Dietary Flavonoids as Xanthine Oxidase Inhibitors: StructureâAffinity

Article pubs.acs.org/JAFC

Dietary Flavonoids as Xanthine Oxidase Inhibitors: Structure−Affinity and Structure−Activity Relationships Suyun Lin,† Guowen Zhang,*,† Yijing Liao,† Junhui Pan,† and Deming Gong§ †

State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang 330047, China School of Biological Sciences, The University of Auckland, Auckland 1142, New Zealand

J. Agric. Food Chem. Downloaded from pubs.acs.org by UNIV OF NEBRASKA-LINCOLN on 08/25/15. For personal use only.

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ABSTRACT: The flavonoid family has been reported to possess a high potential for inhibition of xanthine oxidase (XO). This study concerned the structural aspects of inhibitory activities and binding affinities of flavonoids as XO inhibitors. The result indicated that the hydrophobic interaction was important in the binding of flavonoids to XO, and the XO inhibitory ability increased generally with increasing affinities within the class of flavones and flavonols. The planar structure and the C2C3 double bonds of flavonoids were advantageous for binding to XO and for XO inhibition. Both the hydroxylation on ring B and the substitution at C3 were unfavorable for XO inhibition more profoundly than their XO affinity. The methylation greatly reduced the inhibition (0.75−3.07 times) but hardly affected the affinity. The bulky sugar substitutions of flavonoids decreased the inhibition (1.69−1.99 times) and lowered the affinities (4.20−9.22 times) to different degrees depending on the conjunction site. KEYWORDS: flavonoids, xanthine oxidase, inhibition, structure−activity relationship, molecular modeling

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INTRODUCTION Xanthine oxidase (XO), a highly versatile flavoprotein enzyme, is ubiquitous among species (from bacteria to human) and within various tissues in mammals. In humans, XO is the key enzyme in catalyzing the oxidative hydroxylation of hypoxanthine and xanthine to produce uric acid and subsequent reduction of O2 at the flavin center with generation of reactive oxygen species (ROS), either superoxide anion radical or hydrogen peroxide.1 It has been suggested that an increased level of uric acid is a critical element in the development of hyperuricemia and gout. Furthermore, there is overwhelming acceptance that XO is associated with pathological conditions involving inflammation, metabolic disorders, cellular aging, reperfusion damage, atherosclerosis, hypertension, and carcinogenesis.2,3 Hence, XO is well characterized as a drug target for the treatment and management of diseases involving high enzyme activity level.4 At present, allopurinol is the mainly used inhibitor to treat gout clinically.5 However, rare adverse effects including bone marrow depression, hepatotoxicity, and Stevens Jones syndrome, collectively known as allopurinol hypersensitivity syndrome, have been reported, especially in patients with renal insufficiency.6,7 Therefore, there is an urgent need to develop new effective, less toxic, and more affordable inhibitors. Researchers have recently attempted to discover new XO inhibitors with low side effects, especially “multifunctional inhibitors” with diverse efficacy.3,5 The potential for plants to yield new therapeutic agents has motivated extensive investigation in screening novel natural XO inhibitors.8−10 Among them, flavonoids as a kind of natural product, have attracted increasing attention because of their low toxicity and various activities including antioxidation,11 anticancer,12 antiallergic,13 inhibition of advanced glycation products,14 and cardio-cerebrovascular protection.15 Flavonoids are widely distributed in plants and permeate into the human diet through beverages, vegetables, fruits, grains, tea, and wine and other © XXXX American Chemical Society

plant-derived foods. Flavonoids are characterized by a C6−C3− C6 skeleton labeled with rings A, B, and C (Table 1) and are divided into subclasses depending on the level of oxidation and the pattern of substitution of the C ring, whereas individual compounds within a class differ in the pattern of substitution of the A and B rings.16 Due to their relationship with some health effects, flavonoid-rich products have become increasingly popular. Consumption of these dietary supplements may result in higher intake levels of flavonoids than those ingested with a normal diet. However, whether the enhanced intake will bring beneficial effects on human health are to be determined. Moreover, flavonoids are known to inhibit a number of enzymes such as phosphodiesterase, Ca2+ ATPase, aldose reductase, lipoxygenase, cyclooxygenase,17 and human P450 enzymes. For example, the active flavonoid jaceosidin inhibited cyclooxygenase-2 activity in a concentration-dependent manner with an IC50 value of 2.8 mM.17 Kimura et al. found that the amentoflavone was a potent inhibitor of human cytochrome P450 3A4 and 2C9 with IC50 values of 0.07 and 0.03 μM, respectively.18 The inhibition of some flavonoids toward XO has been widely studied by several workers using in vitro, in vivo, and molecular simulation methods.19−21 For example, apigenin, as a kind of common flavonoid, possessed a strong inhibition activity against XO with respect to xanthine with an inhibition constant (Ki) value of 0.61 ± 0.31 μM.19 Luteolin has been found to inhibit XO activity with a Ki of 1.9 ± 0.7 μM.9 Kaempferol was reported to reversibly inhibit the activity of XO in a competitive manner with a Ki value of 6.77 ± 1.02 μM and a binding constant on the order of 104 L mol−1.10 Additionally, Van Hoorn et al. predicted the XO inhibition of Received: July 10, 2015 Revised: August 15, 2015 Accepted: August 18, 2015

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DOI: 10.1021/acs.jafc.5b03386 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry


Table 1. Chemical Structures of Dietary Flavonoids and Their Affinities for XO and XO Inhibitory Activities

a

Structure has no bicyclic benzopyranone ring. bC2−C3 is saturated, com = competitive, mix = mixcompetitive.

flavonoids on the basis of their structure22 and concluded that the hydroxyl groups on the C-5 and C-7 of flavnoids were favorable for XO inhibition.21,22 However, few investigations have examined the interactions of flavonoid−XO and the inhibitory activities of flavonoids against XO. In this study, the inhibitory activities against XO of 20 flavonoids with C5−OH and C7−OH, which mimic the structural frame of xanthine (Figure 1), were determined

low side effects. It also provides new insights into clinical research of flavonoids as effective XO inhibitors.

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MATERIALS AND METHODS

Materials. XO (35.7 units mL−1, from bovine milk) and xanthine were both purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and then prepared as stock solutions with 50 M Tris-HCl buffer (pH 7.4). The stock solutions of XO and xanthine were freshly prepared just before the experiments. Luteolin, rutin, quercetin, morin, myricetin, myricetrin, and epicatechin were from Aladdin Reagent Int. (Shanghai, China). Chrysin, apigenin, galangin, kaempferol, diosmetin, hyperin, and curcumin were purchased from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China), and phloretin was from Shanghai Yuanye BioTechnology Co., Ltd. They were all dissolved in absolute ethyl alcohol to prepare stock solutions (5.0 × 10−3 mol L−1), and the final alcoholic concentrations in the experiments were 50), compared with rutin (α = 57.29) and baicalin (α = 50.13), naringenin and silybin exhibited much weaker inhibition (α = 140.4 and 189.6, respectively). It could be found that these compounds all possessed a bulky substituent group. Phloretin, curcumin, and epicatechin did not even show XO inhibition activity. Several studies have suggested the inhibitory ability of flavonoids on XO activity.21,26,27 To further characterize the binding region in XO, the kinetic assay of some potent inhibitors (α ≤ 50) was conducted and Lineweaver−Burk double-reciprocal plots were applied. The results from the experiments and previous studies are summarized in Table 1. Allopurinol is a known competitive inhibitor. As shown in Figure 2a, inhibition of XO by chrysin was competitive, which was characterized by similar Vmax and distinctly different Km values, which was also seen for other flavones (apigenin and luteolin), suggesting binding of flavones to the XO active center, thereby replacing xanthine. Apigenin, kaempferol, and luteolin exhibited competitive inhibition (plot not shown), which conformed to the previous results that they could bind to XO at a single binding site and the binding was driven mainly by hydrophobic interactions.10,23,28 Genistein and quercetin also exhibited competitive inhibition as reported previously.19 Myricetin19 showed mixed type inhibition (Figure 2b) as well as morin,22 which can be concluded from different Vmax and Km values. These results indicated that some flavonoids are competitive or mixed competitive inhibitors of XO. Quenching Effect of Flavonoids on XO Fluorescence. The fluorescence spectra of XO quenched by flavonoids were investigated. All flavonoids tested could quench the fluorescence of XO sharply with increasing concentrations. As representative cases, the fluorescence spectra of XO in the presence of chrysin (Figure 3a), phloretin (Figure 3b), myricetin (Figure 3c), and genistein (Figure 3d) are displayed. There were no obvious shifts in the peak at 340 nm of XO fluorescence for chrysin and genistein, whereas the maximum peak (λem = 340 nm) of XO fluorescence was obviously blueshifted in the presence of phloretin and myricetin, suggesting that they were placed in a more hydrophobic environment;29 that is, the molecular conformation of XO was affected. Individually, phloretin exhibited a slight affinity for XO, but it was a very weak inhibitor toward XO, and this feature could also be seen in the curumin−XO and epicatechin−XO systems.

(1)

where F0 and F represent the fluorescence intensities in the absence and presence, respectively, of flavonoids, [Q] is the concentration of flavonoids, Kq is the quenching rate constant, τ0 is the average lifetime of the fluorophore in the absence of the quencher (its value for XO was 2.80 × 10−9 s),10 and KSV is the Stern−Volmer quenching constant. The binding constants (Ka) and the number of binding sites (n) were calculated according to the following formula:25 log

F0 − F 1 = n log K a − n log (F − F )[P ] F [Q t] − n 0 F t 0

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(2)

All of the fluorescence data in this study were corrected for absorption of excitation light and reabsorption of emitted light according to the literature.10 Molecular Modeling. The molecular docking studies were carried out by the MGL tools 1.5.6 with AutoGrid4.0 and AutoDock4.0. It docked flexible ligand into the rigid protein conformationally and exhibited the detail of binding. The bovine milk XO (PDB code 3ETR) complexed with lumazine (substrate) was downloaded from the Protein Data Bank (http://www.rcsb.org/pdb). The 3D structures of ligands (flavonoids and allopurinol) were depicted in Chem3D Ultra 8.0. Before Gasteiger charges were assigned to the macromolecule file, all of the water molecules were removed and the hydrogen atoms were added. A dimension grid box (110 Å × 100 Å × 110 Å) was set up to enclose the active site with grid spacing of 0.37 Å. Docking calculations were performed with the default parameters except that the number of GA runs was set at 100 times. From the outcomes, the best-scoring docked model (the lowest docking energy) of the ligand was proposed as the feasible binding mode of the inhibitor in the active site of XO predicted by this program. The outputs were further rendered with Pymol to obtain visible combination models. Statistical Analysis. All data for enzyme kinetic assays and plotting were obtained using Origin 8.0. Data were analyzed by oneway ANOVA for statistical significance using the SAS statistical package (SAS Institute, Cary, NC, USA). A p value of ≤0.05 was considered statistically significant. C


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Figure 4. (a) Quenching ratio (F/F0) of XO fluorescence spectra with addition of chrysin (Ch), myricetin (My), genistein (Ge), phloretin (Ph), and rutin (Ru). (b) Stern−Volmer plots for XO fluorescence quenching by these flavonoids at 298 K.

Figure 2. (a) Lineweaver−Burk plot for chrysin. The inhibition of XO by chrysin is competitive, as can be seen from similar Vmax and distinctly different Km values. (b) Lineweaver−Burk plot for myricetin. Inhibition of XO by myricetin shows mixed type inhibition, as can be concluded from different Vmax and Km values.

genistein, and myricetin, respectively, whereas the intensities of XO showed much slower decreasing rates with the addition of phloretin and rutin. Chrysin at 10.0 μM quenched the XO fluorescence by 69.4%; genistein, myricetin, and rutin quenched XO fluorescence by 51.9, 48.4, and 31.2%, respectively, but the corresponding concentration of phloretin quenched the XO

Moreover, different quenching ratios (F/F0 ) of XO fluorescence with increasing flavonoid concentration were observed (Figure 4a). The fluorescence intensities of XO decreased sharply with increasing concentrations of chrysin,

Figure 3. Quenching effects of chrysin (a), phloretin (b), myricetin (c), and genistein (d) on XO fluorescence spectra at 298 K. λex = 280 nm. c(XO) = 0.5 μM. c(flavonoids) = 0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0, 16.0, 18.0, and 20.0 μM for curves 1−11, respectively; curve m in every panel represents the individual spectrum of the corresponding flavonoid. D


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Journal of Agricultural and Food Chemistry fluorescence by only 29.1%. These phenomena implied that the quenching of XO fluorescence might rely on the structures of flavonoids. Figure 4b shows the Stern−Volmer plots for XO fluorescence quenching by chrysin, genistein, myricetin, rutin, and phloretin. The linear plots indicated that the KSV and Kq values of XO fluorescence quenching by flavonoids could be assessed from eq 1. The calculated Kq values (0.98−2.50 × 1013 L mol−1 s−1) were much higher than the maximum scatter collision quenching constant of various quenchers with a biopolymer (2.0 × 1010 L mol−1 s−1), indicating that the fluorescence quenching of XO by flavonoid was probably initiated by static quenching resulting from the formation of the flavonoid−XO complex.25,30 These results are in accordance with previous studies,10,23 suggesting that the static fluorescence quenching of XO by morin, kaempferol, luteolin, and quercetin originated from the formation of the complexes. Nature of Flavonoid−XO Noncovalent Interaction. Generally, the noncovalent interactions between flavonoids and proteins are caused by four major forms, namely, hydrogen bonding, van der Waals interaction, hydrophobic interaction, and electrostatic interaction.29,30 The nature of the flavonoid− XO interaction was studied by investigating the molecular property−affinity relationship. Herein, the hydrogen bond acceptor/donor numbers and the lipophilicity of flavonoids were investigated. The relationships between the hydrogen bond acceptor/donor numbers (obtained from PubChem Public Chemical Database) of flavonoids and the affinities for XO (lg Ka) are shown in Figure 5a. The affinities for XO obviously decreased with increasing hydrogen bond acceptor numbers of flavonoids, which implied that the hydrogen-

bonding force was not the main force in the binding of flavonoids to XO.31 For an in-depth investigation of whether or not the lipophilicity played a main role in the binding of flavonoids to XO, the lipophilicity of the compounds under study was assessed by their partition coefficient values (XlgP3) according to the PubChem Public Chemical Database. As shown in Figure 5b, there was a relationship between XlgP3 values and lg Ka values for flavonoids. Generally, the binding affinities increased with an increase in the XLogP3 values. These results indicated that the hydrophobic force instead of lipophilicity may be the main acting force for driving the binding of flavonoids to XO, which was supported by the previous studies in some flavonoids.32−34 In terms of these results, the noncovalent interaction between flavonoids and XO seemed to be different from the previously reported interaction of flavonoid−β-globulin35 but similar to the interactions of flavonoid binding to serum albumin, hemoglobin,29 common plasma proteins,33 and milk proteins.36 Relationship between Affinities and XO Inhibitory Activities. XO is a dimer; each monomer contains an active site, which is believed to be surrounded by many amino acid residues.37 Glu802, Glu1261, and Arg880 residues in the molybdenum center of the gorge play key roles in catalyzing xanthine oxidation,38 whereas some residues at the entrance of the cavity, such as Leu648, Phe649, Phe914, Phe1009, Val1011, Phe1013, and Leu1014, modulate the entry of small molecules including substrates or inhibitors into the center.10,23,37 For the inhibition on the catalytic activity of XO, flavonoids might directly enter the gorge to cover the active site or might just bind to the vicinity and hinder the entrance for xanthine. Thus, the relationship between the affinities for XO and their XO inhibition was discussed. Figure 6a shows that there was no obvious relationship between the affinities and inhibition for flavonoids,39 but the inhibitory activities increased generally with the increasing affinities within the class of flavones and flavonols. These results might be due to the fact that the higher binding affinity increases the opportunity to enter and affect the catalytic site, whereas the inhibition finally rested with the direct interaction between the flavonoids and the active site. Interestingly, it was recently found that several clinically significant drug−enzyme complexes were similarly high-affinity active site-directed inhibitors that compete with substrates.40 Relationship between Topological Polar Surface Area (TPSA) and XO Inhibitory Activities. The TPSA is defined as the surface sum over all polar atoms. TPSA has been shown to be a descriptor characterizing passive molecular transport through membranes, which has been successfully used to predict the human intestinal absorption, bioavailability, Caco-2 monolayer permeability, and blood−brain barrier penetration.41 It has been reported that molecules with a TPSA value of >140 Å2 showed low human intestinal absorption and tended to be poor in permeating cell membranes.42,43 The TPSA values were obtained from the PubChem Public Chemical Database. Figure 6b shows the relationship between TPSA and XO inhibitory activities of flavonoids. The relationship was not evident, but it might be roughly divided into four types considering their inhibitory activities. Type I was the most favorable group with high inhibitory activities (low α value) and penetration abilities (low TPSA), and type II was the less preferred group with good penetration abilities but low inhibitory activities (high α value). Although the flavonoids in type III group had great inhibitory activities on XO in vitro, little absorption may make them

Figure 5. (a) Relationships of the hydrogen bond acceptor/donor number of flavonoids with the affinities (lg Ka) for XO. The hydrogen bond acceptor/donor numbers were taken from the PubChem Public Chemical Database. (b) Relationship of apparent binding constants (lg Ka) with partition coefficient (XLogP3) of flavonoids. The partition coefficient (XLogP3) was taken from the PubChem Public Chemical Database. E


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Structure−Activity of Flavonoids on XO Inhibition and Affinity for XO. Hydroxylation. Previous investigations have indicated that the patterns of the hydroxyl and methoxyl groups on the rings of flavonoids are involved in the bioactivities and binding affinities for proteins.44−47 The effects of hydroxylation of flavonoids on their affinities for XO and XO inhibition are shown in Table 2. Baicalein with an extra C6− OH exhibited 9.84 times lower XO inhibition than chrysin, but their affinities did not change too much, indicating that the hydroxylation on the C6 (ring A) of flavones weakened the inhibitory activity obviously and the binding affinities slightly. Moreover, luteolin and quercetin with additional hydroxyl groups on position C3′ (ring B) showed 1.50 and 0.70 times lower inhibition than apigenin and kaempferol, respectively, whereas their affinity decreased by 0.25 and 0.28 times, respectively. Both the hydroxylation on C4′ (ring B) of chrysin (→apigenin) and galangin (→kaempferol) weakened their XO inhibition abilities by nearly 1.4 times. The hydroxylation on C5′ (ring B) of quercetin (→myricetin) decreased its inhibition and affinity by, respectively, 2.66 and 0.14 times.48 These results indicated that the hydroxyl groups in the B ring may be particularly important for flavonoids in inhibiting XO and binding to XO. Consequently, the α values of galangin, kaempferol, quercetin, and myricetin increased gradually, indicating that the extra hydroxyl substituents in the B ring may be unfavorable for the inhibitory activity of flavonols. These results could be also concluded within flavones (Table 1). This may be explained by the fact that ring B was reported to stabilize in the hydrophobic region of XO;26,28 therefore, the enhancive hydroxy groups were unfavorable for the stability of flavonols. Moreover, galangin (kaempferol) exhibited lower affinity and inhibition than chrysin (apigenin), implying that the presence of a hydroxyl group at position C3 of the flavone significantly reduced the ability to bind to XO as well as its inhibition on XO. This effect was also obtained in the inhibition of flavonoids toward lipoxygenase39 and human aromatase (estrogen synthetase).49 Methylation. As shown in Table 2, the affinity of isorhamnetin for XO was weaker than that of its unmethoxylated form (kaempferol) by 0.37 times, indicating that an additional methoxy group at C5′ (ring B) decreased their binding affinities for XO. C4′-methylation of luteolin (→ diosmetin) hardly affected the affinity for XO, with similar values of lg Ka, but in both conditions, their inhibition on XO obviously decreased. However, a previous study found that the methylation of hydroxyl groups in flavonoids enhanced their binding affinities for HSA by 2−16 times.33 Here, the methylation of flavonoids exhibited great effect on the

Figure 6. (a) Relationship of XO inhibitory activities (α) of flavonoids with affinities (lg Ka) for XO. (b) Relationship of TPSA with XO inhibitory activities (α) of flavonoids. (c) Relationship of TPSA with the affinities (lg Ka) of flavonoids for XO. The TPSA values were obtained online (www.molinspiration.com/cgi-bin/properties).

ineffective in treating gout. Type IV was supposed to be the most unfavorable inhibitor. Moreover, the binding affinity of flavonoids with XO was investigated (Figure 6c). TPSA values were found to generally decrease with increasing lg Ka flavonoids for XO, which was similar to the relationship as reported earlier between flavonoids and HSA.35,43

Table 2. Effects of Hydroxylation and Methylation of Flavonoids on Their Affinities for XO and XO Inhibitory Activities subclass

structural alteration

flavones

3′H → OH 4′H → OH 6H → OH 3H → OH 4′OH → CH3

flavonols

4′H 3′H 5′H 5′H

→ → → →

OH OH OH OCH3

effects (times) affinity

activity

apigenin → luteolin chrysin → apigenin chrysin → baicalein chrysin → galangin luteolin → diosmetin

example

0.25↓ 0.25↓ 0.02↓ 0.04↓ ≈

1.50↓ 1.41↓ 9.84↓ 3.72↓ 3.07↓

galangin → kaempferol kaempferol → quercetin quercetin → myricetin kaempferol → isorhamnetin

0.31↑ 0.28↓ 0.14↓ 0.37↓

1.45↓ 0.70↓ 2.66↓ 0.75↓

F


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structure,19 which was beneficial for inhibiting XO.21,26 The fact that the benzopyran ring resembles the moieties of allopurinol (Figure 1) also might be a reason.22 Generally, some of the structural elements that influence the inhibition of flavonoids against XO could be concluded. When the affinities and inhibitory activities were considered simultaneously, it was notable that the affinities of flavonoids changed consistently with the inhibitory activities, but not proportionally. The typical structure properties of flavonoids affecting the inhibitory effect against XO are illustrated in Figure 8. The


inhibition toward XO, whereas the effect on the affinity for XO was uncertain. Glycosylation. The dietary flavonoids in nature occur mostly as β-glycosides. The flavonols are found mainly as 3- and 7-Oglycoside, although the 4′-position may also be glycosylated in some plants.29,32 As shown in Figure 7, compared with

Figure 7. Effect of glycosylation of flavonoids on their affinities for XO and XO inhibitory activities. Figure 8. Structural elements that influence the inhibitions of 5,7dihydroxyflavone to XO. The up arrows represent an increase in the inhibition; the down arrows represent a decrease in inhibition.

quercetin, its galactoside (hyperin) showed a lower binding affinity and inhibition by 1.69 and 4.20 times, respectively. The inhibitions on XO of rutin and baicalin were about 5.87 and 9.22 times lower than that of quercetin and baicalein, respectively. Then by comparing their corresponding lg Ka, it was found that glycosylation of flavonoids lowered the affinity for XO depending on the conjugation site and the class of sugar moiety. The decreasing affinity for XO after glycosylation may be caused by the nonplanar structure; steric hindrance and hydrophilicity were other possible causes for the decreased affinity.28,39 The behavior of depressed inhibition against XO could be explained by the fact that the increase in the size of the flavonoid compound may also increase the possibility of repulsive interactions in the active site, thus inhibiting the formation of the flavonoid−XO complex. Moreover, the presence of several hydroxyls in the galactoside group may act as an unstable element inside the highly nonpolar region, which finally resulted in a lower inhibitory activity and affinity, which was consistent with previous results. In addition, the previous studies reported that the glycosylation of dietary flavonoids decreased the affinities for plasma protein in vitro.50 The present result that glycosylation decreased the inhibitory activities against XO was similar to the conclusion in a recent review that glycosylation of flavonoids decreased the inhibitory activities on both α-amylases and α-glucosidases.51 Hydrogenation of the C2C3 Double Bond. The flavonoids with chromone (flavones, flavonols, and isoflavones) exhibited stronger inhibition with smaller values of α than those without (flavanone, others). The hydrogenation of the C2C3 double bond decreased the inhibiting ability as well as the binding affinity of flavonoids. For example, naringenin showed a much higher α value and slightly lower lg Ka value than apigenin (Table 1), indicating that the hydrogenation of the C2C3 of flavonoids decreased the inhibitory activities on XO more fiercely than the affinities for it. Phloretin and curumin without dual rings even showed no inhibition on XO, but still exhibited affinity. This may be because the C2C3 double bond in conjugation with a 4-oxo group played an important role in the flavonoids in maintaining a plane molecular

hydrogenation of the C2C3 double bond on flavonoids decreased the inhibition on XO; hydroxylation of 3C (ring C) and the glycosylation of 3COH were unfavorable for the inhibition. The hydroxylation and methoxylation on ring B also decreased the inhibitory activity; moreover, the hydroxylation of 6C (ring A) and glycosylation of 7COH significantly decreased the inhibitory ability. Computational Docking Analysis. From kinetic assay results, the active site of XO is specific for effective flavonoids, and the main focus will be the dockings of flavonoid into the Mo-pterin domain of XO. Herein, typical flavonoids (chrysin, apigenin, galangin, quercetin, rutin, genistein) were compared in their binding conformations to obtain conclusions on structural elements required for effective inhibition toward XO by performing a computational docking study. Allopurinol, as a well-known inhibitor, was used as a reference compound. Figure 9a shows allopurinol and flavonoids occupied the active cavity of XO (yellow), which was a long and narrow channel leading to the black catalytic site (Mo-pterin center). This once again supported that the mimic planar structure as xanthine was essential for potent inhibitory activity on XO.19,22 The close-up views of computer-generated model (Figure 9b−e) reveal that all of the flavonoids plugged the channel (represented as Leu648, Phe649, Leu873, Phe914, Phe1009, Thr1010, Val1011, Phe1013, and Leu1014) and covered the access of designated substrate LUZ by interacting with the surrounding residues with different orientations. It was obvious that once the channel was mostly occupied by inhibitor, it might block the landing of substrate and ultimately prevent its oxidation. Figure 9b shows a close-up view of the active cavity of XO along with allopurinol. It docked in the Mo-pterin domain, showed a close position to LUZ, and formed hydrogen bonds with Lys 771 and Glu802.52 In Figure 9c, we superimposed two flavones of apigenin and chrysin, showing that their benzopyranone rings overlapped and both bound in the channel leading to the buried cofactor G


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Figure 9. (a) Section view of flavonoids (stick) occupying the catalytic site cavity (surface view: yellow) of XO. The black domain is the surface of coenzyme factor (Mo-pterin cofactor). The close-up view of interaction of flavonoids with hydrophobic residues (shown in black ball and gray stick representation and labeled with their numbers) lining the access channel to Mo (green ball) center are shown as (b) allopurinol (Al), (c) chrysin (Ch) and apigenin (Ap), (d) galangin (Ga) and quercetin (Qu), and (e) geninstein (Ge) and rutin. Backbone segments are shown in cyan, and the residues of forming hydrogen bond with the corresponding flavonoids are in full color (Asn768, Glu802, Lys771).

Figure 10. Key interactions of chrysin (a), apigenin (b), galangin (c), quercetin (d), genistein (e), and rutin (f) with the key residues in the active cavity of XO. Pink circles indicate hydrophobic amino acids, and green circles indicate hydrophilic amino acids. The blue short-dash lines indicate hydrogen bonds to side-chain residues. The orange lines represent π-interactions.

The overlay of binding poses of flavonol in the XO active site is displayed in Figure 9d; galangin and quercetin had almost identical binding modes at the active sites with each having ring A and C docked into the channel. Their polar 3C−OH stretched to the space surrounded by nonpolar Phe649, Leu648, and Leu1014. Thus, the weaker inhibition and

with slight difference. Chrysin showed hydrogen bonding of 5COH and 4CO groups with side chain of Lys771 and Ser876 residues (Figure 10a), whereas hydrogen bonds were observed between 4CO and the ether group of apigenin with the side chains of Glu802 and Lys771 residues, respectively (Figure 10b). H


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Journal of Agricultural and Food Chemistry interaction of flavonol relative to flavone could be explained by the unsteadiness of polar hydroxyl in the hydrophobic region.26 The destabilization of polar hydroxyl stretching into the hydrophobic region may cause the lower binding affinity; thus, the more hydroxyl groups in ring B within one subclass, the weaker inhibitory ability it exhibited (Table 1). The hydrogen bonds between 3C−OH of galangin and Asn768 residue (Figure 10c) and between 3′C−OH of quercetin and His875 residue (Figure 10d) might be the reason for their different orientations of ring B. Three common features of binding with XO existing in both flavone and flavonol (Figure 10a−d) could be concluded. First, several hydrogen bonds and π−π interactions existed in their docking into the active pockets.37 For example, there were π−π interactions between ring A and the aromatic residue of Phe1013 and Val1011; interactions between ring B and Phe1013 and Phe649 are shown in the chrysin−XO system (Figure 10a). Several hydrogen bonds were observed, including C5 hydroxyl bound to Lys771 (Figure 10a), Asn768 (Figure 10c), or Ser876 (Figure 10d) and C7 hydroxyl bound to Glu 802 (Figure 10d). This might support the previous result that C5 and C7 hydroxyl groups of flavonoid assist in the inhibitory activity against XO.19,22 Second, the 4CO group (ring C) of both flavonoids formed hydrogen bonds with the side chains of the residue within the cavity. A third significant feature of both flavonoids was shown by inserting ring A and ring C into the hydrophobic cavity instead of ring B. Additionally, each flavonoid docked into the active site of XO uniquely. Genistein is a constitutional isomer of apigenin with a difference in the phenolic group substitution on ring C. The simulation revealed that the binding of genistein was in the opposite orientation to apigenin, with ring B inserting into the active site and approaching the Mo-center (Figure 9e). Herein, rutin had a comparable binding position to genistein with its benzopyranone bicyclic ring stretching beyond the hydrophobic pocket and ring C inserting into the Mo-pterin domain, but it exhibited fairly low affinity to the enzyme. These phenomena could be explained by the fact that the bulky rutinoside moiety located outside the active cavity, which was supposed to badly destabilize its binding; that is, the bulky rutinoside created a steric clash with the surrounding residues, thereby preventing productive binding to XO. 20 Moreover, the π−cation interactions of ring B (genistein) and the NH2 of Arg880 (Figure 10e) and π−π interactions between these flavonoids and Phe1013, Phe1009, and Phe649 (Figure 10e,f) also contributed to the tethering of the two flavonoids to the active sites. All together, our structural and experimental data indicated that these potent flavonoids occupy the active substrate-binding sites in the enzymatic pocket, although with different orientations, physically excluding the landing of substrate, further intervening in the xanthine oxidation activity of XO and hindering the redundant mechanism for uric acid. In summary, this work reinforced flavonoids as effective inhibitors of XO in vitro. It was also possible to establish a structure−activity relationship for the inhibitory activity of flavonoids against XO. Data analysis indicated that the hydrophobic force was the main force of flavonoids to bind with XO. The inhibitory activities increased generally with increasing affinities within the class, especially for flavones and flavonols, which might be because the higher affinity increases the chance to enter the catalytic gorge, but the inhibition finally depends on the direct interaction between the flavonoid and the active site. The typical structure properties of flavonoids

affecting the inhibitory effect against XO are illustrated in Figure 8. The hydrogenation of the C2C3 double bond on flavonoids decreased the inhibition on XO, hydroxylation of C3 (ring C) and glycosylation of 3C−OH were unfavorable for the inhibition, and the hydroxylation and methoxylation on ring B also decreased the inhibitory activity; moreover, the hydroxylation of C6 (ring A) and glycosylation of 7C−OH siginificantly affected the inhibitiory ability. The structure−activity relationship coupled with the results of computer modeling revealed several key factors of flavonoid binding in the active site of XO and inhibition against XO. Specifically, due to their mimic rings (A and C rings) of the xanthine (Figure 1), these compounds bound to the active site effectively. The C2C3 that maintains a planar structure of flavonoids was essential for potent inhibitory activity on XO.19,22 The hydroxyl moiety at C7 and C5 contributed favorable hydrogen bonds and interactions between inhibitors and the active site. However, C3−OH exhibited weaker inhibition, which could be due to the destabilization of polar hydroxyl stretching into the hydrophobic region of the active site and causing lower binding affinity. The methylation of flavonoids exhibited a great effect on the inhibition toward XO, whereas their effect on the affinity for XO was uncertain. The bully substitution of glycosyl on aromatic rings of flavonoids created a steric clash with the surrounding residues, hindering a lower binding affinity for XO. Generally, the occupancy of the active site by the potent flavonoids reduced the landing of substrate to the active sites and further intervened in the xanthine oxidation activity of XO and hindered the redundant mechanism for uric acid. These results provide a molecular basis regarding the binding nature and structural requirements of phytochemical flavonoids to inhibit XO, which may be useful for developing new potential drugs for XO blockade. Thus, flavonoid-containing fruits and vegetables should be included in the diet of patients suffering gout. These examples also illustrate how knowledge of the inhibition mechanism can promote the development of novel therapies. However, the results from this in vitro study may be different from the results obtained from in vivo studies due to metabolic conversion processes; therefore, cell culture and animal studies including these chemicals should be further initiated. In fact, there was a study showing that flavonoids (genistein, apigenin, quercetin, rutin, and astilbin) had a significant effect on XO activities in vivo (serum XO).20 However, it is still necessary to conduct more experiments in vitro and vivo to illuminate if the flavonoids are suitable candidates for replacing allopurinol for the treatment of gout.

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AUTHOR INFORMATION

Corresponding Author

*(G.Z.) Phone: +86-791-88305234. Fax: +86-791-88304347. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (No. 21167013, 31460422, and 31060210), the Natural Science Foundation of Jiangxi Province (20142BAB204001 and 20143ACB20006), the Joint Specialized Research Fund for the Doctoral Program of Higher Education (20123601110005), the Program of Jiangxi ProvinI


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