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Experimental Parasitology (2015)

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Experimental Parasitology
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x p r

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In vivo and in vitro auranon activity against Trypanosoma cruzi:

Possible new uses for an old drug
Marco Tlio Alves da Silva a, Izaltina Silva-Jardim a,1, Gisele Bulhes Portapilla a,2,
Gustavo Machado Alvares de Lima a,3, Fernanda Cristina Costa a,b,4,
Fernanda de Freitas Anibal c, Otavio Henrique Thiemann a,b,*
a Instituto de Fsica de So Carlos, Universidade de So Paulo, Caixa Postal 369, 13560-590, So Carlos, SP, Brazil
b Departamento de Gentica e Evoluo, Universidade Federal de So Carlos, So Carlos, Brazil
c
Departamento de Morfologia e Patologia, Universidade Federal de So Carlos, caixa postal 676, 13565-905, So Carlos, SP, Brazil

H I G H L I G H T S G R A P H I C A L A B S T R A C T

We tested the drug auranon in

Trypanosoma cruzi cells.
We show that auranon is more
potent than benznidazole.
We show the auranon effect on
the six Trypanosoma cruzi DTUs.
Auranon is shown as a potential
Trypanosoma cruzi drug.

A R T I C L E I N F O A B S T R A C T

Article history: Chagas disease, Sleeping Sickness, Nagana and Leishmaniasis are serious infections caused by protozoa
Received 7 April 2014 of the order Kinetoplastidae. They were described over a century ago by seminal work of different physician-
Received in revised form 5 January 2015 researchers and, despite the initial discoveries, few drugs have been made available for the treatment
Accepted 21 May 2015
of these infections. The drugs available present serious ecacy and toxicity problems. Moreover, the emer-
Available online
gence of resistant strains has rendered the development of novel chemotherapeutic strategies a priority.
Auranon is currently in use to treat rheumatoid arthritis in humans. Previous reports showed that this
Keywords:
compound presents activity against Trypanosoma brucei and Leishmania cells. In Trypanosoma cruzi cells,
Trypanosoma cruzi
Chemotherapy auranon resulted in a more potent compound than benznidazole in vitro when tested in different DTUs.
Auranon In vivo experiments, although not decreasing T. cruzi parasitemia, decreases host mortality. Therefore,
we propose auranon as a potential alternative for a new chemotherapy in Chagas disease with the added
advantage of already being approved for use in humans.
2015 Elsevier Inc. All rights reserved.

* Corresponding author. Fax: 55-16-33738991.

E-mail address: thiemann@ifsc.usp.br (O.H. Thiemann).
1
Present address: Departamento de Cincias Biolgicas, Universidade Estadual de Santa Cruz, Campus Soane Nazar de Andrade, 45662900 Ilheus, BA, Brazil.
2 Present address: Universidade de So Paulo, Faculdade de Cincias Farmacuticas de Ribeiro Preto, Avenida do Caf, s/n Monte alegre 14040-903, Ribeirao Preto, SP,

Brazil.
3 Present address: Laboratrio de Qumica Medicinal e Computacional(LQMC) Instituto de Fsica de So Carlos, Universidade de So Paulo, Caixa Postal 369, 13560-590

So Carlos, SP, Brazil.

4
Present address: Centro Nacional de Pesquisa em Energia e Materiais, Laboratrio Nacional de Biocincias, Rua Giuseppe Mximo Scolfaro 10.000 Plo II de Alta Tecnologia,
13083970, Campinas, SP, Brazil.

http://dx.doi.org/10.1016/j.exppara.2015.05.012
0014-4894/ 2015 Elsevier Inc. All rights reserved.

Please cite this article in press as: Marco Tlio Alves da Silva, et al., In vivo and in vitro auranofin activity against Trypanosoma cruzi: Possible new uses for an old drug, Experi-
mental Parasitology (2015), doi: 10.1016/j.exppara.2015.05.012
 ARTICLE IN PRESS
2 M.T.A. Silva et al./Experimental Parasitology (2015)
1. Introduction
Gold compounds have been used as anti-neoplastic, antirheu-
matic and antimicrobials. In 1935, studies have shown that gold salts
reduced inammatory reactions, especially those related to rheu-
matoid arthritis. In 1985, the drug Auranon gold (2,3,4,6-tetra-O-
acetyl-1-thio-beta-Dgluco piranosato-S) triethylphosphine [D SKF
Ridaura -39 162] entered the pharmaceutical marked with signif-
icant ecacy in the treatment of rheumatoid arthritis (Finkelstein
et al., 1976; Fricker, 1996; Kean et al., 1997; Navarro, 2009). To date,
there have been no reports which cast concern on carcinogenicity
or other long-term safety of auranon treatment with respect to
the dangers of prolonged immunosuppressive therapy (Glennas et al.,
1997; Horton, 1983; Itokazu et al., 1995; Kean et al., 1997).
Dual inhibition of inammatory pathways and thiol redox
enzymes by auranon makes it a new candidate for cancer therapy
and treating microbial infections (Madeira et al., 2012). Studies in-
dicated that gold compounds modied various functions of the
immune system and as effective inhibitors of several proteases (par-
ticularly cysteine proteases), factors involved in the progression of
rheumatoid arthritis. Auranon acts as a thioredoxin reductase (TrxR)
inhibitor in mammalian cells, whose activity causes important
changes in the intracellular redox balance and induces severe oxi-
dative stress and cytotoxic effects in vitro (Cox et al., 2008). Others
studies have identied the effect of auranon inhibition of Plasmo-
dium falciparum and Schistosoma mansoni TrxR, resulting in the
parasites growth inhibition in vitro (Kean et al., 1997; Navarro, 2009;
Sannella et al., 2008).
Lobanov et al. (2006) described auranon activity against Try-
panosoma brucei brucei and Trypanosoma cruzi cells as a
selenoproteins inhibitor. The selenoproteins are proteins that contain
the 21st amino acid selenocysteine and have been identied as
oxiredutases, contributing to the cellular redox balance. However,
Ilari et al. (2012) has shown that auranon strongly inhibited
trypanothione reductase (TR), a non-selenocysteine enzyme, and
demonstrated that the Au(I) atom of auranon binds to the cyste-
ine residues involved in the catalytic triad (Ilari et al., 2012).
Auranon has been reported to have in vitro activity against several
human parasites such as P. falciparum, with an IC50 of 0.14 uM
(Sanella et al., 2008); Taenia crassiceps, with an IC50 of 3.8 uM
(Martnez-Gonzlez et al., 2010); Toxoplasma gondi, with an IC50 of
0.28 uM (Andrade et al., 2014); Trypanosoma brucei, with an IC50
<1 uM (Lobanov et al., 2006), and is still considered a potential agent
against schistosomiasis and leishmaniasis (Navarro, 2009).
We report here a detailed investigation of auranon anti-
parasitic activity in different developmental forms of T. cruzi, as well
as evaluating the in vivo ecacy in reducing the parasitemia of Swiss
mice infected with T. cruzi.
2. Methods
2.1. Drug dilution
Auranon (Sequoia Research Products) and benznidazole (Sigma-
Aldrich) were dissolved in dimethylsulfoxide (DMSO) and the
corresponding culture media to obtain the desired experimental con-
centrations. In all experiments, the nal DMSO concentration was
kept lower than 1% v/v (Navarro, 2009).
2.2. T. cruzi cultures
Five T. cruzi DTU (Discrete Taxonomic Units) epimastigotes (DTU
I, strain Triatoma lenti; DTU II, strains SI-9 and Y (SILVA &
NUSSENZWEIG 1953); DTU III, strain QMM-II; DTU V, strain QMM-
I; DTU VI, strain Tulahuen (Pizzi et al., 1952)); were grown at 28 C
Please cite this article in press as: Marco Tlio Alves da Silva, et al., In vivo and in vitro auranofin activity against Trypanosoma cruzi: Possible new uses for an old drug, Experi-
mental Parasitology (2015), doi: 10.1016/j.exppara.2015.05.012
in liver infusion tryptose (LIT) medium (Fernandes and Castellani,
1966) supplemented with 10% heat inactivated fetal bovine serum.
2.3. T. cruzi viability assays
An in vitro assay to measure T. cruzi epimastigote viability using
MTT assay was performed according to Muelas-Serrano et al. (2000)
and Cotinguiba et al. (2009). Briey, the parasites were harvested
during the exponential growth phase. Auranon was added to a 96-
well tissue culture plate in different nal concentrations and T. cruzi
cells (1 107 parasites/mL) were added into each well and the same
quantity of LIT medium was added into the control wells. The plates
were incubated at 28 C for 72 h after which period 10 L of a
2.5 mg/mL MTT/0.22 mg/mL PMS solution was added to each well
and the plates were incubated for 75 min at 28 C. The reaction was
interrupted by the addition of 100 L of 10% of sodium dodecyl
sulfate (SDS)/0.1% HCl solution and the plates were maintained at
room temperature 30 min prior to determine the absorbance at
595 nm. The 50% inhibitory concentration (IC50) values were de-
termined by linear regression analysis from biological triplicates.
2.4. Cell culture and in vitro infection with T. cruzi (Tulahuen
strain) trypomastigote
Human broblast (Normal Neonatal Dermal Fibroblast, Hovatta
et al. (2003)) cell line was infected with T. cruzi Tulahuen strain
trypomastigotes at a 1:5 (cell:parasite) ratio. The cells were cul-
tured in 10% heat inactivated fetal bovine serum supplemented RPMI
1640 medium at 37 C and 5% CO2 atmosphere. They were incu-
bated at 37 C in humidied air and 5% CO2. After 57 days, the
culture medium of the trypomastigote infected cells was collect-
ed and the trypomastigotes maintained in RPMI 1640 at a nal
density of 1 107 parasites/ml. Trypomastigote viability assays were
performed using the CellTiter 96 AQueous Non-Radioactive Cell
Proliferation Assay (Promega), following the manufacturers
instructions.
2.5. Amastigote auranon activity assay
Human broblasts were seeded into 96-well plates (TPP) at a con-
centration of 105 cells per well and incubated for 24 h. Different
concentrations of AF were added in each experimental well. Cells
without treatment were used as controls. Following incubation for
24 or 72 h, the cells were checked for AF effects on their viability
by using the MTS test (CellTiter 96 AQueous Non-Radioactive Cell
Proliferation Assay (Promega)).
The auranon effect against T. cruzi intracellular forms was evalu-
ated using trypomastigotes expressing -galactosidase (Buckner et al.,
1996) and the assay was carried out according to Sulsen et al. (2013).
For this assay, 96-well plates were seeded with nonphagocytic
human brobalst cells at 5 103 per well in 100 L of culture medium
and were incubated for 24 h at 37 C in 5% CO2 atmosphere. Cells
were washed and infected with transfected trypomastigotes ex-
pressing -galactosidase at a parasite/cell ratio of 10:1. After 24 h
culture, the plates were washed twice with PBS to remove unbound
parasites and different auranon concentrations were added in
150 L of fresh complete RPMI medium without phenol red (Gibco).
Controls included infected untreated cells (100% infection control)
and uninfected cells (0% infection control). The assay was devel-
oped by the addition of chlorophenolred-b-D-galactopyranoside
(CPRG) (100 mM) and 1% Nonidet P40, 5 days later. Plates were then
incubated for 4 h at 37 C and the absorbance was measured at
595 nm in a microplate reader (SpectraMax, Molecular Devices). Per-
centage inhibition was calculated as 100 {[(absorbance of treated
infected cells)/(absorbance of untreated infected cells)] 6100}.
 ARTICLE IN PRESS
M.T.A. Silva et al./Experimental Parasitology (2015)
2.6. In vivo trypanocidal activity of auranon
Mice used for experiments were four to six weeks old BALB/c
female mice and bred under specic pathogen-free (SPF) from School
of Pharmaceutical Sciences of Ribeiro Preto USP, were housed
under controlled condition, allowed to acclimatize for 2 days, and
given a commercial rodents chow and sterile water ad libitum. The
animals were inoculated with 10 000 trypomastigote forms from
a previous infected mouse by intraperitoneal injection and ran-
domly assigned to 4 groups of ve animals each: group 1 Animals
treated with auranon (100 mg/kg); group 2 Animals treated with
benznidazole 10 mg/kg; group 3 Animals treated with benznidazole
100 mg/kg; group 4 Control group treated with ethanol 15%. All
compounds were solubilized with 15% ethanol. Treatment with
10 mg/kg and 100 mg/kg of benznidazole (Sigma-Aldrich) and
100 mg/kg of auranon started 2 hours after trypomastigote infec-
tion and daily for 14 days (total of 15 injections) by gavage. Auranon
concentration in the host has a peak between 1 and 2 hours post
ministration (Kean et al., 1997); therefore, we decided to base our
protocol on this knowledge, maximizing its effect against the T.
cruzi bloodstream forms. The level of parasitemia was determined
every two days by the Brener method (Brener, 1962) for 20 days
using the tail blood vessels bleeding technique (Stoltz and Bendall,
1975).
3. Results
3.1. Auranon activity is similar in epimastigote form of different T.
cruzi strains and DTUs
T. cruzi epimastigotes from ve Discrete Taxonomic Units,
DTUs (Zingales et al., 2009), Triatoma lenti DTU I, strains SI-9 and
Y DTU II (Silva and Nussenzweig, 1953), strain QMM-II DTU III,
strain QMM-I DTU V and strain Tulahuen DTU VI (Pizzi et al., 1952),
were tested for their sensitivity to auranon and benznidazole. Our
results (Table 1) show that auranon has higher trypanocidal ac-
tivity when compared with benzonidazole in all 5 DTUs studied
and is 40 times and 11 times more active than benznidazole in Y
strain (DTU II) and Triatoma lenti strain (DTU I) respectively. Strains
of DTI III, V and VI presented intermediate sensitivity, and among
the strains tested, DTU I showed the highest resistance to both drugs
(Table 1). Although the sensitivity to auranon did not vary greatly
among DTUs, benznidazole activity presented large variations. When
Y or Triatoma lenti and SI-9 strains were compared, it was possible
to detect that the last strain is at least two times more sensitive
to benznidazole. The effects observed between Y and SI-9
strains suggest that drugs resistance may not be homogeneous within
DTUs.
Table 1
IC50 values to T. cruzi forms and human broblast treated with benznidazole and auranon.
Cell Strain Benznidazole
IC50 (uM)
T. cruzi (epimastigote) Triatoma lenti 18.65 0.9
SI-9 8.07 0.48
Y 17.62 1.71
QMM-II 11.94 0.96
QMM-I 16.66 0.14
Tulahuen 12.52 1.96
T. cruzi (Trypomastigote) Tulahuen 12.56 0.49
Tulahuen 4.21 0.19
T. cruzi (amastigote) Tulahuen 17.61 0.24
Human broblast HFF-1 1039.76 94.4
Please cite this article in press as: Marco Tlio Alves da Silva, et al., In vivo and in vitro auranofin activity against Trypanosoma cruzi: Possible new uses for an old drug, Experi-
mental Parasitology (2015), doi: 10.1016/j.exppara.2015.05.012
3
3.2. T. cruzi trypomastigotes are more sensitive to auranon
than benznidazole
Only the tulahuen strain was used in the trypomastigote and
amastigote assays for two reasons. First, no large variations sensi-
tivity was detected among DTUs tested and second, tulahuen
allowing a colorimetric assay based in the expression of
-galactosidase, increasing the sensitivity and reproducibility of the
assay, being the recommended in vitro test to trypanocidal drug ac-
tivity (Romanha et al., 2010). IC50 values reveal that auranon has
a higher trypanocidal activity than benznidazole, as shown in Table 1;
however, the difference between the two compounds is less pro-
nounced than for the epimastigote form.
3.3. Auranon IC50 values are similar in T. cruzi amastigote form and
mammal cell
Auranon IC50 for T. cruzi amastigote, Tulahuen strain, was
0.56 0.07 M, only 2.35 times lower than the IC50 value obtained
for this drug in human broblasts (1.32 0.06 M). Benznidazoles
IC50 of 17.61 0.24 M was 31-fold higher than auranon (Table 1);
however, it was less toxic to the host cell line (1,039 94 uM), around
59-fold the benznidazole IC50 for amastigote form (Table 1). Con-
stant microscope observation revealed no signicant morphological
changes in the human broblasts at lower auranon concentra-
tion used in the test. Although the Safety Index (SI) of 2.35 found
for auranon is lower than the recommended for trypanocidal trials
of novel compounds (Romanha et al., 2010), we decided to contin-
ue investigating its activity in vivo since it has been proven safe in
mammalian toxicity trials and the compound is active against
other trypanosomatids such as T. brucei (Lobanov et al., 2006) and
L. major (Ilari et al., 2012), therefore representing a potential lead
compound.
3.4. Auranon reduced T. cruzi mice mortality
Auranon has shown no signicant effect in reducing T. cruzi Y
strain parasitemia levels on infected mice at the acute phase, when
compared to the untreated control animals. Benznidazole treat-
ment, at 10 mg/kg and 100 mg/kg, shows a pronounced decrease
in parasitemia, consistent with other reports in the literature
(Daz-Chiguera et al., 2012) (Fig. 1). An intriguing observation is the
higher infected animal survival upon auranon treatment when com-
pared with untreated animals (Fig. 2). The control group (untreated
animals) presented 60% of deaths 15 days post infection, whereas
animals treated with auranon remain alive in that period (Fig. 2);
similar results were observed for benznidazole-treated animals.
Benznidazole treatment with 10 mg/kg resulted in a 100%
Auranon Time LD50 Benznidazole/
(hours) LD50 Auranon (folds)
IC50 (uM)
1.46 0.14 72 12.77
0.7 0.05 11.52
0.43 0.08 40.97
0.77 0.15 15.50
0.67 0.03 24.86
0.49 0.04 25.55
3.27 0.64 24 3.84
1.09 0.05 48 3.86
0.56 0.07 120 31.44
1.32 0.06 787.12
 ARTICLE IN PRESS
4 M.T.A. Silva et al./Experimental Parasitology (2015)
Fig. 1. Parasitemia curve of mice infected with T. cruzi, strain Y. It was used 4 groups
of animals, control, untreated; benznidazole 10, animals treated with 10 mg/kg of
benznidazole, benznidazole 100, treated with 100 mg/kg benznidazole, auranon,
treated with 100 mg/kg of auranon.
survival rate while benznidazole at a 100 mg/kg dose showed 60%
survival rate, probably as a result of toxic effects of benznidazole
(Fig. 2).
4. Discussion
A growing interest in the development of effective drugs for the spe-
cic therapy of protozoan parasitic infections such as Chagas disease,
sleeping sickness and leishmaniasis has produced a vast amount of data
and generated potential new targets for chemotherapy. However, more
than a century after the identication and description of these impor-
tant diseases, the treatment of these parasite infections remains
unsatisfactory and expensive. Recently, a variety of novel approaches
using drugs already established for other illnesses has emerged based
on an increasing understanding of the parasites biochemical, physio-
logical and metabolic aspects. Several of those advances were results
of the genomic revolution caused by the data emerging from the genome
efforts on Trypanosoma and Leishmania.
Fig. 2. Survival rate of mice infected with T. cruzi, strain Y, and treated with
benznidazole and auranon. It was used 4 groups of animals, control, untreated;
benznidazole 10, animals treated with 10 mg/kg of benznidazole, benznidazole 100,
treated with 100 mg/kg of benznidazole, auranon, treated with 100 mg/kg of
auranon.
Please cite this article in press as: Marco Tlio Alves da Silva, et al., In vivo and in vitro auranofin activity against Trypanosoma cruzi: Possible new uses for an old drug, Experi-
mental Parasitology (2015), doi: 10.1016/j.exppara.2015.05.012
Auranon has been reported for its activity against some bac-
teria, cancer cell lineages and parasites (Angelucci et al., 2009; Chen
et al., 2009; Jackson-Rosario and Self, 2009; Jackson-Rosario et al.,
2009; Kean et al., 1997; Kuntz et al., 2007; Lobanov et al., 2006;
Martnez-Gonzlez et al., 2010; Sannella et al., 2008), including very
recently to Leishmania cells (Sharlow et al., 2014). The mechanism
of action of this drug has been proposed to be a result of the in-
teraction of Gold with the Cys residues of Thioredoxin-Glutathione
Reductase (TGR) and likely the interaction of Gold and the cata-
lytically active selenium-containing Sec residue of TGR selenoprotein
(Angelucci et al., 2009). Recently, a screening for commercial drugs
with potential activity against Giardia lamblia determined the IC50
of auranon around 46 uM and demonstrated in vivo a signi-
cant parasite reduction and the inhibition of enzyme thioredoxin
oxidoreductase (Tejman-Yarden et al., 2013). It is important to
comment that G. lamblia does not have the selenocysteine synthe-
sis and incorporation pathway; therefore, in this parasite auranon
must be acting independent of selenocysteine. In other cases, it was
demonstrated that auranon inhibit the selenoprotein thioredoxin
glutathione reductase of Schistosoma japonicum, enzyme with various
funcions, showing thioredoxin reductase, glutathione reductase and
glutaredoxin activities (Song et al., 2012). In Leishmania infantum,
auranon presented IC 50 of 9.68 1.02 and interacts with
Trypanothione reductase, a non-selenoenzyme, preventing binding
and reduction of trypanothione (Ilari et al., 2012). One important
aspect of T. cruzi infections that is often overlooked in several drug-
screening approaches is the complexity and diversity of the parasite
population in nature. At the moment, six different groups of the
parasite have been classied (Zingales et al., 2009), dened as Dis-
crete Taxonomic Units or DTUs. Those DTUs have correlation with
molecular markers, geographic distribution and the clinical man-
ifestation (gastrointestinal mega-syndrome or cardiomyopathy) it
causes. Therefore, it is reasonable to predict that compounds tested
against T. cruzi would potentially have different ecacies against
the different DTUs and therefore may be more suitable for intro-
duction in different geographic regions or with a dose administered
according to the prevalent DTU and its susceptibility. Our experi-
ments show that this hypothesis is correct, as previously
described (Zingales et al., 2009), and benznidazole is more potent
against DTU II (strain Si9), moderately potent against DTU III (strain
QMM-II) and DTU VI (Tulahuen), and less potent for DTU I (strain
T. lenti), DTU II (strain Y) and DTU V (strain QMM-I). Comparative-
ly, auranon is more homogeneous in its potency against the different
DTUs tested, which is a signicant advantage in the Chagas Disease
treatment.
The most important problems in new trypanocidal drugs dis-
covery are the low or non-activity of the molecules against
intracellular forms, even to compounds very promising against free
parasite forms. Insect and bloodstream forms of T. cruzi exhibit great
sensitivity to auranon in vitro, compared to benznidazole, but it
was not observe parasitemia decreased in vivo or reduced of
amastigote forms in mammalian cells. In the systems studied pre-
viously, auranon was identied to act in important oxidative stress
components, as probably occurs in T. cruzi as well. It has been dem-
onstrated that macrophage infection of T. cruzi involves an increase
in the expression of the parasites oxidative stress response enzymes.
Proteomic studies suggest that iron-dependent superoxide dismutase,
trypanothione synthase (TcTS) and members of the tryparedoxin
peroxidase family (TXNPx) are up-regulated in the metacyclic stage,
trypomastigote and replicative amastigote blood-stages (Parodi-Talice
et al., 2007). Therefore, it is probable that the overexpression of these
enzymes is able to reduce the auranon trypanocidal activity, as ob-
served by the lack of a reduction in the number of amastigotes in
vitro or the host parasitemia in vivo. However, auranon resulted
in the survival of the host upon T. cruzi infection similar to the effect
obtained by benznidazole, with the added advantage of the lower
 ARTICLE IN PRESS
M.T.A. Silva et al./Experimental Parasitology (2015)
mammalian host toxicity of auranon. Our results show that
auranon is a viable drug for the development of new therapies for
the treatment of T. cruzi infection and is available due to its current
use in human therapy.
Acknowledgements
This work was supported in part by the research grants 98/
14138-2 (FAPESP) and 550514/2011-2 (CNPq). Trypanosoma cruzi
strains (Triatoma lenti, SI-9, QMM-II, QMM-I) were genotyped and
were a kind gift from Dr Joo Aristeu da Rosa, Faculdade de Cincias
Farmacuticas, Universidade Estadual Paulista, UNESP.
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