Experimental Parasitology 130 (2012) 183–188

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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

a r t i c l e i n f o a b s t r a c t

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
Arginase
trin. The IC50 values for quercetin, quercitrin and isoquercitrin were estimated to be 3.8, 10 and 4.3 lM,
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-
Isoquercitrin tor Mn2+ 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
interact with Asp129, which is involved in metal bridge formation for the cofactors Mn2þ 2þ
A 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. All rights reserved.

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

Polyamines are essential for cell proliferation and the production
of trypanothione, which is involved in reactive oxygen species (ROS)
detoxification (Colotti and Ilari, 2011). Arginase hydrolyzes
L-arginine to L-ornithine and urea as the first, rate-limiting step of
polyamine synthesis in Leishmania and Trypanosoma brucei, but it
is absent in Trypanosoma cruzi. In Leishmania, arginase produces
L-ornithine, which is then decarboxylated by ornithine decarboxyl-
ase (ODC) to generate putrescine, which continues down the
polyamine synthesis pathway. Inhibition of ODC by 1,4-diamino-
2-butanone induces parasite cell death (Vannier-Santos et al.,
2008). In mammals, arginase is present in large quantities in the
liver, where it catalyzes the final reaction of the urea cycle. There
are two known isoforms of arginase in mammals: arginase A1 and
A2 (hepatic or extra-hepatic). L-Arginine is also converted to nitric
⇑ Corresponding author. Fax: +55 19 3565 4117.
E-mail addresses: edsilva@usp.br (E.R. da Silva), cmaquiaveli@usp.br
(C.d.C. Maquiaveli), pupolin@usp.br (P.P. Magalhães).
A1 in macrophages can be stimulated by TH2 lymphocytes. The
resulting increase in arginase A1 activity leads to the consumption
of L-arginine and to a decrease in NO synthesis that favors the prolif-
eration of Leishmania in macrophages (Wanderley and Barcinski,
2010). T. cruzi, which lacks arginase, establishes a host–parasite rela-
tionship by producing cruzipain, which induces the synthesis of
arginase A2 in host cardiomyocytes (Aoki et al., 2004) or arginase
A1 in macrophages (Cuervo et al., 2008). Thus, the parasite drives
the production of putrescine in infected cells and ingests putrescine
for its own polyamine biosynthesis (Heby et al., 2007). Comparison
of the active sites of human andLeishmania (Leishmania) amazonensis
arginase (da Silva et al., 2002) has shown differences that can be
exploited for rational drug design.
Quercetin has a leishmanicidal effect on the amastigote stage of
Leishmania (Leishmania) donovani (Tasdemir et al., 2006) and L. (L.)
amazonensis (Muzitano et al., 2006a,b), and it reduces parasite
burden in L. (L.) donovani-infected Hamster spleen cells (Sen
et al., 2008). The IC50 values for growth inhibition of L. (L.) donovani
amastigotes are 1.0 and 17.7 lg/mL, respectively, for quercetin and

0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
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
an IC50 of 8 lg/mL (Muzitano et al., 2006a,b). In addition, quercetin
has shown IC50 of 8.3 lg/mL against T. brucei rhodesiensi and
IC50 > 30 lg/mL against T. cruzi, a trypanosomatid that lacks argi-
nase (Tasdemir et al., 2006).
In this work, we tested the leishmanicidal flavonoid aglycone
quercetin and two glycoflavones, quercitrin (quercetin 3-O-a-L-
rhamnopyranoside) and isoquercitrin (quercetin 3-b-D-glucoside)
(Fig. 1). Quercetin interacted with arginase’s substrate, L-arginine,
and its cofactor manganese. Through docking, we simulated the
interactions of quercetin, quercitrin and isoquercitrin with L. (L.)
amazonensis arginase.
2. Materials and methods
2.1. Chemicals and reagents
Quercetin hydrate – P95% (Aldrich 337951), quercetin 3-b-D-
glucoside – P90% (HPLC) (Sigma 17793), quercitrin hydrate –
P78% (Sigma Q3001) and CelLytic™ B Cell Lysis reagent (Sigma
08740) were purchased from Sigma–Aldrich. The Enzymatic Urea
Kit was purchased from QUIBASA (Belo Horizonte, MG, Brasil).
2.2. Analysis of quercetin interaction with the substrate and cofactor of
arginase
The ability of quercetin to chelate metal was directly assessed
through spectral characterization of each flavonoid by measuring
shifts in band I (300–480 nm) and band II (210–290 nm). Band I
and band II absorptions were related to the B and A ring (Fig. 1),
respectively (Leopoldini et al., 2006; Sen et al., 2008). Flavonoid
solutions (50 lM) were added to equimolar concentrations of
Mn2+ or L-arginine in buffer (50 mM glycine, pH 9.6) at room tem-
perature. Shifts in the absorption spectra of the flavonoids after
complex formation with a metal or the L-arginine substrate were
analyzed with the U-2810 UV–VIS spectrophotometer (Hitachi).
2.3. Expression and purification of recombinant arginase
Recombinant arginase without a histidine tag was expressed
from L. (L.) amazonensis as described previously by da Silva et al.
(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
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
reached until OD 0.6–0.8 and recombinant enzyme expression was
induced by adding 1 mM IPTG and 10 mM MnSO4 for 3 h. Cells
were harvested from 50-mL cultures and lysed in 1 mL of buffer
A (MOPS 100 mM pH 7.2, NaCl 500 mM) containing 1 mM PMSF;
10% CelLytic B was added, and the cells were gently agitated for
15 min at room temperature. After lysis, the homogenate was
centrifuged at 12,000g. Arginase in the supernatant was purified
in a 1-mL HiTrap Chelating Sepharose (GE Health Care) column
charged with Ni2+. Arginase activation was performed in the col-
umn at room temperature (25 °C) by slowly loading 3 mL of
50 mM MnSO4 in buffer A (Silva and Floeter-Winter, 2010). Argi-
nase was eluted by loading 3 mL of buffer A containing 50 mM
Imidazole. The eluted solution was mixed with one volume of glyc-
erol and stored at 20 °C. All procedures were performed by inject-
ing buffers into the column with a syringe.
2.4. Inhibition kinetics and IC50 determination
Reactions were carried out in buffer (50 mM CHES pH 9.5) con-
taining variable concentrations of L-arginine substrate (6.25, 12.5,
25, 50 and 100 mM) at pH 9.5, and 5 lM inhibitor. The substrate
concentrations were achieved by serial dilution in water. Two
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
and 100 lL of 500 mM CHES buffer, pH 9.5, and 195 lL of water.
Each reaction mixture contained 40 lL of substrate, obtained by
serial dilution, 30 lL of mix 1 and 30 lL of mix 2, such that the final
concentrations were 5 lM inhibitor, 50 mM CHES and the desired
concentration of L-arg. The reaction mixtures were incubated for
15 min at 37 °C. Arginase activity was determined using the Berth-
elot enzymatic-colorimetric assay method (Fawcett and Scott,
1960), which detects urea production. Briefly, 10 lL of the reaction
mixture was transferred to 750 lL of reagent 1 (20 mM phosphate
buffer, pH 7, containing 60 mM salicylate, 1 mM sodium nitroprus-
side and >500 IU urease). The reagent 1 mixture was incubated at
37 °C for 10 min, and then, 750 lL of reagent 2 (10 mM sodium
hypochlorite and 150 mM NaOH) was added before incubating at
37 °C for 10 min. Spectrophotometric measurements were per-
formed at 600 nm using a Hitachi 2810U spectrophotometer. The
control experiments were performed under the same conditions,
but in the absence of the inhibitor.
IC50 measurements were performed with inhibitor concentra-
tions obtained by the following two serial dilutions: the first set
of dilutions contained 1250, 125, 12.5 and 1.25 lM inhibitor, and
the second set of dilutions contained 250, 25, 2.5, 0.25 lM inhibi-
tor. All assays were performed in duplicate. The reaction was
carried out with 50 mM of the substrate, L-arginine, at pH 9.5,
and 50 mM CHES buffer at pH 9.5.
2.5. Data analysis
Inhibition data were analyzed using GraphPad Prism 3.0 soft-
ware. The transformation of IC50 into Ki was carried out using a
web-based tool (http://botdb.abcc.ncifcrf.gov/toxin/kiConverter.
jsp) that uses the following equation for classical inhibition:
uncompetitive Ki = IC50/(KM/S + 1) (Cer et al., 2009). For all tests,
differences with p < 0.05 were considered significant. Analysis
was performed by ANOVA using GraphPad Prism 3.0 software.
 E.R. da Silva et al. / Experimental Parasitology 130 (2012) 183–188
2.6. Computational analysis
Computational analysis used a previously described model of L.
(L.) amazonensis arginase based on rat liver arginase complexed
with AOH ((S)-2-amino-7oxoheptanoic acid) (PDB, ID code 1T5F)
(da Silva et al., 2002; Shin et al., 2004). The receptor target (L. (L.)
amazonensis arginase model) and docking ligands were prepared
using Chimera (Pettersen et al., 2004). The molecular surface of
the target was generated based on the algorithm developed by
Richards (1977). Sphere generation was performed using the sph-
gen algorithm; the spheres were distributed with dock6 and
selected using ‘‘spheres_selector’’. Grid generation was achieved
using Grid, which is distributed as an accessory to DOCK (Kuntz
et al., 1982). Flexible Dock (Moustakas et al., 2006) was used to
verify interactions between the target arginase and natural chem-
icals. Results obtained by docking were visualized and analyzed on
Chimera alpha version 1.4 (build 28870).
3. Results
3.1. The substrate L-arginine and the cofactor manganese interact with
quercetin
The interaction of Mn2+ with quercetin suppressed band II
(240–285 nm) at 1:1 ratio of flavonoid to metal or a 1:1 ratio of fla-
vonoid to substrate at pH 9.5. Band II was restored in the presence
of EDTA (25 lM), which allowed for interaction with Mn2+ (Fig. 2).
Quercitrin and isoquercitrin did not produce any change in bands I
and II under similar conditions. At pH 7.5, the quercetin bands I
and II were unchanged in the presence of Mn2+ or L-arg. The spec-
trum of quercetin simultaneously incubated with Mn2+ and L-arg
was identical to that obtained with the quercetin–Mn2+ interaction
and suggests that the same mode of interaction exists between the
substrate and the cofactor in the presence of the inhibitor. This re-
sult was reinforced by the lack of interaction between quercitrin
and the substrate, between quercitrin and Mn2+, between isoqu-
ercitrin and the substrate and between isoquercitrin and Mn2+, be-
cause the presence of a rhamnoside or glucoside linked to the
hydroxyl group of the C ring (Fig. 1) causes a steric barrier to com-
plex formation. In fact, Jun (Jun et al., 2007) proposed that this hy-
droxyl group and the keto group, which form the C ring, are
responsible for chelating Mn2+.
3.2. Docking
Molecular docking was performed to predict and visualize the
interaction between the selected flavonoids and arginase. Table 1
summarizes the predicted interactions of flavonoids with amino
acids in the active site pocket. The two hydroxyls from the catechol
group of all flavonoids donate two hydrogen bonds to Asp141.
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.
185
Asp141 occupy the same position as Asp128 which is responsible
for coordinating the Mn2þ A cofactor in the active site of rat arginase
(Kanyo et al., 1996). Quercitrin also donates a hydrogen bond to
the carbonyl of Val193 and accepts a hydrogen bond from
Thr257. Isoquercitrin accepts two more hydrogen bonds from
Asn152 and Thr257 (Fig. 3). Thr257 and Asn152 occupy the same
position as Thr246 and Asn139 in the rat liver arginase active site,
where these two amino acids interact with nor-NOHA (Cox et al.,
2001), a competitive inhibitor of Leishmania (Leishmania) mexicana
arginase (Riley et al., 2011).
3.3. Inhibition of arginase
Recombinant and native arginase were characterized by us pre-
viously (da Silva et al., 2008) and exhibit the same KM and Vmax at
pH 9.5. The kinetics performed at pH 7.5 showed an increase in the
KM and Vmax decreased (Table 2). Quercetin, quercitrin and a third
related molecule, isoquercitrin, were tested for their ability to inhi-
bit L. (L.) amazonensis arginase. Table 3 summarizes the data and
quality of the estimated IC50 and Ki for quercetin, quercitrin and
isoquercitrin. Fig. 4 shows the best fit data for the sigmoidal (Log-
EC50) function for the three flavonoids tested. Fig. 5 shows the
Michaelis–Menten and double reciprocal (1/v, 1/S) Lineweaver–
Burk plot, which shows how the enzyme’s apparent KM varies in
the presence of each inhibitor. Quercetin is a mixed inhibitor
whereas quercitrin and isoquercitrin are uncompetitive inhibitors
of arginase. The inhibitions between the three flavonoids tested
in this work were not significantly (p > 0.05).
4. Discussion
The polyamine biosynthesis pathway has been explored as a
target for the treatment of trypanosomatid infection (Colotti and
Ilari, 2011; Riley et al., 2011). Arginase is the first enzyme of poly-
amine biosynthesis in Leishmania and drugs that target this
enzyme help to treat Leishmania infection (Iniesta et al., 2005,
2001, 2002; Kropf et al., 2005; Taylor-Robinson, 2001).
Disruption of the arginase gene in L. (L.) mexicana (Roberts et al.,
2004) and Leishmania (Leishmania) major (Reguera et al., 2009)
shows that parasite development is blocked in the absence of argi-
nase but restored by adding ornithine or putrescine (Reguera et al.,
2009).
Natural compounds such as flavonoids have shown activity
against Leishmania amastigotes in macrophage infection (Muzitano
et al., 2006a,b; Tasdemir et al., 2006). When used against L. (L.)
donovani, quercetin reduces intracellular amastigote load by 70%
at 45 lM (Mittra et al., 2000). The plant Kalanchoe pinata, which
contains quercetin, quercitrin and other flavonoids, shows activity
against cutaneous (Muzitano et al., 2009) and visceral leishmania-
sis (Gomes et al., 2010).
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.•
. .A DAH.•
. .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
Arginase KM (mM) Vmax (lmol urea min1 mg protein1) KM (mM) Vmax (lmol urea min1 mg protein1)
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-
Inhibitor IC50 (lM) R2 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
tin and two quercetin-derived compounds, quercitrin and Mn2+. 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/
for coordinating Mn2þ A and Mn2þB 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
Fig. 4. Dose–response curve for arginase inhibition by flavonoids: quercetin (), 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 (), 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-
noid aglycone quercetin and its derivative quercitrin exhibit low
toxicity against L6 cells, with IC50 values of 37 and >90 lg/mL,
respectively (Tasdemir et al., 2006). Quercetin also shows no cyto-
toxic effects on Caucasian hepatocyte or lung carcinoma cells
(HepG2 and A549 epithelial cell lines) at concentrations as high as
200 lM (Katalinić et al., 2010). Synthetic inhibitors of arginase such
as nor-NOHA (Nx-hydroxy-nor-L-arginine) and ABH (2(S)-amino-6-
boronohaxanoic) have a Ki of 106M when tested against L. (L.) mex-
icana arginase, but they fail to inhibit the growth of promastigotes at
this concentration (Riley et al., 2011); these results are similar to
those observed with pentostam and quercitrin, which are active
only against amastigotes (Muzitano et al., 2006a,b). Flavonoid
dimers are bivalent modulators of resistance to pentamidine and
sodium stibogluconate in Leishmania (Wong et al., 2007).
To date, the multiple quercetin targets that have been identified
for the treatment of leishmaniasis include: arginase, which was
identified in this work; ribonucleotide reductase (Sen et al.,
2008); and topoisomerase II (Mittra et al., 2000). These data make
quercetin a candidate ‘‘promiscuous’’ drug against leishmaniasis,
like the ‘‘promiscuous’’ anticancer drugs described by Hampton
(2004). ‘‘Magic bullet’’ drug design, which focuses on single biolog-
ical targets, might not be the ideal approach for complex illnesses
such as cancer and cardiovascular disease (Frantz, 2005), and it
should not be considered as an ideal model for the treatment of
leishmaniasis.
The success of quercetin may be due to the inhibition of two
enzymes, arginase and ribonucleotide reductase (Sen et al.,
2008), in addition to the promotion of DNA cleavage by inducing
topoisomerase II, which leads to kDNA linearization (Mittra et al.,
187
2000). It remains to be determined whether quercetin can be used
as a ‘‘promiscuous’’ drug prototype for leishmaniasis treatment.
The information presented here can be used to develop drugs
against leishmaniasis. Moreover, quercetin and derived flavonoids
can be considered prototypes for rational drug design based on the
inhibition of arginase or other quercetin targets.
Acknowledgment
This work was supported by the Foundation for the Support of
Research of the State of São Paulo (FAPESP), Brazil.
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