The Leishmanicidal Flavonols Quercetin and Quercitrin Target

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Experimental Parasitology 130 (2012) 183–188

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Experimental Parasitology

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The leishmanicidal ﬂavonols quercetin and quercitrin target Leishmania (Leishmania) amazonensis arginase ⇑ Edson Roberto da Silva a, , Claudia do Carmo Maquiaveli b, Prislaine Pupolin Magalhães c

a Departamento de Ciências Básicas, Universidade de São Paulo, Faculdade de Zootecnia e Engenharia de Alimentos, Av. Duque de Caxias Norte, 225, CEP 13635-900 Pirassununga, SP, Brazil b Programa de pós-graduação em Fisiologia, Departamento de Fisiologia, Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Av. Bandeirantes, 3900 Monte Alegre, CEP 14049-900 Ribeirão Preto, SP, Brazil c Programa de pós-doutorado Universidade de São Paulo, Faculdade de Zootecnia e Engenharia de Alimentos, Av. Duque de Caxias Norte, 225, CEP 13635-900 Pirassununga, SP, Brazil

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Article history: Polyamine biosynthesis enzymes are promising drug targets for the treatment of leishmaniasis, Chagas’ Received 7 June 2011 disease and African sleeping sickness. Arginase, which is a metallohydrolase, is the first enzyme involved Received in revised form 24 November 2011 in polyamine biosynthesis and converts arginine into ornithine and urea. Ornithine is used in the Accepted 19 January 2012 polyamine pathway that is essential for cell proliferation and ROS detoxification by trypanothione. The Available online 1 February 2012 flavonols quercetin and quercitrin have been described as antitrypanosomal and antileishmanial compounds, and their ability to inhibit arginase was tested in this work. We characterized the inhibition of Keywords: recombinant arginase from Leishmania (Leishmania) amazonensis by quercetin, quercitrin and isoquerci- Leishmania trin. The IC values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3 M, Arginase 50 l Quercetin respectively. Quercetin is a mixed inhibitor, whereas quercitrin and isoquercitrin are uncompetitive Quercitrin inhibitors of L. (L.) amazonensis arginase. Quercetin interacts with the substrate L-arginine and the cofac- 2+ Isoquercitrin tor Mn at pH 9.6, whereas quercitrin and isoquercitrin do not interact with the enzyme’s cofactor or Polyamine substrate. Docking analysis of these flavonols suggests that the cathecol group of the three compounds 2þ 2þ interact with Asp129, which is involved in metal bridge formation for the cofactors MnA and MnB in the active site of arginase. These results help to elucidate the mechanism of action of leishmanicidal flavonols and offer new perspectives for drug design against Leishmania infection based on interactions between arginase and flavones. Ó 2012 Elsevier Inc. Open access under the Elsevier OA license.

1. Introduction oxide and citrulline by nitric oxide synthase. Production of arginase

A1 in macrophages can be stimulated by TH2 lymphocytes. The Polyamines are essential for cell proliferation and the production resulting increase in arginase A1 activity leads to the consumption of trypanothione, which is involved in reactive oxygen species (ROS) of L-arginine and to a decrease in NO synthesis that favors the prolif- detoxification (Colotti and Ilari, 2011). Arginase hydrolyzes eration of Leishmania in macrophages (Wanderley and Barcinski, L-arginine to L-ornithine and urea as the first, rate-limiting step of 2010). T. cruzi, which lacks arginase, establishes a host–parasite rela- polyamine synthesis in Leishmania and Trypanosoma brucei, but it tionship by producing cruzipain, which induces the synthesis of is absent in Trypanosoma cruzi.InLeishmania, arginase produces arginase A2 in host cardiomyocytes (Aoki et al., 2004) or arginase L-ornithine, which is then decarboxylated by ornithine decarboxyl- A1 in macrophages (Cuervo et al., 2008). Thus, the parasite drives ase (ODC) to generate putrescine, which continues down the the production of putrescine in infected cells and ingests putrescine polyamine synthesis pathway. Inhibition of ODC by 1,4-diamino- for its own polyamine biosynthesis (Heby et al., 2007). Comparison 2-butanone induces parasite cell death (Vannier-Santos et al., of the active sites of human andLeishmania (Leishmania) amazonensis 2008). In mammals, arginase is present in large quantities in the arginase (da Silva et al., 2002) has shown differences that can be liver, where it catalyzes the final reaction of the urea cycle. There exploited for rational drug design. are two known isoforms of arginase in mammals: arginase A1 and Quercetin has a leishmanicidal effect on the amastigote stage of A2 (hepatic or extra-hepatic). L-Arginine is also converted to nitric Leishmania (Leishmania) donovani (Tasdemir et al., 2006) and L. (L.) amazonensis (Muzitano et al., 2006a,b), and it reduces parasite

⇑ Corresponding author. Fax: +55 19 3565 4117. burden in L. (L.) donovani-infected Hamster spleen cells (Sen E-mail addresses: [email protected] (E.R. da Silva), [email protected] et al., 2008). The IC50 values for growth inhibition of L. (L.) donovani (C.d.C. Maquiaveli), [email protected] (P.P. Magalhães). amastigotes are 1.0 and 17.7 lg/mL, respectively, for quercetin and

0014-4894 Ó 2012 Elsevier Inc. Open access under the Elsevier OA license. doi:10.1016/j.exppara.2012.01.015 184 E.R. da Silva et al. / Experimental Parasitology 130 (2012) 183–188

Fig. 1. Chemical structures of tested ﬂavonoids.

quercitrin. When tested against L. (L.) amazonensis, quercitrin has centrifuged at 12,000g. Arginase in the supernatant was purified an IC50 of 8 lg/mL (Muzitano et al., 2006a,b). In addition, quercetin in a 1-mL HiTrap Chelating Sepharose (GE Health Care) column 2+ has shown IC50 of 8.3 lg/mL against T. brucei rhodesiensi and charged with Ni . Arginase activation was performed in the col- IC50 >30lg/mL against T. cruzi, a trypanosomatid that lacks argi- umn at room temperature (25 °C) by slowly loading 3 mL of nase (Tasdemir et al., 2006). 50 mM MnSO4 in buffer A (Silva and Floeter-Winter, 2010). Argi- In this work, we tested the leishmanicidal flavonoid aglycone nase was eluted by loading 3 mL of buffer A containing 50 mM quercetin and two glycoflavones, quercitrin (quercetin 3-O-a-L- Imidazole. The eluted solution was mixed with one volume of glyc- rhamnopyranoside) and isoquercitrin (quercetin 3-b-D-glucoside) erol and stored at 20 °C. All procedures were performed by inject- (Fig. 1). Quercetin interacted with arginase’s substrate, L-arginine, ing buffers into the column with a syringe. and its cofactor manganese. Through docking, we simulated the interactions of quercetin, quercitrin and isoquercitrin with L. (L.) 2.4. Inhibition kinetics and IC50 determination amazonensis arginase. Reactions were carried out in buffer (50 mM CHES pH 9.5) containing variable concentrations of L-arginine substrate (6.25, 12.5, 2. Materials and methods 25, 50 and 100 mM) at pH 9.5, and 5 lM inhibitor. The substrate concentrations were achieved by serial dilution in water. Two 2.1. Chemicals and reagents mixes were prepared: mix 1 contained 10 lL of 500 lM inhibitor and 290 lL water, and mix 2 contained 5 lL of enzyme solution Quercetin hydrate – P95% (Aldrich 337951), quercetin 3-b-D- and 100 lL of 500 mM CHES buffer, pH 9.5, and 195 lL of water. glucoside – P90% (HPLC) (Sigma 17793), quercitrin hydrate – Each reaction mixture contained 40 lL of substrate, obtained by P78% (Sigma Q3001) and CelLytic™ B Cell Lysis reagent (Sigma serial dilution, 30 lL of mix 1 and 30 lL of mix 2, such that the final 08740) were purchased from Sigma–Aldrich. The Enzymatic Urea concentrations were 5 lM inhibitor, 50 mM CHES and the desired Kit was purchased from QUIBASA (Belo Horizonte, MG, Brasil). concentration of L-arg. The reaction mixtures were incubated for 15 min at 37 °C. Arginase activity was determined using the Berth- 2.2. Analysis of quercetin interaction with the substrate and cofactor of elot enzymatic-colorimetric assay method (Fawcett and Scott, arginase 1960), which detects urea production. Briefly, 10 lL of the reaction mixture was transferred to 750 lL of reagent 1 (20 mM phosphate The ability of quercetin to chelate metal was directly assessed buffer, pH 7, containing 60 mM salicylate, 1 mM sodium nitroprus- through spectral characterization of each flavonoid by measuring side and >500 IU urease). The reagent 1 mixture was incubated at shifts in band I (300–480 nm) and band II (210–290 nm). Band I 37 °C for 10 min, and then, 750 lL of reagent 2 (10 mM sodium and band II absorptions were related to the B and A ring (Fig. 1), hypochlorite and 150 mM NaOH) was added before incubating at respectively (Leopoldini et al., 2006; Sen et al., 2008). Flavonoid 37 °C for 10 min. Spectrophotometric measurements were per- solutions (50 lM) were added to equimolar concentrations of formed at 600 nm using a Hitachi 2810U spectrophotometer. The 2+ Mn or L-arginine in buffer (50 mM glycine, pH 9.6) at room tem- control experiments were performed under the same conditions, perature. Shifts in the absorption spectra of the flavonoids after but in the absence of the inhibitor. complex formation with a metal or the L-arginine substrate were IC50 measurements were performed with inhibitor concentra- analyzed with the U-2810 UV–VIS spectrophotometer (Hitachi). tions obtained by the following two serial dilutions: the first set of dilutions contained 1250, 125, 12.5 and 1.25 lM inhibitor, and 2.3. Expression and purification of recombinant arginase the second set of dilutions contained 250, 25, 2.5, 0.25 lM inhibitor. All assays were performed in duplicate. The reaction was Recombinant arginase without a histidine tag was expressed carried out with 50 mM of the substrate, L-arginine, at pH 9.5, from L. (L.) amazonensis as described previously by da Silva et al. and 50 mM CHES buffer at pH 9.5. (2008). Briefly, the arginase clone was grown in SOB medium (20 g of tryptone, 5 g of yeast extract and 0.5 g of NaCl, 2.5 mL of 2.5. Data analysis 1 M KCl and distilled water to 1 L) and after sterilization and sub- sequent cooling, were added 10 mL of 1 M MgCl2. Culture cells was Inhibition data were analyzed using GraphPad Prism 3.0 soft- reached until OD 0.6–0.8 and recombinant enzyme expression was ware. The transformation of IC50 into Ki was carried out using a induced by adding 1 mM IPTG and 10 mM MnSO4 for 3 h. Cells web-based tool (http://botdb.abcc.ncifcrf.gov/toxin/kiConverter. were harvested from 50-mL cultures and lysed in 1 mL of buffer jsp) that uses the following equation for classical inhibition:

A (MOPS 100 mM pH 7.2, NaCl 500 mM) containing 1 mM PMSF; uncompetitive Ki =IC50/(KM/S + 1) (Cer et al., 2009). For all tests, 10% CelLytic B was added, and the cells were gently agitated for differences with p < 0.05 were considered signiﬁcant. Analysis 15 min at room temperature. After lysis, the homogenate was was performed by ANOVA using GraphPad Prism 3.0 software. E.R. da Silva et al. / Experimental Parasitology 130 (2012) 183–188 185

2.6. Computational analysis Asp141 occupy the same position as Asp128 which is responsible 2þ for coordinating the MnA cofactor in the active site of rat arginase Computational analysis used a previously described model of L. (Kanyo et al., 1996). Quercitrin also donates a hydrogen bond to (L.) amazonensis arginase based on rat liver arginase complexed the carbonyl of Val193 and accepts a hydrogen bond from with AOH ((S)-2-amino-7oxoheptanoic acid) (PDB, ID code 1T5F) Thr257. Isoquercitrin accepts two more hydrogen bonds from (da Silva et al., 2002; Shin et al., 2004). The receptor target (L. (L.) Asn152 and Thr257 (Fig. 3). Thr257 and Asn152 occupy the same amazonensis arginase model) and docking ligands were prepared position as Thr246 and Asn139 in the rat liver arginase active site, using Chimera (Pettersen et al., 2004). The molecular surface of where these two amino acids interact with nor-NOHA (Cox et al., the target was generated based on the algorithm developed by 2001), a competitive inhibitor of Leishmania (Leishmania) mexicana Richards (1977). Sphere generation was performed using the sph- arginase (Riley et al., 2011). gen algorithm; the spheres were distributed with dock6 and selected using ‘‘spheres_selector’’. Grid generation was achieved 3.3. Inhibition of arginase using Grid, which is distributed as an accessory to DOCK (Kuntz et al., 1982). Flexible Dock (Moustakas et al., 2006) was used to Recombinant and native arginase were characterized by us pre- verify interactions between the target arginase and natural chem- viously (da Silva et al., 2008) and exhibit the same KM and Vmax at icals. Results obtained by docking were visualized and analyzed on pH 9.5. The kinetics performed at pH 7.5 showed an increase in the

Chimera alpha version 1.4 (build 28870). KM and Vmax decreased (Table 2). Quercetin, quercitrin and a third related molecule, isoquercitrin, were tested for their ability to inhi- 3. Results bit L. (L.) amazonensis arginase. Table 3 summarizes the data and quality of the estimated IC50 and Ki for quercetin, quercitrin and 3.1. The substrate L-arginine and the cofactor manganese interact with isoquercitrin. Fig. 4 shows the best fit data for the sigmoidal (Log- quercetin EC50) function for the three flavonoids tested. Fig. 5 shows the Michaelis–Menten and double reciprocal (1/v, 1/S) Lineweaver– 2+ The interaction of Mn with quercetin suppressed band II Burk plot, which shows how the enzyme’s apparent KM varies in (240–285 nm) at 1:1 ratio of flavonoid to metal or a 1:1 ratio of fla- the presence of each inhibitor. Quercetin is a mixed inhibitor vonoid to substrate at pH 9.5. Band II was restored in the presence whereas quercitrin and isoquercitrin are uncompetitive inhibitors of EDTA (25 lM), which allowed for interaction with Mn2+ (Fig. 2). of arginase. The inhibitions between the three flavonoids tested Quercitrin and isoquercitrin did not produce any change in bands I in this work were not significantly (p > 0.05). and II under similar conditions. At pH 7.5, the quercetin bands I 2+ and II were unchanged in the presence of Mn or L-arg. The spec- 4. Discussion 2+ trum of quercetin simultaneously incubated with Mn and L-arg was identical to that obtained with the quercetin–Mn2+ interaction The polyamine biosynthesis pathway has been explored as a and suggests that the same mode of interaction exists between the target for the treatment of trypanosomatid infection (Colotti and substrate and the cofactor in the presence of the inhibitor. This re- Ilari, 2011; Riley et al., 2011). Arginase is the first enzyme of poly- sult was reinforced by the lack of interaction between quercitrin amine biosynthesis in Leishmania and drugs that target this and the substrate, between quercitrin and Mn2+, between isoqu- enzyme help to treat Leishmania infection (Iniesta et al., 2005, ercitrin and the substrate and between isoquercitrin and Mn2+, be- 2001, 2002; Kropf et al., 2005; Taylor-Robinson, 2001). cause the presence of a rhamnoside or glucoside linked to the Disruption of the arginase gene in L. (L.) mexicana (Roberts et al., hydroxyl group of the C ring (Fig. 1) causes a steric barrier to com- 2004) and Leishmania (Leishmania) major (Reguera et al., 2009) plex formation. In fact, Jun (Jun et al., 2007) proposed that this hy- shows that parasite development is blocked in the absence of argi- droxyl group and the keto group, which form the C ring, are nase but restored by adding ornithine or putrescine (Reguera et al., responsible for chelating Mn2+. 2009). Natural compounds such as flavonoids have shown activity 3.2. Docking against Leishmania amastigotes in macrophage infection (Muzitano et al., 2006a,b; Tasdemir et al., 2006). When used against L. (L.) Molecular docking was performed to predict and visualize the donovani, quercetin reduces intracellular amastigote load by 70% interaction between the selected flavonoids and arginase. Table 1 at 45 lM(Mittra et al., 2000). The plant Kalanchoe pinata, which summarizes the predicted interactions of flavonoids with amino contains quercetin, quercitrin and other flavonoids, shows activity acids in the active site pocket. The two hydroxyls from the catechol against cutaneous (Muzitano et al., 2009) and visceral leishmania- group of all flavonoids donate two hydrogen bonds to Asp141. sis (Gomes et al., 2010).

Fig. 2. Evaluation of chelation/interaction capacity of QUE with manganese (a) and arginine (b). Spectral shifts in band I (300–480 nm) and band II (210–290 nm) of the ﬂavonoid were determined in the absence or in the presence of equimolar Mn2+ or arginine. 186 E.R. da Silva et al. / Experimental Parasitology 130 (2012) 183–188

Table 1 Interactions of ﬂavonoids with L. (L.) amazonensis arginase.

Flavonoid Donor (D) Acceptor (A) Hydrogen Distances (Å)

D...•ADAH...•A

Quercetin O6 Asp141 OD1 H9 2.656 1.756 O7 Asp141 OD1 H10 3.017 2.053 Quercitrin O10 Asp141 OD1 H19 2.750 1.778 O11 Asp141 OD1 H20 2.699 1.733 O9 Val193 O H18 3.326 2.533 Thr257 OG1 O4 Thr257 HG1 3.275 2.463 Isoquercitrin O11 Asp141 OD1 H19 2.770 1.801 O12 Asp141 OD1 H20 2.702 1.742 Asn152 ND2 O5 Asn152 HD21 3.402 2.412 Thr257 OG1 O4 Thr257 HG1 3.268 2.470

Fig. 3. L. (L.) amazonensis arginase with docked structures of quercetin (A), quercitrin (B) and isoquercitrin (C). Manganese atoms are represented by magenta spheres.

Table 2 Kinetic parameters of the native and recombinant arginase from L. (L.) amazonensis.

pH = 9.6a pH = 7.5

1 1 1 1 Arginase KM (mM) Vmax (lmol urea min mg protein ) KM (mM) Vmax (lmol urea min mg protein ) Native 23.9 ± 0.96 192.3 ± 14.3 ND ND Recombinant 21.5 ± 0.90 144.9 ± 8.9 66 ± 7 34 ± 4

ND = not determined. a da Silva et al. (2008).

Table 3 Comparison of IC50 and Ki for arginase inhibition by quercetin, quercitrin and apoptosis (Mittra et al., 2000). kDNA and mitochondria were altered isoquercitrin. when the polyamine pathway was inhibited by 1,4-diamino-2- 2 Inhibitor IC50 (lM) R Ki (lM) Type of inhibition butanone (Vannier-Santos et al., 2008). In fact, polyamines are Quercetin 4.30 ± 0.03 0.99 – Mixed responsible for trypanothione biosynthesis, which is characteristic Quercitrin 10.00 ± 0.08 0.95 6.99 Uncompetitive of trypanosomatids. Blocking trypanothione synthesis alters kDNA Isoquercitrin 3.80 ± 0.04 0.99 2.65 Uncompetitive organization and mitochondria through oxidative stress (Colotti and Ilari, 2011). Quercetin interferes with iron metabolism by chelating the metallic ion, and it inhibits ribonucleotide reductase (Sen et al., 2008). Quercetin interacts with Mn2+ at pH 9.5 (Fig. 2), This study describes the effect of the flavonoid aglycone querce- whereas the derivatives isoquercitrin and quercitrin do not chelate 2+ tin and two quercetin-derived compounds, quercitrin and Mn . The interaction of the quercetin with L-arginine at pH 7.5 only isoquercitrin, on arginase activity. The IC50 values for arginase inhi- was observed at concentration of 250 mM of substrate (data not bition by quercetin, quercitrin and isoquercitrin were previously show). The difference observed between flavonoids for mechanism determined to be 4.3, 10.0 and 3.8 lM, respectively. of inhibition and the interaction with substrate and cofactor (Mn2+) Docking analyses show that all flavonoids tested interact with can be due to absence of carbohydrate moiety in quercetin.

Asp129, which is an important conserved amino acid responsible In T. cruzi, which lacks arginase, the IC50 for quercetin (>30 lg/ 2þ 2þ for coordinating MnA and MnB in the active site of arginase mL) is 30 times greater than for L. (L.) donovani (IC50 1 lg/mL); the (Kanyo et al., 1996). IC50 in T. brucei rhodesiensi is 8.3 lg/mL. Quercitrin has IC50 values At higher concentrations, such as 150 lM, quercetin linearizes of 17.7 and 27.9 lg/mL for L. (L.) donovani and T. brucei, respectively 40% of minicircle kDNA by inducing topoisomerase II, leading to (Tasdemir et al., 2006). The data presented here show that quercetin E.R. da Silva et al. / Experimental Parasitology 130 (2012) 183–188 187

Fig. 4. Dose–response curve for arginase inhibition by ﬂavonoids: quercetin (Ç), quercitrin (d) and isoquercitrin (h). The nonlinear regression show r2 > 0.99 for quercetin and isoquercitrin and for quercitrin r2 > 0.94.

Fig. 5. Michaelis–Menten (A) and Lineweaver–Burk plot (B) for inhibitors: quercetin (Ç), isoquercitrin (h), quercitrin (d) and control (.). The nonlinear regression for the Michaelis–Menten plot and the linear regression for the Lineweaver–Burk plot show r2 > 0.99 and r2 > 0.98, respectively.

and derived flavonoids are strong inhibitors of arginase. The flavo- 2000). It remains to be determined whether quercetin can be used noid aglycone quercetin and its derivative quercitrin exhibit low as a ‘‘promiscuous’’ drug prototype for leishmaniasis treatment. toxicity against L6 cells, with IC50 values of 37 and >90 lg/mL, The information presented here can be used to develop drugs respectively (Tasdemir et al., 2006). Quercetin also shows no cyto- against leishmaniasis. Moreover, quercetin and derived flavonoids toxic effects on Caucasian hepatocyte or lung carcinoma cells can be considered prototypes for rational drug design based on the (HepG2 and A549 epithelial cell lines) at concentrations as high as inhibition of arginase or other quercetin targets. 200 lM(Katalinic´ et al., 2010). Synthetic inhibitors of arginase such x as nor-NOHA (N -hydroxy-nor-L-arginine) and ABH (2(S)-amino-6- Acknowledgment 6 boronohaxanoic) have a Ki of 10 M when tested against L. (L.) mexicana arginase, but they fail to inhibit the growth of promastigotes at This work was supported by the Foundation for the Support of this concentration (Riley et al., 2011); these results are similar to Research of the State of São Paulo (FAPESP), Brazil. those observed with pentostam and quercitrin, which are active only against amastigotes (Muzitano et al., 2006a,b). Flavonoid References dimers are bivalent modulators of resistance to pentamidine and sodium stibogluconate in Leishmania (Wong et al., 2007). Aoki, M.P., Guiñazú, N.L., Pellegrini, A.V., Gotoh, T., Masih, D.T., Gea, S., 2004. Cruzipain, a major Trypanosoma cruzi antigen, promotes arginase-2 expression To date, the multiple quercetin targets that have been identified and survival of neonatal mouse cardiomyocytes. Am. J. Physiol. 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