Identification and characterization of flavonoids as sialyltransferase inhibitors

Biochemical and Biophysical Research Communications 382 (2009) 609–613

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Identification and characterization of flavonoids as sialyltransferase inhibitors Kazuya I.P.J. Hidari a,*, Kin-ichi Oyama b, Go Ito c, Miho Nakayama c, Makoto Inai c, Shiho Goto a, Yugo Kanai a, Kei-ichi Watanabe a, Kumi Yoshida d, Takumi Furuta c,1, Toshiyuki Kan c, Takashi Suzuki a a

Department of Biochemistry, University of Shizuoka, School of Pharmaceutical Sciences, Japan and Global COE Program, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan Chemical Instrument Facility, Research Center for Material Science, Nagoya University, Japan c Department of Synthetic Organic and Medicinal Chemistry, University of Shizuoka, School of Pharmaceutical Sciences, Japan and Global COE Program, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan d Graduate School of Information Science, Nagoya University, Japan b

a r t i c l e

i n f o

Article history: Received 28 February 2009 Available online 19 March 2009

Keywords: Flavonoid Sialyltransferase inhibitor

a b s t r a c t Sialyltransferases biosynthesize sialyl-glycoconjugates involved in many biological and pathological processes. We investigated and characterized synthetic flavonoid derivatives as sialyltransferase inhibitors. We first examined 54 compounds by solid-phase enzyme assay using b-galactoside a2,6-sialyltransferase 1 (ST6Gal I) and b-galactoside a2,3-sialyltransferase. Several compounds inhibited sialyltransferase enzyme activity regardless of sialyl-linkage reactions. Among them, two compounds showed inhibitory activity against ST6Gal I with IC50 values less than 10 lM. Three characteristic features of flavonoids were determined by structure-inhibitory activity relationships. First, a double bond between C2–C3 linkages is required for the activity. Second, increasing hydrophilic properties on the B-ring markedly augmented the inhibitory effect. Third, a hydrophobic functional group introduced on the hydroxyl groups of the A-ring enhanced the inhibitory activity. Kinetic analysis using human ST6Gal I indicated a mixed inhibition mechanism of the compounds. In conclusion, the flavonoids identified could be applied for control of cellular expression of sialic acid. Ó 2009 Elsevier Inc. All rights reserved.

Introduction Sialic acid-containing glycoconjugates are involved in a number of biological and pathological events, such as differentiation, tumor metastasis, and inflammation. Carbohydrate structures containing sialic acid residues play critical roles in cell–cell recognition and cell–pathogen interactions [1–5]. However, the biological and pathological significance of endogenous sialic acid-containing glycoconjugates have not been elucidated. To date, 20 families of sialyltransferases involved in the biosynthesis of sialic acid-containing glycoconjugates have been identified [6–8]. b-Galactoside a2,6-sialyltransferase and a2,3-sialyltransferases including ST6Gal I and ST3Gal I–VI generate terminal Neu5Aca2-6Galb1-R and Neu5Aca2-3Galb1-R residues, respectively [9–11]. Specific inhibitors of sialylation could enable us to address their biological significance as well as to control pathological manifestations, such as inflammation and infection by microorganisms. The sialyltransferase inhibitors reported to date can be classified into three categories. First, agents derived from a donor substrate, CMP-Neu5Ac, have been well documented [12–16]. As expected, * Corresponding author. Fax: +81 54 264 5723. E-mail address: [email protected] (K.I.P.J. Hidari). 1 Present address: Institute of Chemical Research, Kyoto University, Japan. 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.03.082

these analogs, such as CDP and CTP, showed competitive inhibition against sialyltransferases with Ki less than 50 lM. Recently, several compounds based on neuraminyl substitution by hetaryl rings were also synthesized and characterized. According to the mechanism of inhibition, these compounds are expected to inhibit all types of sialyltransferase regardless of sialyl-linkage reactions, such a2,3, a2,6, and a2,8. Some of these agents showed potent inhibition against ST6Gal I with Ki less than 100 nM. However, it is very difficult to apply these agents to cellular and animal experiments as they are highly water-soluble and cannot pass easily through cellular membranes. Second, acceptor substrate analogs have been generated by modification of carbohydrate residues [17]. In comparison to the donor substrate analogs, these agents show narrower inhibitory specificity. It is thought that the entire structures of the acceptor sugar chains are recognized by sialyltransferases. Third, competitive inhibitors of the sialyltransferases were discovered by the screening libraries of natural chemical compounds as well as peptides generated by combinatorial chemistry methods [18,19]. The compounds obtained differ from the structures of the enzyme substrates, and the details of the molecular mechanisms of these inhibitory actions have yet to be clarified. The flavonoids widely present in plants have been reported to show many biological functions, including anti-tumor, anti-

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inflammatory, anti-bacterial, and anti-oxidant effects [20–25]. The chemical structures of flavonoids consist of two phenyl groups, i.e., an A-ring and a B-ring, connected through three carbon atoms (C-ring) (Fig. 1). In absorption model experiments using a cultured cell line derived from the intestinal epithelium, flavonoids were shown to be absorbed into the cells within several hours [26]. These chemical compounds have promising activities not only in vitro but also in vivo. In this study, we investigated and characterized flavonoid compounds, which had been chemically synthesized, as sialyltransferase inhibitors. ELISA-based sialyltransferase assay demonstrated that several compounds showed inhibitory activities against both b-galactoside a2,6-sialyltransferase and b-galactoside a2,3-sialyltransferase. Kinetic analysis revealed that these compounds show mixed inhibitory actions on ST6Gal I. These findings indicated that the flavonoids identified here belong to an alternative class of sialyltransferase inhibitors.

Roskilde, Denmark). The enzyme assays were carried out in the wells according to the procedure of Weinstein et al. [10,11] with slight modifications. The reaction mixture in inhibitor screening experiments had the following final concentrations in a total volume of 50 ll: 15 lM or 250 lM CMP-Neu5Ac for ST6Gal I or ST3Gal III, respectively, 0.5% (w/v) Triton CF-54, 2 mM MnCl2, 25 mM Mes buffer, pH 6.5, compounds at the indicated concentrations and enzymes. In kinetic experiments, CMP-NeuAc was used up to a final concentration of 100 lM. The enzyme activity was determined in triplicate in each experiment. (II) HPLC-based galactosyltransferase assay: The enzyme assay for PA-oligosaccharide as an acceptor was carried out according to the procedures reported previously [37] with slight modifications. The reaction mixture had the following final concentration in a total volume of 25 ll: 150 lM UDP-Gal, 0.25% (w/v) Triton CF-54, 40 mM MnCl2, 0.2 mM PA-sugar chain 012, 100 lM compound 5, 40 mM cacodylate buffer, pH 6.5, and enzymes. Galactosylated products were resolved on an HPLC system equipped with an ion-exchange column, PALPAK Type N (6.4 mm  25 cm) (Takara Bio, Inc.). Chromatography was performed with a single solvent system of CH3CN/500 mM triethylamine-acetate buffer (pH 7.3, 60: 40 by vol.) at a flow rate of 1 ml/min.

Materials and methods Materials. Recombinant rat b-galactoside a2,6-sialyltransferase 1 (ST6Gal I) and b-galactoside a2,3-sialyltransferase (ST3Gal III), and bovine N-acetylglucosamine b-1,4-galactosyltransferase 1 (B4GalT1) were purchased from Merck Biosciences (Darmstadt, Germany). Recombinant human ST6Gal I was prepared as described previously [27]. a1-Acid glycoprotein was obtained from Sigma–Aldrich, Inc. (St. Louis, USA). To prepare desialylated a1-acid glycoprotein as an acceptor substrate for inhibitor screening, a1-acid glycoprotein at a concentration of 10 mg/ml was incubated at 37 °C for 3 h with sialidase (5 U/ml) from Arthrobacter ureafaciens, which was kindly provided by Marukin Bio, Inc. (Kyoto, Japan). Pyridylaminated (PA) sugar chains were purchased from Takara Bio, Inc. (Kyoto, Japan). Biotin-conjugated lectins, Maackia amurensis agglutinin (MAA) and Sambucus sieboldiana agglutinin (SSA), were purchased from Seikagaku Corporation (Tokyo, Japan). All other chemicals were of the highest purity available. Flavonoid derivatives. 3-Hydroxyflavone (compound 3), (+)-catechin (compound 40), and ()-epicatechin (compound 45) were purchased from Sigma–Aldrich Japan. (Tokyo, Japan) Quercetin (compound 1) and rutin (compound 6) were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Naringenin (compound 47) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Other compounds that had been synthesized previously were also used [28–35]. Glycosyltransferase assays. The activities of sialyltransferase and galactosyltransferase were measured by the following assay methods. (I) ELISA-based sialyltransferase assay: Quantitative enzyme assay for immobilized glycoproteins as acceptors was carried out according to the procedure of Mattox et al. [36]. Desialylated a1acid glycoprotein as an acceptor at a concentration of 10 lg/ml was immobilized on 96-well plastic plates (Maxisorp; Nunc Inc.,

Core structure a

Results and discussion Flavonoids, which belong to a class of natural polyphenols, can be broadly classified according to skeletal structure into flavone, flavonol, flavanone, and flavanon 3-ol. As shown in Fig. 1, chemically synthesized flavonoid derivatives with three types of core structure (Core a–c) were used as test compounds. Table 1 summarizes the whole structures and sialyltransferase inhibitory effects of these compounds. In an initial experiment, several compounds showed sialyltransferase inhibitory effects. Among these, compounds 37 and 39 showed the most potent inhibitory activities against both ST6Gal I and ST3Gal III with IC50 less than 10 lM. We further examined the chemical determinants of the flavonoid derivatives that showed the inhibitory effects. The chemical compounds that showed inhibitory activity commonly had the Core a framework, and neither Core b nor c. This finding clearly indicated that double bond formation between C2 and C3 positions on the C-ring is essential for the inhibitory effects of the compounds on sialyltransferases. As seen in compounds 36–39, sialyltransferase inhibition increased markedly with increasing number of hydroxyl groups introduced on the B-ring. These observations suggested that hydrophilic properties on the B-ring are indispensable for the inhibitory activities of flavonoid derivatives. Introduction of glucose on the C4´ position of the B-ring abolished the activity, meaning that large hydrophilic substitution on B-ring may have an adverse effect. Recently, we synthesized and evaluated dideoxy-epigallocatechin gallate (DO-EGCG) for the ability to inhibit virus infection [31]. Deoxygenation of A-ring is very effective to

O

7

A

2

3

4

8 5’ 7

6’

C

6 5

2’

4’

B

8

3’

3’

3’ 2’

Core structure c

Core structure b

2’

4’

B O 2

A

C

5

4

6

8 5’ 7

6’ 3

4’

B O 2

A

C

5

4

6

5’ 6’

3

O Fig. 1. Core structures of synthetic polyphenol derivatives used in this study. Core a represents the main structure of flavone or flavonol derivatives. Cores b and c are the main structures of flavanone and flavanone-3-ol, respectively.

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K.I.P.J. Hidari et al. / Biochemical and Biophysical Research Communications 382 (2009) 609–613 Table 1 Summary of structures and inhibitory activity of compounds against three sialyltransferases. Compound

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

Core strct.

a

b

c

IC50 (lM)

Functional groups on flavonoid core structures 0

0

0

0

C3

C4

C5

C6

C7

C8

C2

C3

C4

C5

C6

OH H OH H H O-Rutinose*1 O-b-D-Glc *2 H H O-b-D-Glc H H H H H H H H H H H H H H H H H H H H H H H H H H OH OH (S)-OH (R)-OAc (S)-OH (S)-OAc *3 (R)-OH (S)-OAc H2 H2 H2 H2 H2 H2 H2 H2

@O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O @O H2 (S)-OAc H2 H2 *3 H2 (R)-OAc @O @O @O @O H2 @O @O H2

OH OH H H OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH OH H H H H OH OBn OBn OAc OAc OH OBn OH OH OH OAc OH OH OAc OH

H H H H H H H H H H H H H H H H H H b-D-Glc H b-D-Glc H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

OH OH H H OCH3 OH OH OH OCH3 OCH3 OH OH O-b-L-Glc OH O-b-D-Glc O-b-L-Glc O-b-D-Glc O-b-L-Glc OCH3 OCH3 OCH3 TBDMS TBDMS TBDMS OCH2OCH3 OH OC2H5 O-n-Pr O-i-Pr O-n-Bu OBn OAc OBz OMs OTs H H H H OH OBn OBn OAc OAc OH OBn OH OCH3 OCH3 OCH3 OCH3 OCH3 OCH3 OCH3

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

OH H H H H OH H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H OH H OH H H H H H H H H H H H H H H H

OH OH H H OH OH OH OH OAc OCH3 OH O-b-D-Glc OH O-b-L-Glc O-b-D-Glc O-b-L-Glc O-b-L-Glc O-b-D-Glc OH O-b-D-Glc O-b-D-Glc OH OTBDMS OCH2OCH3 OCH2OCH3 OCH2OCH3 OH OH OH OH OH OH OH OH OH OH OH OH OH OH OBn OBn OAc OAc OH OBn OH OH OCH3 OAc OAc OPiv OPiv OPiv

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H OH H OH OH OBn OBn OAc OAc OH OBn H H H H H H H H

H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H H

0

rST6

hST6

rST3

– – – – 39.0A – – – – – 43.5A – 25.0A – – – – – 32.9%B – – – – – – – 93.5A – – – – – – – – 66.0A 5.1A 424A 1.9A – – – – – – – – – – – – – – –

– – – – 57.5A – – – – – – – 40.0A – – – – – 49.8%B – – – – – – – NT – – – – – – – – 81.7A 7.1A – 7.5A – – – – – – – – – – – – – – –

– – – – 82.2A – – – – – – – 45.1%B – – – – – 10.7%B – – NT NT NT NT NT NT NT NT NT NT NT NT NT NT 192.2A 1.4A NT NT – – – – – – – – – – – – – – –

rST6, hST6 and rST3 indicate rat ST6Gal I, human ST6Gal I and rat ST3Gal III, respectively. Ac, Bn, Bz and Piv indicate acetyl, benzyl, benzoyl and pivaloyl groups, respectively. Glc, glucose. TBDMS, n-Pr, i-Pr, n-Bu, Ms, and Ts indicate tert-butyldimethylsilyl, n- and i-propyl, n-butyl, methanesulfonyl and p-toluenesulfonyl groups. –, not inhibited significantly up to 50 lM compounds; NT, not tested.

4

3 *1, L-Rham a-1,6-D-Glc; *2, O-6-O-Ac-b-D-Glc; *3,

O

.

O

=

O

A B

IC50 values of compounds at 15 lM and 250 lM CMP-Neu5Ac for ST6Gal I and ST3Gal III, respectively. Relative infectivity in the presence of compounds (50 lM) to control infection.

generate diverse types of bioactive polyphenols. Therefore, deoxyflavonoids such as compounds 36–39 were synthesized and evaluated for the ability to inhibit sialyltransferases. Elimination of all hydroxyl groups from the A-ring enhanced the inhibitory effect. This implied that increasing the hydrophobicity of the A-ring

may reinforce the inhibitory effect in cooperation with the B-ring. Other than compound 13, inhibition was also observed for those with small hydrophobic groups up to –OC2H5 introduced on C7 of the A-ring, such as compound 5, 19, and 27. Limited hydrophobic modification on the C7 position may show a specific effect.

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Table 2 Kinetic parameters of human ST6Gal I in the presence of compounds. Compound 5

Compound 36 1

Conc. (lM)

Km (lM)

Vmax (lM

0 30 50 80

4.77 5.21 11.6 25.6

0.66 0.66 0.59 0.35

1

s

)

Compound 37 1

Conc. (lM)

Km (lM)

Vmax (lM

25 50 75

3.39 7.56 12.3

0.66 0.52 0.24

s

1

)

Conc. (lM)

Km (lM)

Vmax (lM1 s1)

2.0 4.0 6.0

3.02 8.39 15.9

0.73 0.72 0.53

Km values are indicated for CMP-NeuAc in the presence or the absence of compounds.

Hydroxylation on the C3 position did not significantly affect the inhibitory activity. On the other hand, comparison of compounds 5 to 19 indicated that glycosylation on the C6 position may affect linkage-specific inhibition of sialylation, such as a2,3 and a2,6. Taken together, we found three relevant rules for sialyltransferase inhibition of synthetic flavonoid derivatives based on the structure-inhibitory activity relationship. First, a double bond introduced between C2 and C3 positions is essential for the inhibitory action. Second, sialyltransferase inhibition depends on increasing hydrophilicity of the B-ring. Third, hydrophobic functional groups introduced on the hydroxyl groups of the A-ring enhance the inhibitory activity. We chose three representative compounds, 5, 36, and 37, and investigated their inhibitory specificities and kinetic properties. To determine the specific inhibition against sialyltransferases, compound 5 was used to examine whether it can inhibit a galactosyltransferase, B4GalT 1. HPLC analysis demonstrated that B4GalT 1 showed 97.0% activity in comparison to the control in the presence of 100 lM compound 5. This result strongly suggested that flavonoids specifically inhibit sialyltransferases. To elucidate the inhibitory mechanism of action of the compounds, we investigated the kinetic properties of human ST6Gal I in the presence or absence of compounds 5, 36, and 37. Table 2 shows a summary of the kinetic parameters of human ST6Gal I. In the absence of any compounds, the Km value of recombinant human ST6Gal I for CMP-NeuAc was almost equivalent to that of the native enzyme, as described previously [38]. As the enzymatic properties, such as substrate specificity and detergent requirements, of recombinant ST6Gal I are similar to those of the native enzyme [27], kinetic analysis of the recombinant enzyme in the presence of flavonoid derivatives was carried out. All three compounds examined affected both Km for CMPNeuAc and Vmax values. These compounds increased Km values for CMP-NeuAc and decreased Vmax values in a dose-dependent manner. These observations clearly indicated that the flavonoid derivatives show mixed inhibition of sialyltransferases. Similar to our findings, Wu et al. screened a natural compound library and obtained soyasaponin I, which inhibited two b-galactoside a2,3-sialyltransferases in a competitive manner against donor substrate, CMP-NeuAc (IC50 5–10 lM) [18]. However, neither the inhibitory mechanism nor chemical determinants of soyasaponin I for sialyltransferase inhibition have been elucidated. In this study, compounds 37 and 39 showed inhibitory effects equivalent to those of soyasaponin I with IC50 of approximately 5 lM. These flavonoids, which have different chemical structures from the steroid-like soyasaponin I, showed a unique mechanism of inhibition against both b-galactoside a2,3- and a2,6-sialyltransferases with mixed inhibition kinetics. These findings indicate that flavonoids have distinct molecular actions on sialyltransferases from soyasaponin I. Sialyltransferase is a type II membrane protein, with the cytoplasmic tail at the N-terminus, followed by a transmembrane region and a long catalytic domain at the C-terminus. There are several conserved regions, termed sialylmotifs in the catalytic domain [39–43]. Sialylmotif L contributes to binding of the donor substrate, CMP-NeuAc. Motif S is involved in binding of both

CMP-NeuAc and the acceptor substrate. The kinetic properties of the flavonoids strongly suggest interactions with either sialylmotif L or S region. This may be partially responsible for the inhibitory activity of the flavonoid derivatives. Alternatively, they may interact with other amino acid residues that are not directly related to sialylmotifs, possibly resulting in sialyltransferase inhibition by induction of conformational changes in substrate binding sites. Further investigations are required to determine the molecular interaction of the flavonoids with sialyltransferases. In conclusion, we found potent and specific sialyltransferase inhibitors by investigating of chemically synthesized flavonoid derivatives. They showed unique characteristics, such as mixed inhibitory mechanisms and chemical determinants responsible for inhibition. According to the physical, chemical, and biological properties, such as stability, adsorption, and safety, these may become lead compounds to modify sialylation both in vitro and in vivo. Acknowledgments This work was supported by Grants-in-Aid for Scientific Research on Priority Areas (18570135) from the Ministry of Education, Science, Sports, and Culture of Japan. This work was also supported by Takeda Science Foundation. References [1] N.M. Varki, A. Varki, Diversity in cell surface sialic acid presentations: implications for biology and disease, Lab. Invest. 87 (2007) 851–857. [2] M.E. Taylor, K. Drickamer, Paradigms for glycan-binding receptors in cell adhesion, Curr. Opin. Cell Biol. 19 (2007) 572–577. [3] Y. Suzuki, Gangliosides as influenza virus receptors. Variation of influenza viruses and their recognition of the receptor sialo-sugar chains, Prog. Lipid Res. 33 (1994) 429–457. [4] S. Olofsson, T. Bergström, Glycoconjugate glycans as viral receptors, Ann. Med. 37 (2005) 154–172. [5] P.R. Crocker, Siglecs: sialic-acid-binding immunoglobulin-like lectins in cell– cell interactions and signaling, Curr. Opin. Struct. Biol. 12 (2002) 609–615. [6] A. Harduin-Lepers, M.A. Recchi, P. Delannoy, 1994, the year of sialyltransferases, Glycobiology 5 (1995) 741–758. [7] S. Tsuji, Molecular cloning and functional analysis of sialyltransferases, J. Biochem. (Tokyo) 120 (1996) 1–13. [8] A. Harduin-Lepers, V. Vallejo-Ruiz, M.-A. Krzewinski-Recchi, B. Samyn-Petit, S. Julien, P. Delannoy, The human sialyltransferase family, Biochimie 83 (2001) 727–737. [9] U. Grundmann, C. Nerlich, T. Rein, G. Zettlmeissl, Complete cDNA sequence encoding human beta-galactoside alpha-2,6-sialyltransferase, Nucleic Acids Res. 18 (1990) 667. [10] J. Weinstein, U. de Souza-e-Silva, J.C. Paulson, Purification of a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase and a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase to homogeneity from rat liver, J. Biol. Chem. 257 (1982) 13835–13844. [11] J. Weinstein, U. de Souza-e-Silva, J.C. Paulson, Sialylation of glycoprotein oligosaccharides N-linked to asparagine. Enzymatic characterization of a Gal beta 1 to 3(4)GlcNAc alpha 2 to 3 sialyltransferase and a Gal beta 1 to 4GlcNAc alpha 2 to 6 sialyltransferase from rat liver, J. Biol. Chem. 257 (1982) 13845– 13853. [12] W.D. Klohs, R.J. Bernacki, W. Korytnyk, Effects of nucleotides and nucleotide: analogs on human serum sialyltransferase, Cancer Res. 39 (1979) 1231–1238. [13] L.D. Cambron, K.C. Leskawa, Inhibition of CMP-N-acetylneuraminic acid: lactosylceramide sialyltransferase by nucleotides, nucleotide sugars and nucleotide dialdehydes, Biochem. Biophys. Res. Commun. 193 (1993) 585– 590.

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