View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Experimental Parasitology 130 (2012) 183–188 Contents lists available at SciVerse ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 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 article info abstract 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 com- pounds, 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 À20 °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) con- taining 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 inhibi- tor. 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.