Insights about resveratrol analogs against trypanothione reductase of Leishmania braziliensis: Molecular modeling, computational docking and in vitro antileishmanial studies

Article abstract of DOI:10.1080/07391102.2018.1502096

In this work, we combined molecular modeling, computational docking and in vitro analysis to explore the antileishmanial effect of some resveratrol analogs (ResAn), focusing on their pro-oxidant effect. The molecular target was the trypanothione reductase

Full text of DOI:10.1080/07391102.2018.1502096

Journal of Biomolecular Structure and Dynamics
ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20
Insights about resveratrol analogues against
trypanothione reductase of Leishmania braziliensis:
molecular modeling, computational docking and in
vitro antileishmanial studies
Adilson D. da Silva, Juliana A. dos Santos, Patrícia A. Machado, Lara A. Alves,
Larissa C. Laque, Vinícius C. de Souza, Elaine S. Coimbra & Priscila V. S. Z.
Capriles
To cite this article: Adilson D. da Silva, Juliana A. dos Santos, Patrícia A. Machado, Lara A.
Alves, Larissa C. Laque, Vinícius C. de Souza, Elaine S. Coimbra & Priscila V. S. Z. Capriles
(2018): Insights about resveratrol analogues against trypanothione reductase of Leishmania
braziliensis: molecular modeling, computational docking and inꢀvitro antileishmanial studies, Journal
of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2018.1502096
To link to this article: https://doi.org/10.1080/07391102.2018.1502096
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Jul 2018.
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Insights about resveratrol analogues against trypanothione reductase
of Leishmania braziliensis: molecular modeling, computational docking
and in vitro antileishmanial studies
Adilson D. da Silvaa, Juliana A. dos Santosa, Patrícia A. Machadob, Lara A.
Alvesc; Larissa C. Laquec; Vinícius C. de Souzac; Elaine S. Coimbra b and
Priscila V. S. Z. Caprilesc*
aDepartamento de Química, I.C.E., Universidade Federal de Juiz de Fora, Juiz de
Fora, MG, 36036-900, Brazil.
bDepartamento de Parasitologia, Microbiologia e Imunologia, I.C.B., Universidade
Federal de Juiz de Fora, Juiz de Fora, MG, 36036-900, Brazil.
cPrograma de Pós-graduação em Modelagem Computacional, Departamento de
Ciência da Computação, I.C.E., Universidade Federal de Juiz de Fora, Juiz de Fora,
MG, 36036-900, Brazil. *Corresponding author: priscila.capriles@ufjf.edu.br,
priscilacapriles@gmail.com

Insights about resveratrol analogues against trypanothione reductase
of Leishmania braziliensis: molecular modeling, computational docking
and in vitro antileishmanial studies
In this work, we combined molecular modeling, computational docking and in
vitro analysis to explore the antileishmanial effect of some resveratrol analogues
(ResAn), focusing on their pro-oxidant effect. The molecular target was the
trypanothione reductase of Leishmania braziliensis (LbTryR), an essential
component of the antioxidant defenses in trypanosomatid parasites. Three-
dimensional structures of LbTryR were modeled and molecular docking studies
of ResAn1-5 compounds showed the following affinity: ResAn1 > ResAn2 >
ResAn4 > ResAn5 > ResAn3. Positive correlation was observed between these
compounds’ affinity to the LbTryR and the IC50 values against Leishmania sp
(ResAn1 < ResAn2 < ResAn4), which allows for TryR being considered an
important target for them. As the compound ResAn1 showed the best
antileishmanial activity, and docking studies showed its high affinity for NADP
binding site (NS) of TryR, plus having been able to induce ROS production in L.
braziliensis promastigotes treated, ResAn1 probably occupies NS interfering in
the electron transfer processes responsible for the catalytic reaction. The in silico
prediction of ADMET properties suggests that ResAn1 may be a promising drug
candidate with properties to cross biological membranes and high gastrointestinal
absorption, not violating Lipinski’s rules. Ultimately, the antileishmanial effect of
resveratrol analogues can be associated with a pro-oxidant effect which, in turn,
can be exploited as an antimicrobial agent.
Keywords: molecular modeling and docking; resveratrol analogues; reactive
oxygen species (ROS); trypanothione reductase, antileishmanial.

1 Introduction
Leishmaniasis is a group of diseases caused by protozoa of the genus Leishmania,
included in the list of Neglected Tropical Diseases by the World Health Organization,
and prevalent in 98 countries and territories, with more than 350 million people at risk
(WHO, 2017). This protozoan is an intracellular obligate parasite that lives inside
macrophages which produce reactive oxygen species (ROS) to control the parasite
infection. However, the parasite presents several evasion mechanisms against the
microbicidal effect of macrophages, including anti-oxidant defenses.
Trypanothione Reductase (TryR) (E.C. 1.8.1.12) is a homodimer with three
structural binding domains (FAD, NADPH and trypanothione), and its activity is an
essential component of the antioxidant defenses in trypanosomatid parasites (Baiocco et
al., 2013). TryR is NADPH dependent and catalyzes the reduction of trypanothione
disulfide [T(S)2] to trypanothione dithiol [T(SH)2], triggering a cascade of events
responsible to neutralize ROS (Rodrigues et al., 2012).
The review presented by Hefnawy and co-workers (2016) shows that
Leishmania species increased the trypanothione reductase (TryR) rates as a mechanism
of resistance to pentavalent antimonials and miltefosine, the main drugs available for
control of leishmaniasis. Since TryR is a specific protein in Trypanosomatidae family
and has been used by the parasite as a strategy for drug resistance, it has been suggested
as a promising target for the development of new chemotherapeutics to treat
leishmaniasis and Chagas disease (Tovar et al., 1998; Alves-Ferreira et al., 2009; Pita et
al., 2009; Capriles et al., 2010; de Paula da Silva et al., 2012; Beig et al., 2015; Pandey
et al. 2015; Pandey et al. 2017; Vera et al. 2017).
Resveratrol (3,5,4'-trihydroxystilbene) (Figure 1A) is a natural stilbene found in
many plants and foods (Chong et al., 2009), and has been considered a potent

antioxidant. Resveratrol has been mainly related to cancer chemoprevention and anti-
aging effects (Renaud et al., 1992; Baur and Sinclair et al., 2006). In addition to these
well-known biological properties, reports also show that resveratrol and trans-
resveratrol analogues present antileishmanial activity (Kedzierski et al., 2007; Ferreira
et al., 2014; Passos et al., 2015). Our research group also reported the antileishmanial
effect of some resveratrol analogues (ResAn) (De Paula et al. 2013, Coimbra et al.
2016a); however, none of these studies related the antileishmanial effect of the
compounds to their pro-oxidant action.
Figure 1. Chemical structure of resveratrol and its analogues. (A) Resveratrol, (B)
resveratrol analogues (ResAn1-5).
In this work, we combined molecular modeling, computational docking and in
vitro tests to investigate the antileishmanial effect of five resveratrol analogues
(ResAn1-5), exploring their pro-oxidant effect. ResAn1-5 (Figure 1B) were docked
against six models of trypanothione reductase of L. braziliensis (LbTryR), evaluating

their affinity by the catalytic and NADP binding sites. Furthermore, we analyzed the in
vitro ROS production in promastigotes treated with the best antileishmanial compound
ResAn1.
2 Materials and methods
2.1 Molecular Modeling
Templates for three-dimensional (3D) model construction were selected from Protein
Data Bank (PDB) based on local alignment (via BlastP) between deposited proteins and
trypanothione reductase of L. braziliensis (LbTryR – UNIPROT: E9JUH3). 3D
structures were selected based on sequence identity, similarity and maximum query
coverage, on the conformation of catalytic and NADP binding sites and on the presence
of NADP in its binding site.
3D models of LbTryR used in this work were modeled using the program
Modeller9v13 (Sali and Blundell, 1993), according to the protonation and partial charge
of residues C52:A, C57:A and H461:B for each enzymatic step. The enzyme was
prepared in six different conditions: (i) C52:A and C57:A are making disulfide bond,
NADP is absent in this binding site which is occluded by Y198:A (this configuration is
here called "closed form"); (ii) C52:A and C57:A are ionized with negative charges,
considering the presence of NADP and FADH2; (iii) C52:A is protonated and
interacting with H461:B and C57:A is negatively charged and interacting with FAD
(considering the presence of NADP); (iv) similar to (iii) with NADP; (v) similar to (ii)
with FAD instead of FADH2; (vi) similar to (i) with NADP.
All models were assessed using the programs Procheck (Laskowski et al., 1993)
and Molprobity (Chen et al., 2010) for the analysis of stereochemical quality. The

energy values were analyzed using nDOPE and Molpdf methods calculated by
Modeller9v13.
2.1.1 Protein-ligand Docking
Grid Construction. Grids were built using Glide program (Friesner et al., 2004). The
grid size for NADP binding site (NS) was X = Y = 25Å and Z = 27 Å and for catalytic
site (CS) was X = Y = 24 Å and Z = 26 Å. The grid for NS was centered on positions X
= -22.668, Y = 1.310 and Z = 3.860, while the grid of CS was centered in X = -26.042,
Y = 2.547 and Z = 5.976.
Molecular docking. ResAn compounds were prepared using OPLS2005 force field in
pH 7.4 ± 0.2, using Ligprep tool in Glide (Schrödinger LLC. New York). The LbTryR
models were prepared for subsequent grid generation and docking using the Protein
Preparation Wizard tool in Glide. All hydrogens were added, the protonation states and
tautomers of Histidines were optimized, and Chi “flip” assignments for Asparagines,
Glutamines and Histidines were performed. Docking studies were performed in the
Glide Extra Precision (XP) mode using the post-refinement step with the parameters set
to the default values.
Analysis of electrostatic and pharmacophoric profiles of the catalytic and NADP
binding sites. For the comparative studies of the catalytic and NADP binding sites of
each step studied, the electrostatic and pharmacophoric profiles of both sites were
analyzed. The PDB2PQR v1.9 (Dolinsky et al., 2004) and APBS v1.4.1 (Holst, Baker
and Wang, 2000) programs were used in the electrostatic profile analysis. For the
pharmacophoric profile analysis we used the Align-it v1.0.4 plugin from PyMol
software (DeLano, 2002) .

2.2 Resveratrol analogues
The ResAn compounds have been produced using the classic bioisosterism (Calil et al.,
2012; Franco et al., 2012; de Paula et al., 2013; Lima et al., 2013; Polonini et al., 2013;
Santos et al., 2015), by substitution of the central C=C linkage with a C=N double bond
in the stilbenoid nucleus, originating hydrazones (Figure 2). ResAn1-5 were obtained by
condensation of phenylhydrazine or 2,4-dinitrophenylhydrazine with the equimolar
amount of aromatic aldehydes in ethanol as solids after being filtered, washed with
ethanol and dried in an oven (yield 52-94%).
Figure 2. Classic bioisosterism in the stilbenoid nucleus.
Melting points (m.p.) were determined using a MQAPF-301-Microquimica
digital apparatus. Infrared spectra (wave numbers in cm-1) were recorded on a
Schimadzu 8400 series FTIR instrument. 1H NMR spectra were recorded on a Bruker
AC-300 and 13C NMR spectra were recorded on a Bruker AC-300 at 75 MHz. The
chemical shifts (δ) were given in parts per million relative to tetramethylsilane (TMS).
Reagents and materials were obtained from commercial suppliers and were used
without purification. All reagents used were analytical reagent grade.
All compounds were characterized by 1H and 13C NMR and melting points
(m.p.) and were in accordance with data in the literature: ResAn1 (2,4-
dihydroxybenzaldehyde phenylhydrazone; beige solid, m.p.: 155,4 - 154, 7 ºC) (Chary,

2008); ResAn2 (4-carboxybenzaldehyde phenylhydrazone; yellow solid, m.p.: 232,7 –
234,0 ºC) (Hansch, 1977); ResAn3 (4-nitrobenzaldehyde phenylhydrazone; garnet solid,
m.p.: 161,2 – 162,5 ºC) (Cushman, 1991); ResAn4 (4-carboxybenzaldehyde 2,4–
dinitrophenylhydrazone; yellow solid, m.p.: 271,2 - 273,0 ºC) (Chary, 2008); ResAn5
(5-bromo-2-hydroxycarboxybenzaldehyde 2,4-dinitrophenylhydrazone; orange solid,
m.p.: 269,0 - 271,7 ºC) (Chary, 2008).
2.3 Biological assays
2.3.1 In vitro antileishmanial activity
Parasites. Leishmania braziliensis (MHOM/Br/75/M2903) and L. amazonensis
(IFLA/BR/1967/PH8) transfected with the gene of red fluorescent protein (RFP) (Rocha
et al., 2013) were used. Leishmania braziliensis and L. amazonensis-RFP were cultured
as previously described (Coimbra et al., 2016b).
Anti-promastigote assay. Anti-promastigote activity was determined by the colorimetric
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide- Sigma-Aldrich,
St. Louis, MO, USA) method, as previously reported (Coimbra et al., 2016b). Parasites
were not exposed (negative control) or exposed to an increasing concentration of the
compounds (3.1-100.0 µM), diluted in DMSO, for 72 h at 25°C in a BOD incubator.
Viability of the promastigotes was determined by measuring the absorbance at 570 nm
(Multiskan MS microplate reader, LabSystems Oy, Helsink, Finland). Results were
obtained from three independent experiments performed in duplicate and expressed as
the concentration inhibiting parasite growth by 50% (IC50). The IC50 values were
obtained using GraFit Version5 software (Erithacus Software Ltd., Horley, UK).

Miltefosine (Cayman Chemical Company, Ann Arbor, MI, USA) and resveratrol
(Sigma-Aldrich, St. Louis, MO, USA) were used as reference drugs.
Anti-amastigote assays. Macrophages were obtained from BALB/c mice as previously
reported (Coimbra et al., 2016b). Adherent macrophages were infected with L.
amazonensis-RFP or L. braziliensis promastigote forms in the stationary growth phase
for 4 h (33ºC in 5% CO2), in the ratio of 10 promastigotes: 1 macrophage. Non-
internalized promastigotes were eliminated, and solutions of tested compounds in
different concentrations (6.2-100.0 µM) were added and the plates were maintained at
33oC in 5% CO2 for 72 h. For L. amazonensis-RFP, fluorescence intensity of the
cultures was measured using a plate-reader fluorometer (FLx800, BioTek Instruments,
Inc., Winooski, VT, USA) set at 540 nm excitation/600 nm emission. For L. braziliensis
parasites, after staining with Giemsa, the parasite burden was evaluated by counting the
intracellular parasite at an optical microscopy (1000X magnification). Miltefosine
(Cayman Chemical Company, Ann Arbor, MI, USA) and resveratrol (Sigma-Aldrich,
St. Louis, MO, USA) were used as reference drugs. The IC50 values were obtained by
using Probit program. All protocols were approved by the Ethical Committee for
Animal Research of Federal University of Juiz de Fora (056/2013-CEUA).
2.3.2 Cytotoxicity on mammalian cells
Cytotoxicity assays were evaluated on murine peritoneal macrophages. Adherent
macrophages were incubated with the compounds in several concentrations (9.4-150.0
µM), for 72 h at 33°C, and the viability of the macrophages was determined as
previously described (Coimbra et al., 2016b). All the tests were performed in duplicate.
The CC50 values were obtained using GraFit Version 5 software. All procedures were
performed in agreement with the Ethical Principles in Animal Research and according

to protocols approved by the Ethical Committee for Animal Research (054/2013-
CEUA).
2.4 Evaluation of Reactive Oxygen Species (ROS) levels in L. amazonensis
promastigotes
Intracellular ROS levels were determined using 2’,7’-dichlorodihydrofluorescein
diacetate (H2DCFDA- Cayman Chemical Company, Ann Arbor, MI, USA) as
previously reported (Coimbra et al., 2016b). Promastigote forms of L. braziliensis were
untreated or treated with 7.33 or 14.66 µM of ResAn1 (concentrations corresponding to
the 1x and 2x IC50 value after 72 h), at 25°C for 24 h. The parasites were incubated with
H2DCFDA 1 mM; for 30 minutes, and the fluorescence was measured using a
spectrofluorometer (FLx800, BioTek Instruments, Inc., Winooski, VT, USA), at
excitation and emission wavelengths of 485 and 528 nm, respectively. Miltefosine
(28.00 µM) was used as a positive control.
3 Results
To verify whether ResAn compounds have an antileishmanial activity associated with
pro-oxidant effects, the protein trypanothione reductase (TryR) was selected as target
for docking studies, considering it is an important enzyme in the antioxidant defenses of
trypanosomatids.
The mechanism of TryR consists in the following steps (Supplementary Figure
S1), according to Leichus et al. (1992): (i) NADPH binding site (NS) is closed due to an
occlusion by the aromatic ring of Y198, C52 and C57 forming a disulfide bond and
FAD is in oxidized form; (ii) Y198 flips its aromatic ring and NADPH binds to the
oxidized enzyme and reduces FAD to a transient FADH2 that donors an electron to C57
reducing C52-C57 disulfide bond; (iii) A charge transfer between C57 thiloate anion

and oxidized FAD occurs and C52 forms a temporary hydrogen interaction with H461;
(iv) an oxidized trypanothione disulfide binds to the reduced enzyme near C52 and
H461 (catalytic site, CS); (v) C52 nucleophilically attacks trypanothione forming a
disulfide bond; (vi) C57 attacks the disulfide bond on C52 to release a reduced
trypanothione and resume the redox-active C52-C57 disulfide bond. All numerations
are according to TryR of L. braziliensis (LbTryR).
Taking this catalytic mechanism into account, the structure of LbTryR models
was constructed by comparative modeling using three conformations of trypanothione
reductase of L. infantum (LiTryR) as templates, selected considering the configuration
of catalytic and NADP binding sites. Sequence alignment (Supplementary Figure S2)
between LbTryR and LiTryR presenting 84% of identity and 91% of similarity.
Stereochemical analysis and the global quality of the LbTryR models are presented in
Table 1.

Table 1: Stereochemical analysis and the global quality of the LbTryR models.
Catalytic
Step
Template
(PDBID)
Normalized
DOPE1
Molpdf1 Outlier2 Allowed(%)2
Heteroatom
S1 (conf1 = 2JK6(Baiocco,
Y198closed) et al., 2009)
FAD
-0.814
6555.83 2
30424.30 2
99.5
99.8
S1 (conf2 = 2JK6 and
FAD,
Y198open) 4ADW(Baiocco NADP and -0.834
and S6
et al., 2013)
GCG3
FAD,
4APN(Baiocco
et al., 2013)
S2 and S3
NADP and -0.767
JV04
6279.14 0
100
FAD,
S4 and S5
4ADW
NADP and -0.694
GCG3
6468.67 0
99.8
1Result obtained with Modeller9v13 (Sali & Blundell, 1993). 2Consensus result between
programs Procheck (Laskowski, MacArthur, Moss, & Thornton, 1993) and Molprobity
(Chen et al., 2010). 3GCG = Bis(Gamma-Glutamyl-Cysteinyl-Glycinyl)Spermidine,
Trypanothione (Synonym). 4JV0 = 4-[[1-(4-ethylphenyl)-2-methyl-5-(4-
methylsulfanylphenyl)pyrrol-3-yl]methyl]thiomorpholine, a diarylpirrole-based
inhibitor.
After model construction, some residues in these models were modified to
represent structural modifications in each step of the catalytic mechanism of TryR

(Table 2). As example of 3D model, it is presented in Figure 3(A) the final overall
structure of LbTryR in step S1 (conf2) and its diagram map is presented in
Supplementary Figure S3. Figures 3(B) and 3(C) present a detailed view of catalytic site
and NADP binding site, respectively. Residues involved in catalytic activity of LbTryR
are presented in lines. In Figure 3(D), it is presented the overall electrostatic potential
profile of LbTryR. In detail (Figure 3(E) and 3(F)), the opposite profiles of the two sites
studied in this work can be observed. The catalytic site (Figure 3(C)) presents a
negatively charged potential profile (red) while NADP binding site (Figure 3(D)) has a
positive one (blue).

Table 2: Final conformation of each protein model considering structural modifications
to represent each step in the catalytic mechanism of LbTryR.
Catalytic
C52
C57
Y198
FAD
NADP
Step
S1 (conf1)1
SSbond
SSbond
S-
SSbond
SSbond
S-
closed
Open
Open
Open
Open
Open
Open
+
-
S1 (conf2)2
+
+
+
+
+
+
+
S2
S3
S4
S5
S6
FADH2
SH(-H461) S-
SH(-H461) S-
+
+
+
+
S-
S-
SSbond
SSbond
2
1 conf1: Y198 occluding NS (Y198closed). conf2: Y198 not occluding NS
(Y198open).

Figure 3. Structure model and electrostatic potential profile of LbTryR. (A) Cartoon
representation of LbTryR. Monomer A is presented in light blue and monomer B in
light pink. Ligand and heteroatoms are presented in sticks: Trypanothione (GCG -
green), NADP (yellow) and FAD (magenta). In detail are presented (B) the catalytic site
and (C) NADP binding site, with residues involved in catalytic activity of LbTryR
presented in lines. (D) The electrostatic potential profile of LbTryR vary from -5 (red)
to 5 (blue) kT/e. In detail are presented the electrostatic potential profile of (E) catalytic
site and (F) NADP binding site. The image was generated using PyMOL, version 1.8.2,
Schrödinger – LLC.
To analyze the affinity of ResAn1-5 to catalytic (CS) and NADP binding (NS)
sites of LbTryR in each step of catalytic mechanism (prepared according to Table 3),

protein-ligand docking tests using Glide program were performed. For NS, in steps S1
to S3, NADP and Trypanothione (GCG) were removed from their binding sites to
observe the binding affinity of each compound to these sites.
According to results presented in Table 3 and Figures 4 and 5, we can sort the
compounds in decreasing order of highest to lowest affinity as follows: ResAn1 >
ResAn2 > ResAn4 > ResAn5 > ResAn3. As observed in Table 3, all the compounds
have preference to NADP binding site (NS) presenting more negative scores.
Analyzing conformations and pharmacophoric profiles, ResAn1-5 compounds
interact with residues considered as important for biological function of LbTryR
(Baiocco et al., 2009). In the NS (Figure 4) these compounds interact with
FAD/FADH2 and Y198 preventing the Π-Π interaction between FAD and NADPH. It
probably interferes in the electron transfer processes from FAD to C57 avoiding the
reduction of C52-C57 disulfide bond, consequently avoiding the catalytic reaction.
Interestingly, stacking interaction of ResAn1-5 compounds with Y198 in S1
conformation 1 (Figure 4A and 4B) may prevent the phenolic ring of Y198 from
undergoing rotation of 120º around Cα-Cβ bond to accommodate the NADPH, as
described by Baiocco et al. (2009).
In the catalytic site, despite the low affinity interaction (Table 3), ResAn1-5
compounds interact with residues involved in catalytic activity (C52, H461 and E466).
Figure 5 shows that ResAn1-5 form hydrogen bonds with residues H461 (which
activates the C52) and E466 (which stabilizes H461 positive charge during the redox
reaction). These interactions might interfere in direct (by H461) and indirect (by E466)
activation of catalytic cysteine. Only ResAn1 interacted directly with the reduced C52
in S4 conformation (Figure 5A and 5B), probably avoiding the reduction of
trypanothione disulfide bond. Moreover, ResAn2 and ResAn3 form hydrogen bonds

with Y110 (residue involved in the interaction with trypanothione), which points to the
possible role of these compounds in avoiding formation of the complex TryR-substrate.
Analysis comparing the resveratrol and ResAn1 interactions with CS and NS
residues showed that both compounds presented similar affinity (data not presented).
However, only ResAn1 interacts with residues involved with the catalytic activity of
this enzyme, suggesting this analogue as the most indicated for further antileishmanial
assays.
Table 3: Docking results between ResAn1-5 compounds and LbTryR in each step of the
catalytic mechanism.
ResAn1
ResAn2
ResAn3
ResAn4
ResAn5
Site Step
Score
1
Interactio
ns3
Interactio
ns3
Scor
e1
Interacti
ons3
Interactio
ns3
cKi2
Score1 cKi2
Score1
cKi2
Interactions3
cKi2
Score1 cKi2
HB: Y198,
T374; S:
Y198; P:
Y198
HB: T374,
G229;
HB: M333,
R287;
HB:
S1
conf14
HB: G376;
-
-5.155 166.47
-5.208 152.22
-5.421 106.26
-4.017 1,136.31
305.13
278.55
G229;
-5.118 177.20
S: Y198, F230 4.796
S: Y198
S: Y198
S: Y198
HB:
M333;
S: FAD,
Y198
HB:
S1
conf25
S: FAD,
-
R228;
38,097 HB: D330;
-6.019 38.73
-4.661
-4.775
-4.793
383.21 S: FAD, Y198
-1.936
Y198
4.850
S: Y198;
P: Y198
.1
P: Y198
HB:
G197,
V285;
S: Y198;
P:
HB:
HB: E202,
-
M333;
HB: A365;
S: FADH2
S2
-7.002
-7.698
7.37
2.28
-7.028 7.05 HB: A365
316.14
306.68
R287; S:
5.889
48.23
-6.033 37.82 S: FADH2;
HBHaloge
FADH2
n: A365
FADH2
HB:
M333,
A365; S:
FAD
HB:
A365,
I199; S:
Y198
HB: I199;
-5.408 108.61 S: Y198;
P: Y198
HB: R287;
-5.939 44.33
HB: A365;
S: FAD
-
S3
S4
190.21
357.59
S: Y198
5.076
HB:
H461,
E467,
HB: Q466,
HB: G49,
C52;
HB: T463;
-4.301 703.59
HB: S470;
S: H461
-
1,407.
96
T463,
H461;
-4.622 409.29
-3.489 2,770.30
-3.890
S: H461
4.702
S: W21
P336;
S: H461
P: H461

HB: T463,
E466,
HB:
P336;
HB: H461,
2,118. HB: Y110;
26 S: F396
HB: H461;
S: H461
-
1,119.
18
S5
S6
-3.534 2,567.68
-3.648
-3.878 1,436.77
432.00
380.63
-4.026
-3.360
T463
4.590
H461;
P: H461
S: H461
HB:
G942,
N340,
E466;
S: H461
HB: T457,
E466,
45,560 HB: Y110,
40,895.7
-
3,444.
18
-3.220 4,362.21 HB: E466 -1.830
-1.894
7
HB: Y110
.77
H461
4.665
R355
2
1Score: in Kcal/mol according to Glide extra precision mode (XPScore). cKi:
3
4
calculated Ki in µM. HB: hydrogen bond, S: stacking, P: Pi-cation. conf1: Y198
occluding NADP binding site (Y198closed). 5conf2: Y198 not occluding NADP
binding site (Y198open).

Figure 4. Best conformations of ResAn compounds obtained from docking results in NADP
binding site (NS) of LbTryR. Score values of each step are presented in Table 3: (A-B) S1 -
configuration 1, (C-D) S1 - configuration 2, (E-F) S2 and (G-H) S3. LbTryR is presented in
cartoon (grey) and FAD in sticks (grey). ResAn compounds are presented in sticks: ResAn1
(red), ResAn2 (green), ResAn3 (blue), ResAn4 (yellow) and ResAn5 (pink). Figures (B), (D),
(F) and (H) showing the pharmacophoric profile of NS, FAD (or FADH2) and ResAn1.
Pharmacophoric characteristics are represented by spheres: aromatic ring and lipophilic region
(gold), hydrogen bond donor (light blue), hydrogen bond acceptor (purple), hydrogen bond
donor and acceptor (blue), positive charge center (dark blue) and negative charge center (red).
The images were generated using PyMOL, version 1.8.2, Schrödinger - LLC.

Figure 5. Best conformations of ResAn compounds obtained from docking results in
catalytic site (CS) of LbTryR. Score values of each step are presented in Table 3: (A-B)
S4, (C-D) S5 and (E-F) S6. LbTryR is presented in cartoon (grey) and FAD in sticks
(grey). ResAn compounds are presented in sticks: ResAn1 (red), ResAn2 (green),
ResAn3 (blue), ResAn4 (yellow) and ResAn5 (pink). Figures (B), (D) and (F) showing
the pharmacophoric profile of CS, FAD and ResAn1. Pharmacophoric characteristics
are represented by spheres: aromatic ring and lipophilic region (gold), hydrogen bond
donor (light blue), hydrogen bond acceptor (purple), hydrogen bond donor and acceptor
(blue), positive charge center (dark blue) and negative charge center (red). The images
were generated using PyMOL, version 1.8.2, Schrödinger – LLC.

In silico results showed that LbTryR can be a target of ResAn compounds. After
this, we wanted to know whether ResAn compounds could inhibit the in vitro growth of
Leishmania sp, therefore, two Leishmania species: L. amazonensis and L. braziliensis,
both related to American Cutaneous Leishmaniasis were used in this work.
Table 4 shows that two compounds (ResAn1 and 2) were effective against
promastigote forms of L. amazonensis, and three compounds (ResAn1, 2 and 4) showed
antileishmanial effect against L. braziliensis promastigotes. Against amastigote forms,
in L. amazonensis only the compound ResAn1 was effective (IC50 of 21.62 µM), while
for L. braziliensis, all compounds that were effective in promastigote forms were also
active against intracellular amastigotes (ResAn1, 2 and 4) with IC50 values of 21.74,
17.54 and 29.32 µM, respectively (Table1). The compound ResAn1 inhibited the
growth of promastigotes and amastigotes of both Leishmania species, being more
effective than resveratrol. Interestingly, none of the compounds tested showed toxicity
on peritoneal macrophages until the maximal concentration used (150 Μm). Besides,
resveratrol showed a CC50 of 120.22 µM against peritoneal macrophages.
In addition to the expected varying sensitivity differences of the Leishmania
species to resveratrol analogue, the results also showed differences in stage-specific
sensitivity to assayed compounds (Table 4). The variation in sensitivity between
promastigotes and amastigotes of Leishmania species is difficult to explain and may be
related to a range of factors. Unlike promastigotes, amastigotes are obligate intracellular
forms surviving in the host cell parasitophorous vacuoles. So, some differences could
be related to stage-specific sensitivity to drugs, such as inactivation/metabolization of
the drug or low drug uptake of the compound by host cell, assays conditions,
differences in expression and exposure of membrane molecules, among others (Escobar
et al., 2002; Biyani & Madhubala, 2012; Cortes et al., 2015; Mandal et al., 2015). A

paradigm example is the effect of pentavalent antimonials on amastigotes. These
biological stages are several times more susceptible to pentavalent antimonials than
promastigotes, however the exact mode of action is not completely elucidated (Haldar
et al. 2011). In contrast, in a recent paper, the authors showed that L. infantum
promastigotes were more sensible to pentamidine, amphotericin B and miltefosine than
amastigotes (Mandal et al., 2015).
Table 4 - Effect of the resveratrol analogues against Leishmania species and
cytotoxicity in murine macrophages.
Antileishmanial activity - IC50 values (µM)a
Macrophages
CC50 values
(µM)b
Promastigotes Amastigotes
Compounds
ResAn1
L. ama1
L. braz2
L. ama1
L. braz2
21.62
(16.29-
28.70)
41.47 ±
3.10
21.74
7.33 ± 0.92
>150.00
(16.95-27.88)
51.21 ±
0.36
10.27 ±
0.40
17.54
>50.00
>50.00
>50.00
>50.00
>150.00
>150.00
>150.00
>150.00
ResAn2
ResAn3
ResAn4
ResAn5
(14.42-21.33)
>100.00
>100.00
>100.00
>100.00
>50.00
24.04 ±
2.68
29.32
(23.84-36.06)
>100.00
>50.00
42.85
(37.56-
48.88)
124.48
(106.44-
145.59)
2.45
23.23±
2.30
120.22 ±
35.71
75.13± 7.60
Resveratrol
Miltefosine
27.18 ±
4.27
28.07 ±
0.47
7.68
131.99 ± 3.95
(5.99-9.84)
(2.21-2.94)
aValues of inhibitory concentration of 50% of parasites (IC50). bValues of citotoxicity
1
concentration of 50% of macrophages (CC50). L. ama (L. amazonensis), 2L. braz (L.
braziliensis). Miltefosine was used as reference drug.

The relationship between structure and activity antileishmanial of the
compounds, in a decreasing order of IC50 values and increasing activity (ResAn4 <
ResAn2 < ResAn1) is a way to observe how the activity changes with the modifications
of the substituent in the aromatic portion of the original aldehyde, i.e., R2 and R3
groups, and the phenylhydrazine or 2,4-dinitrophenylhydrazine portions. Generally,
phenylhydrazine derivatives were more active than the 2,4-dinitrophenylhydrazine ones
and, additionally, were also more effective against L. braziliensis. The active analogues
(ResAn1, 2 and 4) are substituted in the aromatic portion of the original aldehyde by the
–OH or –COOH groups but, while the –OH substituent group positively influenced the
overall profile antileishmanial of the compound ResAn1 (against L. amazonensis and L.
braziliensis), the –COOH group proved to be more selective in potentiation of activity
against the L. braziliensis. On the other hand, the presence of a substituent –NO2 and –
Br in the aromatic portion of the original aldehyde was unfavorable to antileishmanial
activity.
Computational docking and in vitro antileishmanial results point out ResAn1 as
the most promising compound. Additionally, we tried to verify whether this compound
could also induce intracellular ROS production. The treatment of L. braziliensis
promastigotes with the compound ResAn1 induced a high increase production of
reactive oxygen species (ROS) but only at 14.66 µM (56%, compared to the untreated
control) (Figure 6). Promastigotes treated with miltefosine, used as positive control, also
increased the ROS production.

Figure 6. Generation of ROS in L. braziliensis promastigotes treated with ResAn1.
Parasites were untreated (negative control) or treated with the compound and after this
time the cells were probed with H2DCFDA. Miltefosine was used as positive control.
Fluorescence was measured and the data were expressed as the fluorescence intensity in
arbitrary units of means of three independent experiments. Statistical significance
compared with the negative control is indicated with asterisks (ns=not significant and
**p < 0.001).
4 Discussion
Leishmania has a unique defense mechanism against oxidative stress: the dithiol peptide
named trypanothione [T(SH)2]. This peptide, in reduced state [T(SH)2], scavenge the
ROS generated inside the cell and it is maintained in this state by action of
trypanothione reductase (TryR) (Saudagar et al., 2013). Although structurally and
mechanistically analogous to the mammalian enzyme glutathione reductase, the
protozoan TryR presents some differences in its disulfide binding site. So, TryR is
present only in trypanosomatids and its absence in human makes it an attractive
molecular target for drug design (Freitas-Júnior et al., 2012). TryR has been target for
several drugs, including pentavalent antimonials, the first line drugs used to treat
leishmaniasis (Singh and Sundar, 2014; De Morais et al., 2015).

In this work, the three-dimensional structure of TryR of L. braziliensis (LbTryR)
was modeled and docked against ResAn compounds showing the following affinity:
ResAn1 > ResAn2 > ResAn4 > ResAn5 > ResAn3. These results are very interesting,
since the in vitro antileishmanial activity of these compounds presented a similar
performance, considering the IC50 values (ResAn1 < ResAn2 < ResAn4). Besides the
interaction with the active site of LbTryR, a high affinity of ResAn1 to LbTryR in steps
S2 and S3 (Figure 4 and Table 2) occupying NADP binding site was observed. This
may interfere in the electron transfer processes avoiding the reduction of C52-C57
disulfide bond, responsible for the catalytic reaction.
Resveratrol showed low to moderate antileishmanial activity, with IC50 values
ranging from 23.23 to 124.48 µM, being these values closely resembled to those
reported in the literature (Kedzierski et al., 2007; Ferreira et al., 2011 ou 2014). In this
work, ResAn1 was more effective than resveratrol, highlighting their antileishmanial
activity. It is well known that sensitivity to accessible leishmaniasis therapies varies
among different Leishmania species (Mandal et al., 2015). Bearing in mind that this fact
should be considered in drug screening studies in the present work, ResAn compounds
were assayed against two Leishmania species related to American Cutaneous
Leishmaniasis (Silveira et al., 2009).
Previous works show that resveratrol and some analogues decreased ROS levels
in T. cruzi amastigotes and in L. amazonensis infected-macrophages, suggesting
antioxidant effect of these compounds (Paiva et al. 2012, Passos et al. 2015)
Nevertheless, Alarcon de la Lastra and Villegas (2007) reported that despite antioxidant
effects of resveratrol being more exploited, this drug can also exhibit pro-oxidant
properties. Here, ResAn1 induced increase of ROS production in treated L. braziliensis

promastigotes. This effect can be associated to pro-oxidant effect of ResAn1 or the
ROS levels observed is a consequence of inhibition of LbTryR.
We also performed the analysis of Lipinski’s rules (RO5) for ResAn1 using the
programs
Swiss-ADME
(http://www.swissadme.ch/)
and
admetSAR
(http://lmmd.ecust.edu.cn:8000/predict/) (data not shown). This analysis is important to
qualify the compound as a drug candidate for oral administration. In general, an active
drug should not have more than one violation of the following criteria: at most 5
hydrogen bond (HB) donors and 10 HB acceptors, a molecular weight between 180
g/mol and 500 g/mol, molar refractivity from 40 to 130; surface area no greater than 142
Å2, and an octanol-water partition coefficient Log P not greater than 5 (which is the
important physiochemical property that indicates the ability of molecule to cross
biological membrane) (Lipinsk et al., 2001; Nile et al., 2016).
The analyzes showed that ResAn1 did not have violations of RO5, presenting
the following predicted values: HB donors and acceptor equals to 3, molecular weight
of 228.25 g/mol, molar refractivity equal to 67.97, polar surface area of 64.85 Å2 and
Log P equal to 2.16. These results suggest that ResAn1 may be a promising drug
candidate with properties to cross biological membranes and high gastrointestinal
absorption. These physicochemical properties are related to orally active drugs in
humans, associated with 90% of orally active drugs that have achieved phase II clinical
status (Lipinski, 2004). Moreover, the physicochemical parameters showed that ResAn1
can be associated with acceptable aqueous solubility and intestinal permeability,
comprising the first steps in oral bioavailability (Lipinski, 2004). The selective and
antileishmanial activity of the compound ResAn1 showed in this work, together with
the theoretical predictions, encourage us to support further studies of the oral treatment
against Leishmania sp.

Acknowledgements
This work was supported by "Fundação de Amparo a Pesquisa do Estado de Minas
Gerais" (FAPEMIG), “Conselho Nacional de Desenvolvimento Científico e Tecnológico”
(CNPq) and “Universidade Federal de Juiz de Fora" (UFJF). The authors thank the fellowship
by CNPq and CAPES. The authors are grateful to Reproduction Biology Center (CBR) of the
Federal University of Juiz de Fora for supply of animals. Leishmania amazonensis-RFP was
generously donated by Dr. Rodrigo RP Soares (Rene Rachou Research Center, Oswaldo Cruz
Foundation, BH, Brazil).

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