Flavonoids as Substrates and Inhibitors of Myeloperoxidase

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Flavonoids as Substrates and Inhibitors of Myeloperoxidase: Molecular Actions of Aglycone and Metabolites Yuko Shiba,†,# Takashi Kinoshita,‡,# Hiroshi Chuman,‡ Yutaka Taketani,§ Eiji Takeda,§ Yoji Kato,| Michitaka Naito,⊥ Kyuichi Kawabata,† Akari Ishisaka,† Junji Terao,† and Yoshichika Kawai*,† Department of Food Science, Graduate School of Nutrition and Biosciences, The UniVersity of Tokushima, Tokushima 770-8503, Japan, Theoretical Chemistry for Drug DiscoVery, Institute of Health Biosciences, The UniVersity of Tokushima, Tokushima 770-8505, Japan, Department of Clinical Nutrition, Graduate School of Nutrition and Biosciences, The UniVersity of Tokushima, Tokushima 770-8503, Japan, School of Human Science and EnVironment, UniVersity of Hyogo, Himeji 670-0092, Japan, and DiVision of Nutrition and Health, School and Graduate School of Life Studies, Sugiyama Jogakuen UniVersity, Nagoya 464-8662, Japan ReceiVed March 2, 2008

Myeloperoxidase (MPO), secreted by activated neutrophils and macrophages at the site of inflammation, may be implicated in the oxidation of protein/lipoprotein during the development of cardiovascular diseases. Flavonoids have been suggested to act as antioxidative and anti-inflammatory agents in ViVo; however, their molecular actions have not yet been fully understood. In this study, we examined the molecular basis of the inhibitory effects of dietary flavonoids, such as quercetin, and their metabolites on the catalytic reaction of MPO using a combination of biological assays and theoretical calculation studies. Immunohistochemical staining showed that a quercetin metabolite was colocalized with macrophages, MPO, and dityrosine, an MPO-derived oxidation product of tyrosine, in human atherosclerotic aorta. Quercetin and the plasma metabolites inhibited the formation of dityrosine catalyzed by the MPO enzyme and HL-60 cells in a dose-dependent manner. Spectrometric analysis indicated that quercetin might act as a cosubstrate of MPO resulting in the formation of the oxidized quercetin. Quantitative structure-activity relationship studies showed that the inhibitory actions of flavonoids strongly depended not only on radical scavenging activity but also on hydrophobicity (log P). The requirement of a set of hydroxyl groups at the 3, 5, and 4′-positions and C2-C3 double bond was suggested for the inhibitory effect. The binding of quercetin and the metabolites to a hydrophobic region at the entrance to the distal heme pocket of MPO was also proposed by a computer docking simulation. The current study provides the structure-activity relationships for flavonoids as the anti-inflammatory dietary constituents targeting the MPO-derived oxidative reactions in ViVo. Introduction It has been suggested that oxidation modification of biomolecules plays an important role in the pathogenesis of several diseases including atherosclerosis, cancer, and neurodegenerative diseases. In addition to nonenzymatic oxidation reactions, several enzymes including peroxidases are suggested to catalyze the oxidative modification in some tissues. Myeloperoxidase (MPO1) is the most abundant protein in neutrophils, monocytes, and macrophages (1, 2). In the presence of hydrogen peroxide (H2O2), the ferric (Fe(III))-form MPO is oxidized by a twoelectron equivalent forming the redox intermediate compound I (Reaction 1). The main physiological substrate of compound * To whom correspondence should be addressed. Tel: 81-88-633-9592; Fax: 81-88-633-7089. E-mail: [email protected]. † Department of Food Science, The University of Tokushima. ‡ Theoretical Chemistry for Drug Discovery, The University of Tokushima. § Department of Clinical Nutrition, The University of Tokushima. | University of Hyogo. ⊥ Sugiyama Jogakuen University. # These authors contributed equally to this study. 1 Abbreviations: MPO, myeloperoxidase; DPPH, 1,1-diphenyl-2-picrylhydrazyl; Q3GA, quercetin-3-O-β-D-glucuronide; PBS, phosphate buffered saline; BSA, bovine serum albumin; HPLC, high-performance liquid chromatography; Q3G, quercetin-3-O-β-D-glucoside; TTBS, Tris-buffered saline containing 0.05% Tween 20; QSAR, quantitative structure-activity relationship; SHA, salicylhydroxamic acid.

I is chloride (Cl-), which undergoes a two-electron oxidation to form hypochlorous acid (HOCl) (Reaction 2), a strong chlorinating oxidant. HOCl can chlorinate biomolecules including proteins, lipids, and nucleic acids at the site of inflammation (3–7). Compound I also reacts with various organic substrates (RH), while the heme undergoes two sequential one-electron reduction steps, generating compound II and the ferric-form MPO, respectively (Reaction 3 and Reaction 4).

MP3 + H2O2 f compound I

(Reaction1)

Compound I + Cl- f MP3 + HOCl

(Reaction2)

Compound I + RH f compound II + R· Compound II + RH f MP

3+

+ R·

(Reaction3) (Reaction4)

It has been suggested that the low molecular substrates interact with the hydrophobic pocket of the MPO enzyme and serve as substrates for the MPO (8, 9). L-Tyrosine, an endogenous substrate, is oxidized to the tyrosyl radical, generating the stable dimer dityrosine (1). MPO also catalyzes the oneelectron reaction of NO2- to NO2·, an intermediate that nitrates biomolecules (10, 11). These MPO-derived oxidation products have been detected in human atherosclerotic lesions (12–14). These observations suggest that MPO-derived oxidation reactions may be implicated in the pathogenesis of inflammatory

10.1021/tx8000835 CCC: $40.75  2008 American Chemical Society Published on Web 07/12/2008

FlaVonoids as Substrates and Inhibitors

diseases such as atherosclerosis. Indeed, the formation of atherosclerotic lesions was enhanced in MPO-transgenic mice (15). Numerous compounds have been reported to inhibit MPO (16, 17). One type of inhibition of the production of HOCl involves trapping the MPO as compound II. This type of inhibition is reversible because superoxide and other substrates reduce compound II to its ground state. In recent years, flavonoids have been expected to exert beneficial effects on human health and to prevent or ameliorate several diseases. Epidemiological studies have shown the potential roles of flavonoid intake on lowering the risk for the incidence of cardiovascular diseases and cancer (18–23). Although a number of mechanisms including scavenging and/ or antioxidant properties has been proposed for the beneficial effects of flavonoids in ViVo, the precise molecular mechanisms are still unclear. We have recently shown that a metabolite of quercetin (3,3′,4′,5,7-pentahydroxyflavone), a major flavonoid in the human diet, specifically accumulates in activated macrophages in human atherosclerotic aorta (24). In addition, the strong inhibitory effect of quercetin aglycone on the MPOcatalyzed oxidation reactions was demonstrated in Vitro (25, 26). On the basis of this information, we presumed that inhibition of MPO activity might be one mechanism for the prevention of cardiovascular diseases by flavonoids. During absorption, quercetin, as well as other flavonoids, is metabolically converted to the glucuronidated and/or sulfated forms by intestinal and hepatic enzymes (27). A part of the quercetin can also be methylated on the B-ring catechol. In general, the amount of free quercetin aglycone is under the detection limit in human plasma (28). Therefore, intake of a flavonoid provides a variety of structurally different metabolites in the plasma and tissues. In this study, we examined the inhibitory effects of flavonoids and their analogues including their endogenous metabolites on MPO activity using a biological assay system in Vitro. Furthermore, to understand the molecular actions of the flavonoids and the metabolites, quantitative structure-activity relationship (QSAR) studies and a computer docking simulation for flavonoids were performed.

Experimental Procedures Materials. MPO from human sputum (purity 95%) was obtained from Elastin Products Co., Inc. (Owensville, MO). Quercetin dihydrate and sulfatase H-1 (from Helix pomatia) were purchased from Sigma-Aldrich Co. (St. Louis, MO). L-Tyrosine, fisetin, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Other flavonoids were obtained from Extrasynthese (Genay, France). Quercetin-3β-D-glucuronide (Q3GA) was chemically synthesized as previously reported (29). The mouse monoclonal antibody to CD68 (Clone KP-1, M814) and rabbit polyclonal antibody to MPO (A398) were purchased from Dako Japan (Kyoto, Japan). The mouse monoclonal antibody to dityrosine (mAb1C3) was prepared as previously reported (30). The mouse monoclonal antibody to Q3GA (mAb14A2) was recently developed by immunizing mice with Q3GA-keyhole limpet hemocyanin conjugate as the immunogen (24). Cell Culture. HL-60 cells were obtained from the American type Culture Collection and were grown in Dulbecco’s modified Eagle’s medium (DMEM; Sigma) supplemented with 10% fetal bovine serum, 100 µg/mL penicillin, and 100 units/mL streptomycin in a 5% CO2-containing atmosphere. Immunohistochemical Staining. Paraffin-embedded aortas were obtained from autopsy cases in Nagoya University Hospital, and tissue sections were prepared in 5-µM thickness. Sections were deparaffinized in xylene, hydrated in aqueous ethanol, and incubated in phosphate buffered saline (PBS) containing 1% (w/v) bovine serum albumin (BSA) and 1.35% (v/v) normal serum from the same

Chem. Res. Toxicol., Vol. 21, No. 8, 2008 1601 host species of the secondary antibody for 30 min to block the nonspecific binding of the secondary antibody. To obtain maximal sensitivity of staining, sections were activated in Vector Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, USA) by autoclave at 121 °C for 10 min prior to blocking. The primary antibodies in PBS containing 1% BSA were treated at 4 °C overnight. Immunostaining was performed using the avidin-biotin complex method with the Vectastain ABC-AP (alkaline phosphatase) kit and Vector Alkaline Phosphatase Substrate Kit II (Vector Laboratories, Burlingame, CA, USA). All sections were counterstained with hematoxylin (Wako). Dityrosine Formation Assay. MPO (0.1 units/mL) was mixed with different concentrations of the test compounds and 0.2 mM tyrosine in chelex-100-treated 0.1 M phosphate buffer (pH 7.4). The reaction was initiated by adding 200 µM H2O2, followed by incubation at 37 °C for 1 h. The reaction was terminated by adding catalase (25 µg/mL). The reaction mixture was treated with Ultrafree MC membrane (Millipore, Billerica, MA), and the fluorescence intensity of the filtrates was measured by high-performance liquid chromatography (HPLC) with a fluorescence detector or directly by a Hitachi F-4000 spectrofluorophotometer (Hitachi, Tokyo, Japan). HPLC separation was performed using a TSK-gel ODS80Ts column (4.6 × 150 mm, Tosoh, Tokyo, Japan) equilibrated with 5% acetic acid/methanol (29/1) at a flow rate of 1 mL/min with fluorescence detection at 300 nm excitation/400 nm emission. The IC50 values indicating the 50% inhibitory concentrations were calculated by a KaleidaGraph 3.6J (Synergy Software). Cell-mediated dityrosine formation was evaluated as follows. HL60 cells (1 × 106 cells) were mixed with tyrosine (0.2 mM) and different concentrations of flavonoids in PBS. The reaction was initiated by adding 1 mM H2O2, and the mixture was incubated at 37 °C for 1 h. After termination of the reaction, the cells were precipitated by centrifugation, and the supernatants were then used for the analysis of dityrosine as described above. Animal Experiment. Male CD rats (Crl:CD(SD), 7 weeks, Charles River Laboratories Japan, Inc., Yokohama, Japan) were divided randomly into control and experimental groups. This study was performed according to the guidelines for the care and use of laboratory animals of The University of Tokushima Graduate School, Institute of Health Biosciences. Quercetin-3-β-D-glucoside (Q3G) was dissolved in 0.5% aqueous carboxymethylcellulose and then orally administered (50 mg/kg body weight) to one group. The control group was treated with 0.5% aqueous carboxymethylcellulose. After 30 min, plasma was collected from each rat. Plasma mixed with 5 volumes of methanol was centrifuged, and the supernatants were dried under a nitrogen stream. The residues were dissolved in PBS and then used as the nonprotein plasma fraction for the dityrosine assay. The total content of quercetin derivatives in the plasma was measured by HPLC after β-glucuronidase/sulfatase treatment. Briefly, 50 µL plasma samples were mixed with 25 µL of 2 mg/ mL sulfatase H-1 (from Helix pomatia, 14 units of sulfatase and 300 units of β-glucuronidase activity/mg enzyme) in 50 mM sodium phosphate buffer (pH 5.0) and incubated at 37 °C for 2 h. After incubation, the proteins were removed by adding 5 volumes of methanol followed by centrifugation, and the supernatants were dried under a nitrogen stream. The residues were dissolved in HPLC solvent (solvent A/B ) 85/15) and injected into a Cadenza CDC18 column (3 × 100 mm, Imtakt Corporation, Kyoto, Japan) equilibrated with 15% B at a flow rate of 0.45 mL/min. Solvents A and B were H2O/tetrahydrofuran/trifluoroacetic acid (98/2/0.1) and acetonitrile, respectively. The gradient program was as follows: 0-5 min (15% B), 5-22 min (15-30% B), 22-25 min (30-50% B), and 25-30 min (50-15% B). The peaks for quercetin (retention time ) 20 min) and methylated quercetins (mixture of 3′-methyl and 4′-methyl quercetins, retention time ) 25 min) were monitored at UV 370 nm. On the basis of the peak areas of authentic compounds, the plasma concentrations of quercetin (nonmethylated metabolites) and methylated quercetins (methylated metabolites) were calculated. For HPLC analysis of the intact plasma metabolites, plasma samples were analyzed by a procedure similar to that

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described above without sulfatase H-1 treatment. The samples were injected into a TSK-gel ODS 80Ts column (4.6 × 150 mm) equilibrated with 15% B at a flow rate of 0.8 mL/min. Solvents A and B were 0.5% (v/v) phosphoric acid and acetonitrile, respectively. The gradient program was as follows: 0-2 min (15% B), 2-22 min (15-40% B), 22-24 min (hold), and 24-32 min (40-15% B). Immunoblot Analysis of MPO. The HL-60 cells were washed twice with phosphate-buffered saline (pH 7.0) and lysed with RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1 mM ethylenedinitrilotetraacetic acid) containing 1 mM phenylmethylsulfonyl fluoride. The protein samples were boiled with reducing sample buffer for 5 min. The samples were run on 10% SDS-polyacrylamide gels, transferred to a nitrocellulose membrane (Hybond-ECL, GE Healthcare), incubated at room temperature for 1 h with 5% skim milk in TTBS (Tris-buffered saline containing 0.05% Tween 20) for blocking, washed in TTBS, and treated with rabbit antihuman MPO antibody at 4 °C overnight. After washing, the blots were further incubated for 1 h at room temperature with antirabbit IgG antibody coupled with horseradish peroxidase in TTBS. After washing, the membrane was visualized using an ECL detection reagent. Compound II Reduction Assay. It has been reported that an excess H2O2 converts the MPO ground state into a relatively longlife compound II (31, 32). MPO (0.5 µM, λmax ) 430 nm) was treated with H2O2 (25 µM), resulting in the formation of compound II (λmax ) 456 nm). Flavonoids were added immediately to the compound II solution and monitored after 15 s by a spectrophotometer DU-640 (Beckman). DPPH Radical Scavenging Activity. DPPH (0.25 mM) in 0.1 M Tris-HCl buffer (pH 7.5) was mixed with different concentrations of the test compounds or ascorbic acid. After incubation at room temperature for 20 min, the absorbance of the reaction mixture was measured at 517 nm. The standard curve for ascorbic acid was developed, and the ascorbic acid-equivalent was then calculated. The DPPH radical scavenging activity was expressed as mol DPPH scavenging/mol test compound on the basis of the information that one mole of ascorbic acid may scavenge two moles of DPPH radicals (29). Quantitative Structure-Activity Relationship (QSAR). We determined the molecular geometries of all the compounds in this work using the ab initio molecular orbital method with the level of HF/6-31G* basis set in the Gaussian 03 package (Gaussian, Inc., Wallingford, CT). We estimated the octanol-water partition coefficient log P (C log P) by using Bioloom (BioByte Corp., Claremont, CA). We already reported that the estimated value of C log P is not that much deviated from the measured one (33). Statistical analyses were preformed on the basis of multilinear regression; n represents the number of compounds, r is the correlation coefficient, s is the standard deviation, F is the ratio of regression and residual. Docking simulation between a flavonoid and MPO was performed with a docking program FlexX in SYBYL 6.91 (Tripos, Inc., St. Louis, MI). In the docking procedure, the active site of MPO is defined as amino acid residues within a 6.5Å distance from the heme moiety and the residues locating in the close vicinity of the heme, Gln91, His95, and Arg239 (PDB code, 1DNW). The result of the FlexX docking was visualized in Pymol 0.99 (http://pymol.sourceforge.net/). Surface Plasmon Resonance (SPR) Analysis. To detect interaction between flavonoids and MPO, SPR analysis was performed using Biacore X (GE Healthcare UK Ltd., Buckinghamshire, UK). MPO was immobilized on a CM5 sensor chip (research grade) using the amino coupling kit. Aliquots of 40 µL of flavonoids in Hepes-EDTA buffer (10 mM Hepes of pH 7.4, 3.4 mM EDTA, 0.15 M NaCl, and 0.005% Tween 20) was injected to the flow cell at a flow rate of 20 µL/min. All reactions were carried out at 25 °C. Affinity between flavonoids and MPO was analyzed by BIAevaluation software 4.0.

Shiba et al.

Figure 1. Immunohistochemical staining of human atherosclerotic lesions. (A) Anti-CD68 (a macrophage marker), (B) anti-MPO, (C) antiquercetin-3-glucuronide (Q3GA, a major quercetin metabolite) mAb14A2, and (D) antidityrosine (DiTyr) mAb1C3. Magnification ×20.

Results MPO Colocalizes with Quercetin Epitopes in Human Atherosclerotic Lesions. To examine whether quercetin can interact with MPO in ViVo, immunohistochemical staining of human atherosclerotic lesions, a target of accumulation of MPO (34) and quercetin (24, 35), was carried out (Figure 1). The immunoreactive materials with anti-MPO (panel B) and antiQ3GA (panel C) were colocalized in macrophage-derived foam cells identified by the staining with anti-CD68 (panel A). In addition, dityrosine, a MPO-derived tyrosine dimer, was also colocalized in macrophage cells (panel D). These results suggest that quercetin metabolites could interact with the MPO enzyme and the oxidative product dityrosine in macrophage cells in human aortic tissues. Quercetin and the Metabolites Inhibit MPO-Mediated Dityrosine Formation. We have recently developed a simple HPLC-fluorescence assay system to evaluate the inhibitory effects of various antioxidants on the formation of dityrosine in the MPO/H2O2/tyrosine system (26). The benefit of this system is that it excludes the scavenging activity of chlorinating intermediates such as HOCl because the reaction is performed under chloride-free conditions. Although quercetin was found to be one of the strongest inhibitors in this assay system (26), the inhibitory mechanism and the physiological significance have not yet been elucidated. In this reaction system, the concentration of dityrosine formed was approximately 4.6 µM. As shown in Figure 2A, quercetin indeed inhibited the formation of dityrosine in a dose-dependent manner. However, quercetin is found in human/rodent plasma as the glucuronidated and/or sulfated metabolites rather than the aglycone (free) form. As shown in Figure 2B, Q3GA, a major metabolite in rats and humans (28, 29), also inhibited MPO-derived dityrosine formation in a dose-dependent manner, and the activity was comparable to that of quercetin aglycone. Although it is generally thought that conjugation of flavonoids attenuates their biological activities (24, 29), this result indicates that glucuronide metabolites of quercetin, as well as aglycone, could be the potent inhibitor of MPO reactions. During the MPO/H2O2/tyrosine system, tyrosine was initially oxidized to the tyrosyl radical and subsequently dimerized into dityrosine (Figure 2C). Thus, the following two possible actions of quercetin were suggested: (i) quercetin scavenges the formed tyrosyl radical and/or (ii) quercetin inhibits the catalytic action of the MPO enzyme.


Figure 2. Inhibition assay for MPO-catalyzed tyrosine dimerization reation. (A) HPLC profiles for the reaction mixtures of MPO/H2O2/ tyrosine in the presence of different concentrations of quercetin. (B) Inhibitory effect of quercetin and quercetin-3-glucuronide (Q3GA) on MPO-catalyzed dityrosine formation. (C) Proposed scheme for the formation of dityrosine during the reaction of MPO/H2O2/tyrosine system (ref 1).

To examine whether quercetin metabolite mixtures present in plasma could inhibit MPO reactions, the inhibitory effect of rat plasma extract after oral intake of a quercetin glucoside was examined. Figure 3A shows the HPLC profile for the quercetin metabolites in rat plasma after quercetin-3-glucoside intake. It has been reported that many types of glucuronidated and/or sulfated quercetin metabolites with or without methylation of the B-ring catechol have been found in human/rodent plasma after the intake of quercetin (28, 29). The total quercetin contents with or without methylation were measured as shown in Figure 3B. As shown in Figure 3C, compared with control rat plasma, quercetin-fed rat plasma significantly inhibited the dityrosine formation. These results raise the possibility that quercetin (or other flavonoids) and its metabolites can inhibit the MPOderived oxidation reactions in ViVo. To further examine whether quercetin and its metabolites can inhibit cell-mediated MPO reactions, a dityrosine formation assay was performed using HL-60 cells, a well-known neutrophil/macrophage-like cell line that expresses the MPO enzyme. The HL-60/tyrosine/H2O2 reaction system resulted in a significant formation of dityrosine, whereas the lack of one component failed to generate dityrosine (Figure 3D). The concentration of dityrosine formed in this system was estimated to be approximately 5.9 µM. Quercetin successfully inhibited the HL60-derived dityrosine formation. Furthermore, a major quercetin metabolite Q3GA as well as aglycone also inhibited cellmediated dityrosine formation in a dose-dependent manner

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Figure 3. Quercetin metabolites can inhibit MPO-catalyzed dityrosine formation. (A) HPLC profiles of the plasma extract of a rat fed with quercetin-3-glucoside (Q3G, 50 mg/kg body weight). Plasma was collected 30 min after intake. As comparison with authentic standards, Q3GA, quercetin aglycone (Q), and 3′- or 4′-methylated quercetin (MeQ) were identified. Asterisks indicate the peaks of quercetin metabolites, not found in control plasma. (B) Plasma concentrations of quercetin metabolites determined by HPLC analysis. Samples (n ) 6) were analyzed after treatment with β-glucuronidase/sulfatase and then detected as quercetin (nonmethylated metabolites) and methylated quercetin (methylated metabolites). Control, 0.5% carboxymethylcellulose; Q3G-fed, 50 mg/kg Q3G-administered orally. (C) Inhibitory effect of Q3G-fed plasma extracts on MPO-catalyzed dityrosine formation expressed as % inhibition versus blank (no plasma sample) reaction. *Student’s t test, P < 0.05 (n ) 3). (D) Cell-mediated formation of dityrosine and the inhibition by quercetin. HL-60 cells (1 × 106 cells) were incubated with tyrosine (200 µM) and 1 mM H2O2 in PBS at 37 °C for 1 h in the absence or presence of quercetin (10 µM). (E) Inhibition of cell-mediated dityrosine formation by different concentrations of quercetin and Q3GA. Ascorbic acid was also used as positive control. Data shown in D and E are the means of duplicate determinations.

(Figure 3E). Compared with ascorbate, a representative watersoluble antioxidant, quercetin and glucuronide may be good inhibitors, at least in this experimental model. In these experiments, the protein expression of the MPO enzyme in HL-60 was confirmed by immunoblot analysis (data not shown). We confirmed that MPO expression was not affected by the treatment with quercetin compounds at least under these experimental conditions (data not shown). In the dityrosine formation assays, we used a relatively higher concentration (200 µM or 1 mM) of H2O2 to avoid the consumption of H2O2 by antioxidative flavonoids. We then optimized the H2O2 concentration to clarify the inhibitory effects of flavonoids on the MPO reaction. Inhibition of MPO-Derived Dityrosine Formation by Various Flavonoids. As mentioned above, quercetin and other flavonoids are found as their metabolite forms, in which at least one hydroxyl group was modified by glucuronidation, sulfation, and/or methylation. Therefore, it is important to understand which hydroxyl group contributes to the inhibitory effects of flavonoids on MPO-catalyzed reactions. Several quercetin

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Figure 4. Chemical structures of quercetin and the analogous flavonoids.

analogues (see Figure 4), which lack a different position of a hydroxyl group or a C2-C3 double bond, were then evaluated. IC50, the concentration that inhibited the formation of dityrosine by 50%, was calculated for each flavonoid. Quercetin was the strongest inhibitor (IC50 ) 1.27 µM) among the five flavonoids. The order of the inhibitory effect (IC50, µM) was as follows: quercetin (1.27) > kaempferol (2.08) > fisetin (3.87) > luteolin (4.22) > taxifolin (6.63). Although kaempferol, lacking a 3′OH, exhibited an inhibitory effect comparable to that of quercetin, fisetin and luteolin, lacking 5- and 3-OH, respectively, were relatively weak, showing the significance of the number and position of the hydroxyl group on the inhibitory effects. In addition, taxifolin, which contains the same numbers and positions of hydroxyl groups as quercetin, exhibited weak activity, showing the requirement of a C2-C3 double bond for the activity. The DPPH radical scavenging activity of flavonoids was also measured to examine the contribution of tyrosyl radical scavenging to the inhibition of dityrosine formation by these compounds. Quercetin was also the strongest radical scavenger among the five flavonoids: quercetin (9.5) > luteolin (5.6) > fisetin (3.7) > taxifolin (2.8) > kaempferol (2.6) (mol DPPH trapped/mol). The order of the radical scavenging activity corresponded to the results previously reported (36). It is of interest that the IC50 for dityrosine formation was not fully correlated with the radical scavenging activity, especially for luteolin and kaempferol. To further examine the structure-activity relationship, another 18 related compounds were also tested for dityrosine formation and DPPH radical assay. The results for the total of 23 compounds are summarized in Supporting Information, Table 1. The list contains 16 aglycones, 6 glycosides, and a sulfate. The inhibitory effects of aglycone compounds on dityrosine formation generally correlated with DPPH radical scavenging activity, but the correlation coefficient was not statistically significant (r ) 0.64). Quercetin as the Substrate of MPO. The one-electron reduction of compound I to compound II has been reported as the inhibitory action of phenolic compounds (17), and formed compound II could be further reduced by the substrates in a one-electron reaction to regenerate the native enzyme (Figure 5A). To investigate whether quercetin and other flavonoids act as one-electron reaction substrates, the compound II reduction assay was examined. Compound II is more stable than compound I (37). It has been reported that compound II is also reduced by a one-electron reducing substrate such as tyrosine (31). A good preparation of compound II is obtained by adding a 50-fold excess of H2O2 to the pure native enzyme. The formation of compound II was observed with an absorption maximum at 456 nm. Addition of tyrosine, an established

Figure 5. Quercetin as substrate of MPO. (A) Spectrometric evaluation of one-electron reduction of MPO compound II by the substrates: broken lines, ground state; dotted lines, compound II formed by the addition of excess H2O2; solid lines, immediately after addition of substrates. The absorbance maximum at 456 nm (indicated as a gray line) shows the presence of compound II. Top, tyrosine (50 µM); middle, quercetin (0.25 µM); bottom, luteolin (0.25 µM). Kinetic scheme of MPO reactions is illustrated in a box. (B) Schematic presentation of the oxidation of quercetin and the formation of glutathione adducts, as hypothesized previously (37). (C) Formation of oxidized quercetin detected as the glutathione conjugates. The reaction was performed upon incubation of MPO/H2O2/quercetin in the presence of glutathione (GSH, 0.5 mM) at 37 °C for 20 min and analyzed by HPLC. Two quercetin-GSH adducts (6- or 8-glutathionylated quercetins, ref 38) were overlapped in this HPLC condition.

substrate of MPO, resulted in the disappearance of the absorption maximum at 456 nm (Figure 5A, top spectrum), showing the one-electron reduction of compound II to the ground state. Quercetin completely reduced compound II at a concentration of 0.25 µM (Figure 5A, middle spectrum), suggesting that quercetin is a one-electron reducing substrate of MPO. Under this reaction condition, the reduction was also observed by kaempferol (data not shown). In contrast, luteolin could not reduce compound II at the same concentration (Figure 5A, bottom spectrum). Fisetin could not as well (data not shown). The compound II reduction activity of these flavonoids was closely correlated with the inhibitory efficiency for dityrosine formation. It has been reported that several peroxidases catalyze the formation of oxidized quercetin (quercetin quinone) followed


by the formation of adducts with glutathione (38) (Figure 5B). To examine whether quercetin could be oxidized by MPO in our system, oxidized quercetin was detected by the glutathione (GSH) trapping method. Figure 5C shows the HPLC chromatograms of the reaction mixture of MPO/H2O2/quercetin in the presence of GSH. The major product was detected in the complete reaction system, whereas the product could not be detected in reaction systems lacking GSH, MPO, or quercetin. Formation of the product also requires H2O2. These results suggest the formation of the GSH adduct(s) of oxidized quercetin. Although two isomeric adducts at the 6- and 8-positions of quercetin have been identified (38), we could not separate the two isomers under our HPLC conditions. Quantitative Structure-Activity Relationship (QSAR). In addition to the radical scavenging activity, the possibility of the direct binding of flavonoids with the MPO enzyme is conceivable. We then performed QSAR analyses in order to clarify physicochemical properties that govern the variations in inhibitory potency. It has been reported that aromatic substrate molecules can bind to the hydrophobic heme pocket of myeloperoxidase (8). Therefore, we first examined the significance of log P (C log P) on MPO inhibitory activity (summarized in Supporting Information, Table 1). Eq 1 for the aglycones (i.e., not glycosides and sulfate) shows that the inhibitory effect on dityrosine formation is highly correlated with log P values.

pIC50 ) 1.64 C log P - 0.538 C log P2 + 4.34 n ) 11, r ) 0.862, s ) 0.184, F ) 11.6, C log Popt ) 1.52, Outlier ) 5-deoxykaempferol (1) In these and the following correlation equations, pIC50 is defined as log(1/IC50), n represents the number of compounds, r is the correlation coefficient, s is the standard deviation, F is the ratio of regression and residual, and C log Popt is the optimum C log P value. As described above, it is very probable that the total inhibitory process for the MPO inhibition contains the radical scavenging reaction for the tyrosyl radical. Therefore, eq 1 was further improved by adding the DPPH radical scavenging activity, and then the following eq 2 with a much higher correlation was formulated. pIC50-log RSADPPH ) 1.136 C log P - 0.355 C log P2 + 3.94 n ) 10, r ) 0.816, s ) 0.184, F ) 6.98, C log Popt ) 1.60, Outliers ) 5-deoxykaempferol, kaempferol (2) RSADPPH is the mol DPPH radical trapped/mol test compound. Equation 2 shows that the inhibitory effect of flavonoids on the MPO-derived dityrosine formation is strongly governed by both hydrophobicity and radical scavenging activity. Subtraction of log RSADPPH from pIC50 gives a rough estimate of the contribution of hydrophobicity. Next, the influence of functional groups of flavonoids on the structure-activity relationship was further examined. It has been reported that the catechol group shows lower O-H bond dissociation enthalpies which associate with the free radical scavenging activity (39). The flavonoids with the B-ring catechol group indeed showed lower bond dissociation enthalpies (Supporting Information, Table 1). The map of SOMO (singly occupied molecular orbital) for the quercetin 4′-O· radical, a most easily formed quercetin radical, showed that the distribution of unpaired electrons is delocalized over the B-ring catechol and the C2-C3 double bond (Figure 6A). The QSAR analysis of the DPPH radical scavenging activity also suggested the


Figure 6. Structural requirement for the inhibitory effect of flavonoids. (A) SOMO (singly occupied molecular orbital) for quercetin. The zones in green indicate positive regions, while red zones refer to negative ones. (B) Correlation plot between C log P and pIC50 based on eq 3 with indicator variable (Irecog). (b), Irecog ) 1; (O), Irecog ) 0; gray circle, outlier.

significance of the B-ring catechol and C2-C3 double bond for the activity (data not shown). Thus, the structural requirement of flavonoids for the inhibitory effect on MPO-derived dityrosine formation was investigated by another QSAR analysis. We introduced Irecog as an indicator variable. Irecog takes unity when the compound contains the 3-, 4′-, 5-OHs and C2-C3 double bond, and otherwise zero. Equation 3 shows that the MPOinhibitory activity is nicely expressed with the C log P and Irecog.

pIC50 ) 1.54C log P - 0.486C log P2 + 3.76Irecog + 4.22 n ) 11, r ) 0.938, s ) 0.180, F ) 17.2, C log Popt ) 1.58, Outlier ) 5-deoxykaempferol (3) As expected, eq 3 strongly supports that the existence of the 3-, 4′-, 5-OHs and a C2-C3 double bond significantly contributes to the inhibitory effect. Indeed, the three most effective inhibitors (Figure 6B), quercetin, kaempferol, and myricetin, possess the suggested functional groups (see Figure 4). In general, flavonoid glycosides and sulfates are considerably hydrophilic because glucuronidation/sulfation metabolism is the major detoxification pathway that gives hydrophilicity to exogenous active compounds leading to excretion. Indeed, glycosides and sulfates have lower log P values (≈ 0) compared with those of the corresponding aglycones (Supporting Information, Table 1). However, the inhibitory effects on the MPO inhibition of the glycosides and sulfate are not weak as predicted from their significant hydrophobicity change. QSAR analysis for an extended compound set including glycosides (monosaccharides and disaccharides) and sulfates was performed. In the analysis, we introduced another indicator variable Isugar, which expresses the hydrophilicity and/or steric hindrance of the attached sugar and sulfate moiety. Isugar takes 0 for the aglycones, 1 for the monosaccharides and sulfate, and 2 for the disaccharide. πagl expresses the C log P value of aglycone moiety for each glycoside and sulfate. Finally, the following equation was formulated.

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Figure 7. QSAR result of the inhibitory effect of quercetin metabolites. (A) Correlation plot between C log P and pIC50 - log RSADPPH based on eq 4 with indicator variable (Isugar). Isugar ) 0 (aglycone), ) 1 (monoglycoside or monosulfate), and ) 2 (disaccharide). (B) Illustration for proposed hydrophobic and hydrophilic moieties of a quercetin metabolite (quercetin-3-glucuronide as an example).

pIC50 - log RSADPPH ) 1.05πagl - 0.347πagl2 - 0.161Isugar + 4.03 n ) 16, r ) 0.849, s ) 0.390, F ) 10.3, πagl-opt ) 1.51 Outliers ) 5-deoxykaempferol, kaempferol, 3,7-dihydroxyflavone (4)

Equation 4 shows that the MPO inhibitory effects of the glycosides and sulfates depend on the hydrophobicity of the corresponding parent aglycones (πagl, C log P for parent aglycone). Figure 7A shows the plots for eq 4. If the inhibitory effects of the glycosides and sulfates depend on their hydrophobicity, the plots could be nearly on the regression curve with Isugar ) 0 (dotted curve). Negative coefficient (-0.161) for Isugar in eq 4 indicates that the conjugation of flavonoids attenuates their inhibitory effects; however, the conjugation did not dramatically decrease the inhibitory effects as expected (the plots for Isugar ) 1 and 2). The plots for Isugar ) 1 and 2 systematically deviate from the corresponding parent aglycone (i.e., quercetin or isorhamnetin) to the left side. These results suggested that the hydrophobicity of flavonoid backbone moiety, if conjugated, could be involved in the inhibitory actions for MPO reaction (Figure 7B). Computer Simulation of the Binding of Quercetin and Its Metabolites to MPO. Participation of the hydrophobicity (log P) of flavonoids in QSAR analyses could signify the hydrophobic interaction of flavonoids to MPO because the QSAR analyses were conducted using IC50 values obtained from cell-free MPO-catalyzed reaction in Vitro. It has been indicated that aromatic molecules bind close to the distal heme pocket in MPO (8). The X-ray structure of salicylhydroxamic acid (SHA), a known MPO inhibitor, bound to MPO has been reported, showing the aromatic ring of SHA binding to a hydrophobic region at the entrance to the distal heme pocket between the heme pyrrole ring D and the side chain of Arg239 (9). In addition, the hydroxamic acid moiety is hydrogen-bonded to both the distal His95 and Gln91 amide group but not coordinated

Figure 8. Docking simulation of quercetin and the glycoside to the active site of MPO. (A) Docking model for SHA (yellow) and quercetin (purple) bound to the heme (green) pocket of MPO. (B) Connolly surface calculation for the heme pocket of MPO shown in panel A. (C) Docking model for quercetin-3-glucuronide (Q3GA) bound to the heme (green) pocket of MPO. Three key residues (Q91, H95, and R239), the A-C rings of quercetin structure, and the sugar moiety of Q3GA are also indicated.

to the heme iron. On the basis of this information, the binding of quercetin to the pocket of MPO including the three key residues (Gln91, His95, and Arg239) was studied by computational docking. As shown in Figure 8A, quercetin could be docked similarly to SHA with the B-ring of quercetin oriented to the heme plane and close to the D pyrrole ring. The B-ring of quercetin might be close enough to the D pyrrole ring to achieve stacking, in which the distances were from 2.4 to 3.2 Å (Supporting Information, Figure 1). The Connolly surface map illustration also showed that quercetin can enter through the pocket leading to the active site with the B-ring oriented to the pocket (Figure 8B). Finally, to examine whether endogenous quercetin metabolites could bind to MPO, we simulated the binding of Q3GA to MPO. Figure 8C showed that Q3GA could bind to the active site of MPO with the B-ring oriented to the heme plane in a way similar to that for the aglycone. These results suggested the possible mechanism for the binding of quercetin and the metabolites to the MPO active sites. Binding of Quercetin and the Metabolite to MPO. Finally, we examined whether quercetin and the related compounds indeed bind to MPO by SPR analysis. MPO was successfully


Figure 9. Binding of flavonoids to MPO. (A) The sensorgrams indicated response after injection of quercetin to the flow cell of the MPOcaptured sensor chip. (B) The sensorgrams after injection of 25 µM quercetin, kaempferol, 5-deoxykaempferol (5-deoxy-K), genistein, isorhamnetin (IR), and taxifolin (Taxi). The DMSO-derived solvent peaks were detected immediately after injection because the stock solutions of flavonoids were prepared using dimethylsulfoxide (DMSO) due to the insolubility of isorhamnetin in aqueous buffer,

immobilized on a censer chip using an amino coupling reagent. The immobilization of MPO on a censer chip was confirmed by a specific binding of anti-MPO IgG antibody but not by control IgG (Supporting Information, Figure 2). As expected, it was confirmed that quercetin binds dose-dependently to MPO. In addition, the binding of other flavonoids was also examined. As shown in Figure 9B, quercetin and kaempferol (two highest inhibitors) exhibited significant affinity to MPO. However, the order of the affinity for these flavonoids did not fully correlate with that of IC50 or C log P (see Supporting Information, Table 1), indicating that the results might include the nonspecific binding of flavonoids to the MPO enzyme. We also confirmed the dose-dependent binding of Q3GA to MPO, whereas the resonance unit (RU) was lower than that of the aglycones (Supporting Information, Figure 2).

Discussion In this study, we demonstrated the inhibitory actions of flavonoids on the MPO-catalyzed oxidation using the combination of in Vitro assays and computational approaches, which are powerful for clarifying the structure-activity relationships based on the complicated experimental data. We have shown that quercetin and its related analogues including the metabolites effectively inhibited MPO-catalyzed dityrosine formation. During the reaction, MPO catalyzes the one electron oxidation of tyrosine to a tyrosyl radical that dimerizes to dityrosine. In general, phenolic groups of flavonoids and other phenolic antioxidants possess radical scavenging activity, which has been characterized as the ability to scavenge organic radical reagents such as DPPH. Indeed, DPPH radical scavenging activity of most flavonoids used in this study was observed (Supporting Information, Table 1); however, in addition to the radical


scavenging activity obtained from a simple in Vitro assay system such as a DPPH assay, we should pay more attention to the affinity/interaction between the target molecules under physiological conditions. For example, several flavonoids have been reported to bind to the hydrophobic pocket of serum albumin (40–42). In addition, the hydrophobicity of the compounds may contribute to the interaction with the lipid bilayer of cell membranes. For MPO, it has been reported that the organic substrates can interact with the hydrophobic pockets of the enzyme (8). Indeed, the QSAR results suggested that the optimal hydrophobicity (C log Popt) exists for the effective inhibitory activity of flavonoids on the MPO-derived dityrosine formation. The C log Popt value (≈1.5) also indicated that quercetin (C log P ) 1.27) is a potent inhibitor of the MPO reaction. Upon the QSAR analyses, a few outliers were found, whereas the reason remains unknown at the moment. On the basis of the previous report on the hydrophobic binding of SHA to the MPO active site (9), a docking simulation for the binding of quercetin to the hydrophobic region at the entrance to the distal heme pocket of MPO was performed. In the pocket, the van der Waals surface is created by the heme pyrrole ring D, the β, γ, and δ carbons of Arg239, and three nearby phenylalanines (Phe99, Phe366, and Phe407) could accommodate the aromatic ring of SHA. In addition, Gln91 and His95 are involved in the hydrogen bond with the hydroxyl moiety. Quercetin could be docked in a manner similar to the binding of SHA, a known MPO-binding inhibitor, to the active site, in which the B-ring of quercetin oriented to the heme (Figure 8). The binding of flavonoids to MPO was also experimentally confirmed by SPR analysis (Figure 9). A similar result was also obtained in which tyrosine, another substrate for MPO, could be docked to the pocket with the phenolic hydroxyl group oriented to the heme center (data not shown). The results indicate that the B-ring hydroxyl group(s) of flavonoids may be important for entering the pocket leading to the active site of MPO. QSAR analysis also suggested that the existence of the 3-, 4′-, and 5-OHs, and C2-C3 double bond are required for the inhibitory effect (Figure 6). Quercetin, kaempferol, and myricetin are the representative examples of the structural requirement. We also demonstrated that quercetin could reduce compound II, an active form of MPO, to the ground-state in a one-electron reaction (Figure 5). These results suggest that quercetin and related flavonoids are effective substrates for the MPO enzyme. It has been recently reported that chloride ion enhances the affinity of MPO to indole compounds (43). Similar enhancement might be possible for flavonoids because quercetin could effectively inhibit cellmediated MPO reaction in the presence of physiological chloride ion (Figure 3). In this study, we focused on quercetin, one of the representative flavonoids found in the human diet, and showed its inhibitory effects on MPO reaction. However, over 4000 structurally unique flavonoids have been identified in plant sources (44, 45). A comprehensive database for the structure and biological activity of flavonoids has also been developed (46, 47). This study showed that the utilization of theoretical analysis enables us to understand the structure-activity relationships of flavonoids. The structural requirement for MPO inhibition suggested in this study may be useful for searching and/or screening therapeutic flavonoids and other natural compounds in plant sources for cardiovascular diseases. One critical thing in considering the biological activities of flavonoids in ViVo is that flavonoids are generally circulating as the hydrophilic metabolites with glucuronidation and/or

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sulfation in the plasma and tissues (27). Indeed, the log P values of quercetin glycosides and a sulfate were lower (≈0) than that of the aglycone (Supporting Information, Table 1). In addition, glucuronidation/sulfation generally reduced the biological activity of flavonoid (24, 29). However, the inhibitory effects of several metabolites on dityrosine formation were not so much attenuated (Supporting Information, Table 1). We also demonstrated that Q3GA, a major quercetin metabolite, inhibited the HL-60-mediated dityrosine formation (Figure 3). In addition, the plasma extract of quercetin-fed rats also inhibited the MPO reaction. The plasma concentration of total quercetin metabolites after the intake of onion (a quercetin-rich plant source) was reported to be around 1 µM (47). The fact that the IC50 values of quercetin and Q3GA on MPO inhibition were 1.27 and 3.63 µM, respectively, supports the possibility that plasma concentrations of quercetin metabolites could inhibit the MPO reactions in ViVo. QSAR analysis for quercetin glycoside/sulfate showed that the inhibitory effect could be controlled by the hydrophobicity of the parent aglycone rather than the intact metabolites (Figure 7), suggesting that the partial structure (i.e., flavonoid backbone) can interact with the hydrophobic region of MPO. The docking simulation study also showed that 3-glycosylated quercetin can also bind to the hydrophobic pocket of MPO in a way similar to that of the aglycone (Figure 8C). These results suggest that quercetin metabolites, such as Q3GA, can act as MPO inhibitors in ViVo. We have shown that quercetin glucuronides preferentially accumulated in the macrophage cells expressing MPO in human atherosclerotic lesions (Figure 1). It has been reported that the expression of MPO in macrophage cells may contribute to the formation of atherosclerotic lesions (15). Thus, our study indicates that MPO in macrophage cells might be a potential target molecule of dietary flavonoids for their antiatherosclerotic actions. We investigated the inhibitory effects of flavonoids on the MPO-derived tyrosine dimerization reaction. MPO also catalyzes the production of hypochlorous acid (HOCl), a strong oxidant, upon reaction with H2O2 and Cl-. Although HOCl plays an important role in host defense, it also oxidizes biomolecules including protein, DNA, and lipids at the site of chronic inflammation. HOCl oxidizes LDL and high-density lipoprotein in human arteries, resulting in the formation of atherogenic modified lipoproteins (3, 48, 49). Flavonoids have also been reported to be effective scavengers for HOCl, resulting in the formation of chlorinated flavonoid derivatives (50, 51). For quercetin, the formation of 6-mono and 6,8-dichlorinated quercetins has been reported upon reaction with HOCl (51). Thus, quercetin and other flavonoids might act as multifunctional antioxidants that inhibit the catalytic action of MPO and scavenge oxidants such as HOCl. The relative contribution between the two pathways to the anti-inflammatory effects of flavonoids remains unclear. As far as we know, neither oxidized quercetin nor chlorinated quercetin are detected in biological samples. The antiatherogenic activity of quercetin has been reported by several epidemiological studies and animal experiments. We have previously shown that quercetin intake dramatically reduced the atherosclerotic lesions in hypercholesterolemic rabbits (35). Our results showed that dietary quercetin and other flavonoids might act as anti-inflammatory and antiatherogenic agents through, at least in part, the inhibition of MPO. Acknowledgment. This study was supported in part by a Grant-in-Aid for Young Scientists (to Y. Kawai) from the Ministry of Education, Culture, Sports, Science, and Technology, by the Ministry of Agriculture, Forestry and Fishery Food

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Project, Japan, and by the Center of Excellence Program in the 21st Century in Japan. Supporting Information Available: Calculated distance between quercetin and the heme moiety of MPO, confirmation of immobilization of MPO on a sensor chip, SPR analysis of quercetin-3-glucuronide, and the parameters used in QSAR studies for various flavonoids. This material is available free of charge via the Internet at http://pubs.acs.org.

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Flavonoids as Substrates and Inhibitors of Myeloperoxidase

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