values of 5 and 8 against the Y224F and/or Y224A variants were significantly higher than those against the wild-type enzyme (Table 3). By contrast, the Ki values of 4Hfuro[3,2-b]pyrrole-5-carboxylic acid and 11 against the Y224F and Y224A variants were comparable to those against the wildtype enzyme. Collectively, these results suggested that the Tyr224 residue of human DAO is an important determinant in its interaction with 5 and 8 but not with 4H-furo[3,2-b]pyrrole-5carboxylic acid and 11; however, the molecular details underlying the kinetic effects remain to be elucidated. Inhibitory Activities of Compounds 5, 8, and 11 against Human DAO Expressed in Cultured Mammalian Cells. To assess whether 5, 8, and 11 inhibit the enzymatic activity of human DAO expressed in mammalian cells, human embryonic kidney 293 cells were stably transfected with the Nterminal HA-tagged human DAO expression plasmid. The DAO-overexpressing mammalian cell line 293.HA-hDAO-24 was established. The 293/NEO cell line also was established and used as a control cell line. This cell line was derived from 293 cells after stable transfection with the parental plasmid and was not forced to express DAO. The 293/NEO and 293.HAhDAO-24 cells were cultured for 24 h in the presence of various concentrations of exogenous D-Ala (0, 8.3, or 17 mM), and the concentrations of D- and L-Ala in the cells and culture media were then determined by high-performance liquid chromatography (HPLC). When the 293/NEO and 293.HA-hDAO-24 cells were cultured with 0 mM D-Ala, D-Ala was undetectable or detected only at trace levels in the cells and culture media (Figure 5A,B). By contrast, during incubation of the cells with 8.3 and 17 mM D-Ala, D-Ala markedly accumulated both in the cells and culture media, and the levels were dependent on the D-Ala concentration added in the media. The addition of D-Ala to the media had almost no significant effect on L-Ala levels both in the cells and in the culture medium (Figure 5C,D). Importantly, the D-Ala levels in the 293.HA-hDAO-24 cells incubated with 8.3 and 17 mM D-Ala were significantly lower than those in the 293/NEO cells incubated with the same concentrations of D-Ala (Figure 5A). The D-Ala levels in the culture media containing either 293/NEO or 293.HA-hDAO24 cells did not significantly differ (Figure 5B). Collectively, these results suggested that 293 cells (293/NEO and 293.HAhDAO-24 cells) are able to take up D-Ala from the culture medium and that D-Ala incorporated into the 293.HA-hDAO24 cells is efficiently degraded by DAO expressed in this cell line. The effects of 5, 8, and 11 on D-Ala levels in the 293/NEO and 293.HA-hDAO-24 cells were examined. Specifically, the cells were treated with 5, 8, or 11 for 30 min and then exposed to 17 mM D-Ala for 24 h. The concentrations of D- and L-Ala in the cells were then determined. On the basis of preliminary experiments, the compounds were used at a concentration of 83 μM to exclude direct cytotoxicity. The effect of 4H-furo[3,2b]pyrrole-5-carboxylic acid also was tested as control inhibitor; however, this compound was used at 17 μM because of its

Table 1. IC50 Values of Candidate Compounds against Human DAO, DDO, and Ser Racemase IC50 (μM)a compd benzoate crotonate 4H-furo[3,2-b] pyrrole-5carboxylic acid meso-tartrate malonate 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

DAOb

DDO

Ser racemase

46.9 ± 6.4 (65.7 ± 8.5) 357 ± 101 (598 ± 40) 0.177 ± 0.048 (0.214 ± 0.099)

>10000

>10000

>10000

>10000

>100

>100

>10000 >10000 6269 ± 719 (>10000) >2500 >2500 >10000 26.0 ± 6.6 (49.2 ± 7.6) >1000 >10000 0.440 ± 0.029 (0.943 ± 0.161) >2500 >100 28.5 ± 3.9 (59.8 ± 19.5) >1000 >500 199 ± 22 (298 ± 83) >5000 7905 ± 886 (>10000) 1832 ± 479 (3725 ± 209) 666 ± 184 (1104 ± 128) 900 ± 100 (1450 ± 495) 253 ± 31 (365 ± 123) >10000

3922 ± 275 1355 ± 37 >10000

2820 ± 1251 24.9 ± 0.9 8386 ± 1063

NDc ND ND >10000

ND ND ND 8213 ± 1866

ND ND 7579 ± 1126

ND ND >10000

ND ND >10000

ND ND 5282 ± 455

ND ND >5000

ND ND >5000

ND >10000

ND >10000

>10000

5159 ± 2971

>10000

5723 ± 776

>10000

5015 ± 1981

>10000

>10000

ND

ND

Data are shown as the mean ± standard deviation of three to four independent assays. bIC50 values against DAO are determined with DSer or D-Ala (in parentheses) as the substrates. cND: not determined. a

variant on D-Ala was significantly higher than that of the wildtype enzyme, but the kcat value was lower. Therefore, the catalytic efficiency (kcat/Km) of this variant for D-Ala was markedly lower than that of the wild-type enzyme (Table 2). These results suggested that Tyr-224 is catalytically important for the full enzymatic activity of DAO and are consistent with previous reports on the structural significance of this residue, as described above.55−58 The dose-dependent effects of benzoate, 4H-furo[3,2-b]pyrrole-5-carboxylic acid, 5, 8, and 11 on the enzymatic activities of the Y224F and Y224A variants were examined. All of these compounds inhibited the activities of the Y224F and Y224A variants on D-Ala in a dose-dependent manner, and the IC50 values of these compounds were determined (data not shown). Using the determined Km values for D-Ala and IC50, the inhibitor constant (Ki) values of these compounds against the DAO variants were calculated59 and compared with those against wild-type DAO (Table 3). The calculated Ki value of E

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Figure 2. Absorption spectra of human DAO in the absence or presence of the compounds. Wavelength scans were carried out at 25 °C with the recombinant human DAO (1.7 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 40 μM (4H-furo[3,2-b]pyrrole-5-carboxylic acid) or 200 μM (benzoate, compounds 5, 8, and 11, and malonate) at the indicated times. The scan range was 300−600 nm in the presence of benzoate (A), compound 5 (C), and malonate (F), 320−600 nm in the presence of 4H-furo[3,2-b]pyrrole-5-carboxylic acid (B) and compound 11 (E), and 370− 600 nm in the presence of compound 8 (D).

limited solubility. The treatment of 293/NEO cells with these compounds had almost no significant effect on D-Ala levels in the cells (Figure 6A). By contrast, the treatment of 293.HAhDAO-24 cells with 4H-furo[3,2-b]pyrrole-5-carboxylic acid, 5, and 8 caused a significant increase in D-Ala levels in the cells. The D-Ala content in 293.HA-hDAO-24 cells treated with 11 also appeared to be higher than that in the vehicle-treated cells, but this difference was not significant. The L-Ala levels in 293/ NEO and 293.HA-hDAO-24 cells were essentially unchanged by treatment with any compound (Figure 6B). Therefore, these results suggested that 5, 8, and 4H-furo[3,2-b]pyrrole-5carboxylic acid cross the cell membrane efficiently and reach DAO localized in the peroxisome, resulting in the effective inhibition of the enzymatic activity of DAO expressed in the cells.

derivative of 3-hydroxyquinolin-2(1H)-one, which is a known inhibitor of DAO,40 as described below in detail. The lactone O atom in the chromen ring of 8 is substituted by the lactam NH group in the chemical structure of 3hydroxyquinolin-2(1H)-one (Chart 1). The IC50 value of 3hydroxyquinolin-2(1H)-one against human DAO was reported to be 4 nM (geometric mean [n = 76]; 95% confidence interval = 3−5) under conditions where the final concentration of the substrate D-Ser in the assay is 0.2 mM.40 This IC50 value is relatively lower than that of 4H-furo[3,2-b]pyrrole-5-carboxylic acid (9 nM, geometric mean [n = 10]; 95% confidence interval = 7−13), which was determined under the same conditions.40 By contrast, the IC50 value of 8 against human DAO was 0.440 ± 0.029 μM under the conditions where the final concentration of D-Ser in the assay is 10 mM, and it was relatively higher than that of 4H-furo[3,2-b]pyrrole-5-carboxylic acid (0.177 ± 0.048 μM), which was determined under the same conditions (Table 1). Therefore, it appears that the inhibitory activity of 8 against DAO is relatively weaker than that of 3-hydroxyquinolin2(1H)-one. In the determined structure of human DAO complexed with 3-hydroxyquinolin-2(1H)-one, the lactam NH group of 3-hydroxyquinolin-2(1H)-one interacts with the backbone carbonyl of Gly-313 to form a hydrogen bond (Figure 4F).40 The structural model of human DAO complexed with 8 suggested that a van der Waals interaction is formed between the lactone O atom of 8 and the backbone carbonyl of Gly-313 (Figure 4D), but this interaction is probably weaker than the above-mentioned hydrogen bonding interaction formed by 3-hydroxyquinolin-2(1H)-one. The relatively weaker inhibitory activity of 8 compared with 3-hydroxyquinolin2(1H)-one may be caused by these differences in the binding modes. The results of the kinetic analysis revealed that 5, 8, and 11 inhibit the activity of DAO by competing with the substrate



DISCUSSION Structure-based in silico screening is an efficient approach to identify lead structures that are effective for a given target. In particular, this approach is highly effective when the 3D X-ray crystallographic structure of the target enzyme protein complexed with the known potent inhibitor has been determined because information from the determined structure is helpful in selecting the lead compounds during the in silico screening. Accordingly, a high hit ratio can be achieved. This is true for human DAO,40,42,55 and several compounds were successfully identified in the present study as novel DAO inhibitors based on in silico screening. Compounds 5, 8, and 11 were investigated in more detail because they exhibited relatively high levels of inhibition against DAO but not against other enzymes (DDO and Ser racemase) (Table 1). To our knowledge, there has been no report of characterization of these three compounds as DAO inhibitors. However, 8 is a F

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human DAO complexed with 3-hydroxyquinolin-2(1H)-one, the lactam NH group of 3-hydroxyquinolin-2(1H)-one also interacts with the backbone carbonyl of Gly-313 to form a hydrogen bond (Figure 4F),40 as described above. In the mammalian DAO molecule, a loop formed by residues 216−228 has been postulated to act as an “active-site lid” that opens and closes on substrate/product migration in and out of the active site.58 The side chain of Tyr-224 within the lid covers the bound ligand, and its importance in the binding of 5 and 8, as well as benzoate, was supported by site-specific mutagenesis of this residue. Namely, the Ki values of benzoate, 5, and 8 against the Y224F and/or Y224A variants of human DAO were significantly higher than those against the wild-type enzyme (Table 3). This result suggests that the substitution of Tyr-224 with Phe or Ala decreased the affinity of DAO for these compounds. In contrast, the interaction of 11 with the DAO active site was not significantly influenced by the substitution of Tyr-224 with Phe or Ala residue. Namely, the Ki values of 11 against the Y224F and Y224A variants were comparable to that against the wild-type enzyme (Table 3), suggesting that the Tyr-224 residue of human DAO is not an important determinant in its interaction with 11. The structural modeling of human DAO complexed with 11 and subsequent computational analysis of enzyme−ligand interactions suggested that the side chain aromatic ring of Tyr-224 also contributes to the binding to 11 by means of its interaction with the thiophene ring moiety of 11 (Figure 4E). Thus, the findings obtained in the structural model are incompatible with the abovementioned results of the site-specific mutagenesis of Tyr-224. However, it is notable that Fonda and Anderson (1968) previously examined the inhibitory activities of a series of metaand para-substituted benzoate derivatives against mammalian DAO and found that there is an increase in binding of these inhibitors as the electron-withdrawing property of the substituent increases.46 It was proposed that the aromatic residue of DAO interacts with the aromatic nucleus of the bound ligand primarily through electron donation. Accordingly, the electron-withdrawing substituent is presumed to be essential for the aromatic ligand to interact tightly with the Tyr-224 residue of DAO. Compound 11 has a cyclopentane ring in its chemical structure (Chart 1), and this ring moiety functions as an electron-donor group rather than an electronwithdrawing group. Therefore, the interaction between the side chain aromatic ring of Tyr-224 and the thiophene ring moiety of 11 may possibly be weakened by the electron-donating cyclopentane ring moiety of this compound, thereby explaining the fact that the effect of the substitution of Tyr-224 with Phe or Ala residue on the interaction with 11 was not appreciable. Determination of the crystal structure of DAO complexed with 11 will aid in understanding the binding mode of 11 in the DAO active site. Additional experiments using cultured mammalian cells suggested that 5, 8, and 4H-furo[3,2-b]pyrrole-5-carboxylic acid, efficiently inhibit the enzymatic activity of DAO expressed in the cells. Namely, the treatment of 293.HA-hDAO-24 cells with these compounds caused a significant increase in the intracellular D-Ala levels that is otherwise maintained at low levels by the degradative activity of DAO expressed in the cells (Figure 6A). It thus appears that 5, 8, and 4H-furo[3,2b]pyrrole-5-carboxylic acid, will be possible lead compounds for the development of a clinically useful DAO inhibitor. Moreover, the D-Ala content in 293.HA-hDAO-24 cells treated with 11 also appeared to be higher than that in the vehicle-

Figure 3. 1/v versus 1/[S] plots of the enzymatic activity of human DAO in the absence or presence of the inhibitors. Enzymatic activities on D-Ala were assayed using the recombinant human DAO in the absence or presence of compounds 5 (A), 8 (B), and 11 (C). The inhibitors were used at the indicated final concentrations. The Vmax and Km values for D-Ala were determined (data not shown), and the lines in the figures were drawn by fitting these kinetic parameters to the Lineweaver−Burk equation.54 Data are from a representative result of three independent experiments, and similar results were obtained in all experiments.

(Figure 3). Thus, all of these compounds are novel, active sitedirected DAO inhibitors that can be valuable tools for investigating the structure−function relationships of DAO, including the molecular details of the active site environment of DAO. However, it is notable that binding modes of these compounds in the DAO active site are conceivably different from one another. Compound 8 and 4H-furo[3,2-b]pyrrole-5carboxylic acid apparently have a greater affinity for the active site of DAO than benzoate, 5, or 11 (Table 3). This is presumably due, at least in part, to the difference in the interaction with the backbone carbonyl of Gly-313. In the determined structure of human DAO complexed with benzoate (Figure 4A), no interaction was observed between the bound ligand and the backbone carbonyl of Gly-313.55 Similar findings also were obtained in the structural models of human DAO complexed with 5 and 11 (Figure 4C,E). By contrast, in the determined structure of human DAO complexed with 4Hfuro[3,2-b]pyrrole-5-carboxylic acid (Figure 4B), the pyrrole NH group of 4H-furo[3,2-b]pyrrole-5-carboxylic acid interacts with the backbone carbonyl of Gly-313 to form a hydrogen bond.42 The structural model of human DAO complexed with 8 suggested that a van der Waals interaction is formed between the lactone O atom of 8 and the backbone carbonyl of Gly-313 (Figure 4D), as described above. Therefore, it appears that the interaction with the backbone carbonyl of Gly-313 is vital for the bound ligand to exert the potent inhibitory effect on DAO. Supporting this consideration, in the determined structure of G

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Figure 4. Structural models of human DAO complexed with the inhibitors. The experimentally determined structures of human DAO complexed with benzoate (PDB ID: 2DU8),55 4H-furo[3,2-b]pyrrole-5-carboxylic acid (PDB ID: 3CUK),42 and 3-hydroxyquinolin-2(1H)-one (PDB ID: 3G3E)40 are shown in (A), (B), and (F), respectively. The proposed structural models of human DAO complexed with compounds 5, 8, and 11 are shown in (C), (D), and (E), respectively. The carbon atoms in FAD, bound ligands, and side chains of amino acid residues are colored yellow, cyan, and green, respectively. Other atoms are colored as follows: nitrogen, blue; oxygen, red; sulfur, brown. A loop formed by residues 216−228 (activesite lid) is colored magenta. Black dotted lines denote possible hydrogen bonds or van der Waals interactions, while purple arrows denote possible π−π interactions.

that 5 and 11 have relatively lower cell membrane permeability than 4H-furo[3,2-b]pyrrole-5-carboxylic acid and 8. In addition, the stability of these compounds in the cells may possibly differ from one another. In conclusion, several compounds were identified in the present study as novel DAO inhibitors based on in silico screening. Among them, compounds 5, 8, and 11 have the potential to serve as active site probes to elucidate the structure−function relationships of DAO. These compounds also can be useful lead compounds for drug discovery because they are compounds of relatively low molecular weight. In fact, all these compounds showed high “ligand efficiency” (LE) values ranging from 0.65 ± 0.02 to 0.90 ± 0.01 kcal/mol per non-hydrogen atom (Table 3). LE is defined as the binding free energy per heavy (non-hydrogen) atom and provides a metric for evaluation and comparison of binding potency in relation to molecular size.60 Notably, LE of 5 was comparable to those of

Table 2. Apparent Steady-State Kinetic Parameters of Purified Recombinant Human DAO and Its Variants with DAla as the Substrate enzyme

Km (mM)a

kcat (s−1)a

kcat/Km (M−1 s−1)a

wild-type Y224F variant Y224A variant

1.99 ± 0.85 2.97 ± 0.29 12.1 ± 1.2

6.77 ± 0.65 6.70 ± 0.97 3.90 ± 0.29

3791 ± 1391 2246 ± 120 327 ± 54

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

treated cells (Figure 6A). However, the magnitude of increased D-Ala contents in the cells treated with 5 and 11 was smaller than that observed by treatment with 4H-furo[3,2-b]pyrrole-5carboxylic acid or 8. This difference is conceivably due to the fact that these compounds have a greater affinity for DAO than 5 and 11 (Table 3), as described above. Alternatively, it is likely

Table 3. Ki and LE Values of the Inhibitors against Purified Recombinant Human DAO and/or Its Variants Ki (μM)a compd

wild-type

Y224F variant

Y224A variant

benzoate 4H-furo[3,2-b]pyrrole-5-carboxylic acid 5 8 11

± ± ± ± ±

± ± ± ± ±

± ± ± ± ±

10.9 0.0355 8.16 0.156 9.92

1.4 0.0165 1.25 0.027 3.23

54.0 0.0317 24.7 0.265 14.4

b

15.9 0.0054 5.0b 0.051 3.8

37.5 0.0351 20.6 3.69 4.59

c

1.0 0.0066 2.5b 0.34b 0.25

LE for wild-type (kcal/mol per non-hydrogen atom)a 0.78 0.96 0.90 0.80 0.65

± ± ± ± ±

0.01 0.02 0.01 0.01 0.02

a Data are shown as the mean ± standard deviation of three independent assays. bp < 0.01 (Dunnett multiple comparison test) compared to the wildtype. cp < 0.05 (Dunnett multiple comparison test) compared to the wild-type.

H

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Figure 5. Effects of overexpression of DAO on D-Ala contents in mammalian cells and culture media. The 293/NEO and 293.HA-hDAO-24 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, and the cells were exposed to various concentrations (0, 8.3, and 17 mM) of exogenous D-Ala for 24 h. Amino acids were then extracted from the cells and culture media. The concentrations of D-Ala in the cells (A) and culture media (B), and L-Ala in the cells (C) and culture media (D), were determined by HPLC. Data are shown as the mean ± standard deviation of three independent assays. The double asterisks indicate significant differences (p < 0.01) based on Student’s t test.

points for further optimization. Furthermore, 11 also may have sufficiently high ligand efficiency to warrant further development as a lead compound because a LE of >0.3 kcal/mol per non-hydrogen atom is empirically considered to be suitable for lead optimization.60 Chemical modifications of some moieties of these compounds that should fill the space between the compounds and the enzyme and raise the affinity of these compounds to the active site of the enzyme would be an effective approach for improving the inhibitory activity of these compounds against DAO.



EXPERIMENTAL SECTION

Chemicals. All the chemicals described below were used without further purification. D-Amino acids, proteinogenic L-amino acids, ampicillin, bovine serum albumin, catalase from Aspergillus niger, and G418 were purchased from Sigma-Aldrich (St Louis, MO). FAD, pyridoxal phosphate, ATP, o-phthalaldehyde (OPA), N-acetyl-Lcysteine (NAC), benzoate, crotonate, meso-tartrate, and malonate were purchased from Wako Pure Chemical (Osaka, Japan). 4HFuro[3,2-b]pyrrole-5-carboxylic acid was purchased from Focus Synthesis LLC (San Diego, CA). Boc-L-cysteine and fetal bovine serum were purchased from Novabiochem (Läufelfingen, Switzerland) and Gibco-BRL (Gaithersburg, MD), respectively. The compounds selected by in silico screening were purchased from 12 vendors: 1, 4, 5, and 8 were from Sigma-Aldrich, 2 and 3 were from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), 6 was from Maybridge (Cambridge, UK), 7 was from Peakdale (Chapel-en-le-Frith, UK), 9 was from Butt Park Ltd. (Okehampton, UK), 10 and 12 were from Labotest (Niederschöna, Germany), 13 was from Zelinsky Institute Inc. (Newark, DE), 14 was from Life Chemicals Inc. (Burlington, Canada), 15 was from Ark Pharm, Inc. (Libertyville, IL), 11 and 21 were from Bionet Research Intermediates (Camelford, UK), 16, 17, and 18 were from Enamin Ltd. (Kiev, Ukraine), and 19 and 20 were from Vitas-M Laboratory, Ltd. (Moscow, Russia). The purity of all 10 experimentally validated hits (1, 5, 8, 11, 14, 16−20) was greater than 95% as verified using HPLC, gas chromatography, or nuclear magnetic resonance (NMR) by the vendors or in our hands (1H NMR spectra of all these compounds are depicted in Supporting Information Figure S1). All other chemicals were of the highest grade available and purchased from commercial sources. In Silico Screening of Compounds Inhibiting DAO Activity. The X-ray crystallographic structures of ligand-free human DAO (PDB ID: 2E48)61 and human DAO complexed with respective ligands

Figure 6. Effects of the DAO inhibitors on intracellular D-Ala levels in cultured mammalian cells. The 293/NEO and 293.HA-hDAO-24 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, and the cells were treated with the respective DAO inhibitors (4Hfuro[3,2-b]pyrrole-5-carboxylic acid, or compounds 5, 8, or 11) or the vehicle for 30 min. Subsequently, the cells were exposed to 17 mM of exogenous D-Ala for 24 h, and amino acids were then extracted from the cells. The concentrations of D-Ala (A) and L-Ala (B) in the cells were determined by HPLC. Data are shown as the mean ± standard deviation of three independent assays. The single and double asterisks indicate significant differences (p < 0.05 and p < 0.01, respectively) compared to the vehicle-treated cells, based on the Dunnett multiple comparison test.

4H-furo[3,2-b]pyrrole-5-carboxylic acid and 8 (Table 3). Therefore, it appears that 5, in addition to 4H-furo[3,2b]pyrrole-5-carboxylic acid and 8, constitutes promising starting I

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(PDB ID: 3CUK, 3G3E, 2E49, 2E4A, 2E82, and 2DU8)40,42,55,61 were obtained from the Protein Data Bank (http://www.pdb.org). All structures were prepared for docking simulation analysis using the Protein Preparation Wizard software (Schrö dinger Suite 2009; Schrödinger, LLC, New York, NY). Specifically, hydrogen atoms were added to the proteins, the protonation state of His residues was optimized, the orientations of hydroxyl groups and Asn/Gln residues were corrected, and energy minimization was carried out with the OPLS-AA force field.62 After removing the ligands from the proteins, docking grids were generated around Leu-215, Tyr-224, Tyr-228, Arg283, and Gly-313 residues in the active site using the Glide grid generation module in the Glide software (Schrödinger Suite 2009; Schrödinger). To determine a criterion for the selection of candidate compounds during the in silico screening, a linear discriminant function was constructed as described below. The 51 types of compounds, which had been evaluated as novel DAO inhibitors in the reports of Duplantier et al. (2009) and Sparey et al. (2008),40,42 were classified into two groups based on their reported IC50 values: active (IC50 of ≤5 μM) and inactive (IC50 of >5 μM). The 3D structures of these compounds were built using the LigPrep and Epik softwares (Schrödinger Suite 2009; Schrödinger) by generating low energy tautomeric, stereoisomeric, and ionization states within the range of pH 7.0 ± 2.0, followed by the conformational sampling using the ConfGen software (Schrödinger Suite 2009; Schrödinger) with a maximum number of 10. Subsequently, docking of the resulting 3D structures of the compounds to the target DAO proteins prepared as described above was carried out using the Glide software in the standard precision (SP) mode. The best-docked pose with the lowest Glide score was selected for each compound, and pharmaceutically relevant properties of these compounds were evaluated using the QikProp software (Schrödinger Suite 2009; Schrödinger). Independent variables that could be used to discriminate the active compounds from the inactive ones were selected by a stepwise selection procedure using the freely available statistics computing platform R63 and the R function (http://aoki2.si.gunma-u.ac.jp/R/src/sdis.R). This resulted in the construction of the following linear discriminant function: Activity = −1.59 × (Glide docking-score) − 0.557 × (PSA) + 0.215 × (FISA) − 4.85, where PSA and FISA are the polar surface area and hydrophilic component of the solvent-accessible surface area, respectively. In this function, the compound is defined as active if “Activity” is greater than 0, whereas the compound is defined as inactive if “Activity” is equal to or less than 0. Commercially available 4 million compounds listed in the chemical structure database of Namiki Shoji Co. (http://www.namiki-s.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 four compounds (4H-furo[3,2-b]pyrrole-5-carboxylic acid, iminoDOPA, 3-hydroxyquinolin-2(1H)-one, and benzotetrazole). These four compounds were reported to bind to the active site of mammalian DAO.40,42,61,64 The screen used the Topomer Search and/or Unity 2D search modules in the SYBYL 8.1 software (Tripos, Inc., St. Louis, MO), and 13806 compounds that exhibited good similarity scores were selected as first hit compounds. Subsequently, the 3D structures of these first hit compounds were built and docked to the target DAO proteins, as described above. The best-docked pose with the lowest Glide score was selected for each compound, and the PSA and FISA of the compounds selected were evaluated, as described above. The Glide docking-score, PSA, and FISA were applied to the above-mentioned linear discriminant function, and 429 compounds that were defined as active were selected as second-hit compounds (Supporting Information Table S2). Of these second-hit compounds, 15 compounds (1− 15) were selected further by means of a cluster analysis using the Canvas software (Schrödinger Suite 2009; Schrödinger) and/or visual inspection and were used to test the inhibitory activity against human DAO in this study. Because 5, 8, 11, and 14 exhibited relatively high inhibitory activity against human DAO (Table 1), the structures of these compounds were used as lead structures for further in silico screening as described

below. Furthermore, structure-based molecular design was carried out to find additional candidates using the Muse software (Tripos, Inc.) and 1383 structures were generated as candidates. Subsequently, the 3D structures of these candidate compounds were built and docked to the target DAO proteins, and the potential ability of these compounds to inhibit the DAO activity was evaluated, as described above. Among several compounds that were defined as active in the linear discriminant function (Supporting Information Table S3), only four commercially available compounds (16−19) were selected and used to test the inhibitory activity against human DAO in this study. Because 14 has a novel chemical structure in comparison with known potent inhibitors of DAO, a chemical substructure search was carried out in the chemical structure database of Namiki Shoji Co. (http://www. namiki-s.co.jp/english/) using the JChem Base software (ChemAxon, Budapest, Hungary). In this search, the 1,3,4-oxadiazole structure of 14 was used as the query structure, and 13 analogues of 14 were identified (Supporting Information Table S4). Subsequently, the 3D structures of these analogues were built and docked to the target DAO proteins. The best-docked pose with the lowest Glide score was selected for each compound, and only two commercially available compounds (20 and 21) that exhibited lower Glide docking-score and/or Glide ligand efficiency than those of 14 were selected and used to test the inhibitory activity against human DAO. Construction of Recombinant Protein Expression Plasmids. The construction of expression plasmids for the N-terminally Histagged human DAO (pRSET-His-hDAO) and N-terminally Histagged human DDO (pRSET-His-hDDO) has been described previously.50,65 Y224F and Y224A substitutions were introduced into pRSET-HishDAO by site-directed mutagenesis using the QuikChange II SiteDirected Mutagenesis Kit (Agilent Technologies, Santa Clara, CA) to generate expression plasmids for the N-terminal His-tagged Y224F and Y224A variants of human DAO (pRSET-His-hDAO-Y224F and pRSET-His-hDAO-Y224A, respectively). The following mutagenic oligonucleotides were used (mutated codons are underlined): for Y224F, 5′-CCA GAG AGA GGC ATC TTC AAT TCC CCG TAC ATC-3′ and 5′-GAT GTA CGG GGA ATT GAA GAT GCC TCT CTC TGG-3′; for Y224A, 5′-GAC CCA GAG AGA GGC ATC GCC AAT TCC CCG TAC ATC AT-3′ and 5′-ATG ATG TAC GGG GAA TTG GCG ATG CCT CTC TCT GGG TC-3′. Introduction of the desired mutations was confirmed by sequencing. An entire cDNA clone of human Ser racemase (MGC ID: 103800), which was obtained from human brain,66 was purchased from Invitrogen (Carlsbad, CA). Using this clone as a template, the human Ser racemase cDNA was PCR-amplified. The primers used were as follows: 5′-GGA TCC GAT GTG TGC TCA GTA TTG CAT CTC CTT TG-3′ (forward primer) and 5′-AGA TCT TTA AAC AGA AAC AGA CTG ATA AGA AGC TG-3′ (reverse primer). These primers were designed to contain additional BamHI and BglII sites at the 5′-ends of the forward and reverse primers, respectively. The PCR product was cloned into pT7Blue (Novagen, Madison, WI), and the sequence was confirmed (pT7-hSerR). Subsequently, the 1.0 kb BamHI−BglII fragment containing the entire human Ser racemasecoding sequence of pT7-hSerR was subcloned into pRSET-B (Invitrogen) to construct the N-terminal His-tagged human Ser racemase expression plasmid (pRSET-His-hSerR). An entire cDNA clone of human DAO (MGC ID: 103812), which was obtained from human brain,66 was purchased from Invitrogen. Using this clone as a template, the human DAO cDNA was PCRamplified. The primers used were as follows: 5′-AAG CTT GAG ACA GGC CAT GTA CCC ATA CGA TGT TCC AGA TTA CGC TCG TGT GGT GGT GAT TGG AG-3′ (forward primer) and 5′-GAA TTC TCA GAG GTG GGA TGG TGG CAT TCT G-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 also was designed to contain a HAtag-coding sequence. The PCR product was cloned into pT7Blue, and the sequence was confirmed (pT7-HA-hDAO). Subsequently, the 1.1 kb HindIII−EcoRI fragment containing the entire N-terminal HAtagged human DAO-coding sequence of pT7-HA-hDAO was J

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mM ATP, 1 mM dithiothreitol, and 10 mM L-Ser in a final volume of 150 μL. The mixture was incubated at 37 °C for 10 min, and then 600 μL of 100% (v/v) methanol was added to stop the reaction. Subsequently, the mixture was incubated at −80 °C for 1 h and centrifuged at 20000g for 10 min at 4 °C to remove precipitated proteins. The supernatant (600 μL) was then filtered through a 0.45 μm filter (Millex-LH; Millipore, Bedford, MA), and the filtered solution was appropriately diluted with H2O. The following procedure for the analysis of amino acids is based on the method of Hashimoto et al. (1992).68 Specifically, 10 μL of the diluent was mixed with 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-Lcysteine in 1 mL of 100% [vol/vol] methanol) to fluorescently derivatize the amino acids in the mixture. After a 2 min incubation at room temperature, 10 μL of the sample was injected into the Jasco chromatographic system, which consists of a model PU-2089 pump, a model FP-2025 fluorescence detector, and a model 807-IT integrator (Jasco Corp., Tokyo, Japan). The sample was separated on an octadecylsilyl silica gel column of Mightysil RP-18GP (150 mm × 4.6 mm internal diameter; Kanto Chemical Co., Tokyo, Japan) at a flow rate of 1 mL/min, with a mobile phase of 50 mM sodium acetate buffer (pH 5.4):acetonitrile (82:18). The fluorescence was detected at an excitation wavelength of 344 nm and an emission wavelength of 443 nm. The amounts of D- and L-Ser were determined based on the peak areas in the chromatograms. One unit of enzyme activity was defined as the production of 1 μmol of D-Ser per min under the described assay conditions. For testing the inhibitory activity of candidate compounds, each compound was added to the reaction mixture. The relative inhibitory activity of the compounds was determined by considering the activity of the enzyme in the absence of compound as 100%. The IC50 values of tested compounds were determined using the following formula, as described elsewhere:69,70 IC50 = 10̂(log [A/B] × [50 − C]/[D − C] + log B), where A and B are the higher and lower concentrations nearest to the middle of the curve, respectively, while C and D are the inhibition percentages at the concentrations of B and A, respectively. The kinetic parameters of the recombinant human DAO and its variants were determined under conditions in which the production of 2-oxo acids was linear with incubation time and exhibited Michaelis− Menten type properties. For the determination of Vmax and Km values for D-Ala, different final concentrations (0.5−40 mM) of D-Ala were used as the substrate. The Vmax and Km values were calculated from the Hanes−Woolf plots.71 The kcat values were calculated from the Vmax values and the estimated molecular masses of the recombinant proteins (43631, 43615, and 43539 Da for N-terminal His-tagged human DAO, Y224F variant of human DAO, and Y224A variant of human DAO, respectively). The Ki values of several compounds were determined using the following formula described by Cheng and Prusoff (1973):59 Ki = IC50/(1 + [S]/Km), where [S] is the substrate concentration. These Ki values determined were confirmed by the replots of slope of Lineweaver−Burk plot versus inhibitor concentration.54 The LE values of several compounds were determined using the following formula described by Hopkins et al. (2004):60 LE = −RT ln Ki/Nnon‑hydrogen atoms, where R, T, and Nnon‑hydrogen atoms are the gas constant, the absolute temperature in Kelvin, and the number of nonhydrogen atoms, respectively. Spectrum Analysis of DAO. A plate reader (PowerWave XS, BioTek Instruments, Winooski, VT) was used to measure the absorption spectra of human DAO. Wavelength scans were carried out at 25 °C with the purified recombinant human DAO (1.7 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 40 μM (4Hfuro[3,2-b]pyrrole-5-carboxylic acid) or 200 μM (benzoate, 5, 8, 11, and malonate). The spectra were recorded 0, 2, 15, and 30 min after the compound addition. The scan range was 300−600 nm in the presence of benzoate, 5, and malonate, 320−600 nm in the presence of 4H-furo[3,2-b]pyrrole-5-carboxylic acid and 11, and 370−600 nm in the presence of 8. In these range, the absorption spectrum of each

subcloned into pcDNA3.1(+) (Invitrogen) to construct the Nterminal HA-tagged human DAO expression plasmid (pcDNA-HAhDAO). Expression and Purification of Recombinant Proteins. Escherichia coli strain 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-hDAO and pRSET-His-hDDO, as previously described50,65 with the following modifications. When the culture was grown to A620 = 0.5, the incubation temperature was decreased to 26 °C and then cells were grown for an additional 30 min. After adding 0.5 mM isopropyl-β-Dthiogalactopyranoside, the culture was further incubated at 26 °C for 16 h. Cells were then centrifuged at 10000g for 10 min at 4 °C, and crude extract was prepared using BugBuster protein extraction reagent and Lysonase bioprocessing reagent (Novagen) in the presence of 50 μM FAD and protease inhibitors (Nacalai Tesque, Kyoto, Japan), according to the manufacturer’s instructions. Under the same conditions, cells transformed with pRSET-His-hDAO-Y224F, pRSET-His-hDAO-Y224A, and pRSET-His-hSerR were cultured and the crude extracts were prepared; in the case of the cells transformed with pRSET-His-hSerR, 50 μM pyridoxal phosphate was used instead of FAD. All recombinant proteins were purified by affinity chromatography using a chelating column. Crude extracts were applied to a His GraviTrap column (GE Healthcare Bio-Sciences, Piscataway, NJ) and equilibrated with 20 mM sodium phosphate buffer (pH 7.4) containing 0.5 M NaCl and 10 mM imidazole. Then the column was washed with the same buffer, and bound proteins were eluted using a stepwise gradient of 50−500 mM imidazole. Each fraction (2.0 mL) containing recombinant human DAO, human DDO, Y224F variant of human DAO, or Y224A variant of human DAO was dialyzed twice for 3 h at 4 °C 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. Each fraction (2.0 mL) containing recombinant human Ser racemase was dialyzed twice for 3 h at 4 °C against 1 L of 10 mM Tris-HCl buffer (pH 8.0) containing 5 mM 2-mercaptoethanol and 10% (v/v) glycerol. The dialyzed fractions were recovered and centrifuged at 10000g for 10 min at 4 °C to pellet the proteins denatured during dialysis. The supernatants were pooled as purified enzymes and used immediately for enzyme assays or stored frozen at −80 °C until use. All recombinant proteins were purified to near-homogeneity when examined by SDS-polyacrylamide gel electrophoresis. From 1 L of fermentation broth, the following amounts of purified N-terminal His-tagged proteins were obtained: 4.5 mg of human DAO, 2.3 mg of human DDO, 4.3 mg of Y224F variant of human DAO, 8.5 mg of Y224A variant of human DAO, and 2.5 mg of human Ser racemase. The protein concentrations of the purified enzyme preparations were determined using the Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin as a standard. Enzymatic Activity Assays. The activity of DAO and DDO was determined using a colorimetric assay for 2-oxo acid production as previously described.67 Briefly, appropriate amounts (0.23−4.2 μg) of the purified enzymes were added to a reaction mixture of air-saturated 40 mM sodium pyrophosphate buffer (pH 8.3), 23 U A. niger catalase, 60 μM FAD, and 10 mM amino acids in a final volume of 150 μL. In the case of the reaction of Y224F and Y224A variants of human DAO, 15 and 60 mM D-Ala, respectively, were used as the substrate. The mixture was incubated at 37 °C for 10 min, and then 10 μL of 100% (weight/vol) trichloroacetic acid was added to stop the reaction. The 2-oxo acid product was reacted with 2,4-dinitrophenylhydrazine and quantitated by measuring the A445 against a blank mixture lacking amino acids. One unit of enzyme activity under the described assay conditions was defined as the production of 1 μmol of 2-oxo acid per min. The activity of Ser racemase was determined by measuring D-Ser formed from L-Ser in the reaction mixture. Appropriate amounts (30 μg) of the purified enzyme were added to a reaction mixture of 40 mM Tris-HCl buffer (pH 8.0), 60 μM pyridoxal phosphate, 1 mM MgCl2, 1 K

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compound did not interfere with the measurement of spectral changes of DAO. Cell Lines. Human embryonic kidney 293 cells were cultured in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal bovine serum at 37 °C in 5% CO2/95% air. For preparation of the DAO-overexpressing mammalian cell line, pcDNA-HA-hDAO was transfected into 293 cells using the TransIT-293 transfection reagent (Mirus Bio Corp., Madison, WI) according to the manufacturer’s instructions. The cells were selected for resistance to 800 μg/mL G418, and drug-resistant cells were mixed as a stably transfected cell pool (293/HA-hDAO). Subsequently, several single cell clones were isolated from 293/HA-hDAO cells by the standard limited dilution method.72 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 10000g for 10 min at 4 °C to remove insoluble material. The cell lysates were then subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis using a mouse anti-HA antibody (Anti-HA [mouse IgG1-κ], Monoclonal [HA124], AS; Nacalai Tesque) (1:2,500 dilution) as the primary antibody, and horseradish peroxidaseconjugated antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) (1:5,000 dilution) as the secondary antibody. Because the most intensely positive band was detected in the sample prepared from clone 24, this cell line (293.HA-hDAO-24) was used for further experiments. For preparation of the cell line that acquires resistance to G418 but does not overexpress DAO, 293 cells were transfected with the parental plasmid pcDNA3.1(+) and selected for resistance to G418. Drug-resistant cells were mixed as a stably transfected cell pool (293/ NEO) and were used as control cell line in this study. The 293.HAhDAO-24 and 293/NEO cells were maintained in Dulbecco’s Modified Eagle’s Medium supplemented with 10% (v/v) fetal bovine serum and 800 μg/mL G418 at 37 °C in 5% CO2/95% air. Determination of Amino Acid Contents in Cells and Culture Media. The 293.HA-hDAO-24 and 293/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 mL of fresh medium, and then 100 μL of each compound solution or the vehicle (5% [vol/vol] dimethylsulfoxide) was added. After 30 min incubation, 100 μL of 0− 200 mM D-Ala solution was added, and the cells were cultured for 24 h before extraction of amino acids in the cells and culture media. The amino acids in the cells and culture media were extracted by methanol, as previously described73 with the following modifications. Specifically, the culture media was recovered and centrifuged at 300g for 5 min at 4 °C to remove cell debris. The supernatant (120 μL) was then mixed with 480 μL of 100% (v/v) methanol in a 1.5 mL microtube, and the mixture was incubated at −80 °C for 1 h to extract the amino acids. The cells were washed twice with ice-cold 10 mM phosphate-buffered saline (pH 7.4), collected with a cell scraper, and 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 and culture media were centrifuged at 20000g for 10 min at 4 °C to remove precipitated proteins. The supernatant (500 μL) was then evaporated to dryness, and the residue was dissolved in 50 μL of 200 mM borate buffer (pH 10.2) and filtered through 0.45 μm filter (Millex-LH; Millipore). The filtered solution was appropriately diluted with the same buffer and stored at −80 °C until the analysis of amino acids was performed. The concentrations of D- and L-Ala were determined in the cells and culture media by HPLC using the OPA precolumn derivatization technique, as described by Nimura and Kinoshita (1986).74 An aliquot (10 μL) of the diluent was prepared, and then 30 μL of 200 mM borate buffer (pH 10.2) and 20 μL of OPA/NAC reagent (prepared by mixing 8 mg of OPA with 10 mg of NAC in 1 mL of 100% [vol/ vol] methanol) were added to fluorescently derivatize the amino acids in the mixture. After 2 min incubation at room temperature, 10 μL of the sample was injected into the Jasco chromatographic system. The

sample was separated on an octadecylsilyl silica gel column of Mightysil RP-18GP (150 mm × 4.6 mm internal diameter; Kanto Chemical Co.) at a flow rate of 1 mL/min, with a mobile phase of 70 mM phosphate buffer (pH 6.3):methanol (73:27). The fluorescence was detected at an excitation wavelength of 350 nm and an emission wavelength of 445 nm. The amounts of D- and L-Ala were determined based on the peak areas in the chromatograms.



ASSOCIATED CONTENT

S Supporting Information *

Chemical information on the compounds identified by in silico screening, activity values of 429 compounds that were identified by similarity searching and were defined as active in the linear discriminant function, activity values of 155 compounds that were identified by structure-based molecular invention and were defined as active in the linear discriminant function, Glide docking score and Glide ligand efficiency of 13 analogues of compound 14, 1H NMR spectra for experimentally validated hits. This material is available free of charge via the Internet at http://pubs.acs.org.



AUTHOR INFORMATION

Corresponding Author

*Phone: +81-3-5791-6229. Fax: +81-3-5791-6381. E-mail: [email protected]. Author Contributions ∥

These two authors contributed equally to this work.

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Noriko Sato (Graduate School of Pharmaceutical Sciences, Kitasato University) for performing the NMR analyses. This work was supported in part by a Grant-in-Aid for Scientific Research (24790086) from the Japan Society for the Promotion of Science and a Kitasato University Research Grant for Young Researchers (to M.K.).



ABBREVIATIONS USED DAO, D-amino acid oxidase; DDO, D-aspartate oxidase; FAD, flavin adenine dinucleotide; HPLC, high-performance liquid chromatography; IC50, 50% inhibitory concentration; LE, ligand efficiency; NAC, N-acetyl-L-cysteine; NMDA, Nmethyl-D-aspartate; NMR, nuclear magnetic resonance; OPA, o-phthalaldehyde



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