The 2′,4′-dihydroxychalcone could be explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species

Article abstract of DOI:10.1016/j.bmcl.2015.06.085

Abstract The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is considered as an attractive target to design new antileishmanial drugs and a previous in silico study reported on the importance of chalcones to achieve its inhibiti

Full text of DOI:10.1016/j.bmcl.2015.06.085

Accepted Manuscript
The 2´,4´-dihydroxychalcone could be explored to develop new inhibitors
against the glycerol-3-phosphate dehydrogenase from Leishmania species
Thais G. Passalacqua, Fabio A.E. Torres, Camila T. Nogueira, Leticia de
Almeida, Mayara L. Del Cistia, Mariana B. dos Santos, Luis O. Regasini, Marcia
A.S. Graminha, Reinaldo Marchetto, Aderson Zottis
PII:
S0960-894X(15)00690-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.06.085
BMCL 22875
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
9 March 2015
22 June 2015
23 June 2015
Please cite this article as: Passalacqua, T.G., Torres, F.A.E., Nogueira, C.T., Almeida, L.d., Del Cistia, M.L., dos
Santos, M.B., Regasini, L.O., Graminha, M.A.S., Marchetto, R., Zottis, A., The 2´,4´-dihydroxychalcone could be
explored to develop new inhibitors against the glycerol-3-phosphate dehydrogenase from Leishmania species,
Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.06.085
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The 2´,4´-dihydroxychalcone could be
explored to develop new inhibitors against
the glycerol-3-phosphate dehydrogenase
from Leishmania species
Leave this area blank for abstract info
Thais G. Passalacqua; Fabio A. E. Torres; Camila T. Nogueira; Leticia de Almeida; Mayara L. Del Cistia;
Mariana B. dos Santos; Luis O. Regasini; Marcia A. S. Graminha; Reinaldo Marchetto; Aderson Zottis

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
The 2´,4´-dihydroxychalcone could be explored to develop new inhibitors against
the glycerol-3-phosphate dehydrogenase from Leishmania species
a
a
a
a
Thais G. Passalacqua ; Fabio A. E. Torres ; Camila T. Nogueira ; Leticia de Almeida ; Mayara L. Del
a
b
b
a
c
Cistia ; Mariana B. dos Santos ; Luis O. Regasini ; Marcia A. S. Graminha ; Reinaldo Marchetto ; Aderson
c
1
Zottis *
a
UNESP – School of Pharmaceutical Sciences, Department of Clinical Analyses, Rodovia Araraquara-Jaú, Km 01, Araraquara, SP, 14801-902, Brazil.
UNESP – Institute of Biosciences, Letters and Exact Sciences, Department of Chemistry and Environmental Sciences, Rua Cristóvão Colombo, 2265, Jardim
b
Nazareth, São José do Rio Preto, SP, 15054-000, Brazil.
c
UNESP – Institute of Chemistry, Department of Biochemistry and Chemical Technology, Rua Professor Francisco Degni, 55, Caixa Postal 355, Araraquara,
SP, 14800-060, Brazil.
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The enzyme glycerol-3-phosphate dehydrogenase (G3PDH) from Leishmania species is
considered an attractive target to design new antileishmanial drugs and a previous in silico study
reported on the importance of chalcones to achieve its inhibition. Here, we report the
identification of a synthetic chalcone in our in vitro assays with promastigote cells from
Leishmania amazonensis, its biological activity in animal models, and docking followed by
molecular dynamics simulation to investigate the molecular interactions and structural patterns
that are crucial to achieve the inhibition complex between this compound and G3PDH. A
molecular fragment of this natural product derivative can provide new inhibitors with increased
potency and selectivity.
Keywords:
Leishmania amazonensis
2
´,4´-dihydroxychalcone
Cytotoxicity assay
Glycerol-3-phosphate dehydrogenase
Docking
2
009 Elsevier Ltd. All rights reserved.
Molecular dynamics
The Leishmaniasis is a complex of diseases caused by a
protozoan of the genus Leishmania, which is spread over 98
countries around the world and affects about 12 million people,
endangering other 350 million.[1] It is one of the most neglected
diseases due to the absence of interest from pharmaceutical
industry for developing new drugs against it.[2] This parasitic
disease has been classified into three groups, visceral, cutaneous,
and mucocutaneous Leishmaniasis [3], which have different
clinical manifestations and degree of morbidity and mortality.[4]
Transmission is caused by the flying vector Phlebotomines[5],
and two morphological forms are present during the life cycle of
this parasite, promastigotes and amastigotes.[6] Promastigotes
are injected into the mammalian host during the insect blood
meal[7], and phagocytised by macrophages. After, they transform
into amastigotes form that survive and multiply within the
macrophage phagolysosome.[8] The current therapy of
Leishmaniasis relies upon few drugs[9, 10] that show toxicity
and/or inefficiency[5, 7], which evidence the high necessity of
more efficacious drugs. In this sense, natural products can be a
valuable alternative to provide huge diversity of chemical
structures for biological screening campaigns against Leishmania
species to develop new antileishmanial drugs.
We have identified a 2´,4´-dihydroxychalcone (DHC)
inhibitor against promastigotesforms of Leishmania amazonensis
during our cellular assays. Accordingly, chalcones have been
reported that are promising inhibitors of glycerol-3-phosphate
dehydrogenase[11], a glycosome enzyme that plays a critical role
in the parasite to keep its essential metabolism, and it is
considered as an attractive target to develop new antileishmanial
drugs. The simple structure of chalcones and their ease of
preparation make them an attractive scaffold to attach functional
groups for enhancing their biological activities.[12] Basically,
these molecules are open-chain flavonoids (figure 1) in which the
two aromatic rings join by a three-carbon α, β-unsaturated
carbonyl system.[13]
A
B
C
Figure 1. 2D basic structure of a flavonoid (A), a chalcone (B), and
the leishmanicidal DHC identified in our screening assays with
atoms numbered accordingly (C). Structures sketched with
Marvin.[14]
—
——
*
Corresponding author. Tel.: +55 16 3301-9877 / 3301-9707.
Fax: +55 16 3322-2308; E-mail: zottisad@gmail.com.

Based on these good results we extend the investigation with
DHC and the G3PDH enzyme by docking and dynamics
simulation to reveal the molecular interactions that could drive
the formation of this inhibition complex. A deep understanding
of such interactions is a crucial step to address the synthesis of
new derivatives with increased affinity for G3PDH.
HEPES and 2 mM L-glutamine, and incubated during 4 h at 37
°C in a 5% CO -air mixture. The medium was removed and a
new one was added to the cells which were treated with different
concentrations of DHC and pentamidine. Cells without drugs
were used as negative control. After that, plates were incubated
2
2
for 24 h at 37 °C in a 5% CO -air mixture. Subsequently, the
MTT colorimetric assay was carried out as described above.
Absorbance was read in a 96-well plate reader (Robonik®) at
The DHC (MW 240.26) was synthesized following the
Claisen Schmidt condensation using the described protocol by
Zeraik and collaborators[15], with minor modifications. Briefly,
in a 200 mL vial, 2´,4´-dihydroxyacetophenone (20 mmol) and
potassium hydroxide (0.8 mol) were dissolved in ethanol (80
mL), and the mixture was stirred at 5 ºC for 10 minutes followed
by addition of benzaldehyde (20 mmol in 10 mL of ethanol). The
reaction mixture was stirred at room temperature and monitored
by TLC using hexane:ethyl acetate as the mobile phase (7:3). The
reaction was quenched after 24 h by pouring the mixture into 500
mL of ice-cold water with stirring. A precipitate formed after
quenching with cold water, which was then filtered. The crude
product was purified by flash chromatography with hexane:ethyl
acetate in increasing order of polarity. The compound was
identified using 1H and 13C NMR spectral data obtained from a
Varian DRX-500 spectrometer (11.7 T). Chemical shifts (δ) were
expressed in ppm. Coupling constants (J) were expressed in Hz,
and splitting patterns are described as follows: s = singlet; br s =
broad singlet; d = doublet; t = triplet; m = multiplet; and dd =
doublet of doublets. The white solid was obtained in 20% yield.
5
40 nm. The drug concentration corresponding to 50% of cell
growth inhibition was expressed as the cytotoxic concentration
(
CC50).
Previous study reported that chalcones can exhibit strong
interaction with G3PDH from Leishmania.[11] In that in silico
investigation the crystal structures deposited in Protein Data
Bank[17] with codes 1EVZ, 1M66, 1N1E and 1N1G were used,
all of them with identical residue sequences. A rapid comparison
of the aminoacid sequences of G3PDH from L. mexicana, L.
infantum, L. major, L. brasiliensis, and L. donovani through an
alignment procedure in the UniProt[18] (www.uniprot.org)
corroborated the high conservation degree of their binding site
(data not shown). These evidences prompted us to choose
1N1E[19] (1.90 Å of resolution) as the based target structure for
our computational studies. The crystal structure of human
G3PDH (PDB ID 1X0X)[20] was chosen for structural
superposition and comparison. The steps of reconstruction,
stereochemical corrections and energy minimization of the
G3PDH structure 1N1E were performed by the KoBaMIN
server.[21] The 3D structure of DHC was sketched through
Maestro[22] and docking was performed with AutoDock v4.2
and AutoDock Tools v1.5.6[23] following the default steps.
Briefly, merged non-polar hydrogens were set to DHC structure
and in the modeled 1N1E, flexibility of the DHC was considered
by the number of torsions set automatically, and Gasteiger
charges were applied for both. The map of the region for docking
was assigned through Autogrid considering the binding site of
the adenosine analogue in the 1N1E structure. The grid was set to
1
7
6
9
H NMR (500 MHz, DMSO-d6):δ 8.12 (d; J = 9.0 Hz, H-6’),
.93 (d; J = 15.5 Hz, H-β), 7.86 (dd; J = 7.0, 2.0 Hz, H-2 and H-
), 7.75 (d; J = 15.5 Hz, H- α), 7.43 (m, H-3 – H-5), 6.36 (dd; J =
.0, 2.0, H-5’), 6.22 (d; J = 2.0 Hz, H-3’);13C NMR (125 MHz,
DMSO-d6): δ 190.7 (C-β’), 168.0 (C-4’), 166.1 (C-2’), 143.1 (C-
β), 134.7 (C-1), 132.9, (C-6’), 130.5 (C-4), 128.9 (C-2, C-3, C-5,
C-6), 121.5 (C-α), 112.3 (C-1’), 109.1 (C-5’), 102.7 (C-3’).
Adult male Swiss albino mice (20–35 g) were used in the
experiments. They were housed in single-sex cages under a 12 h
light:12 h dark cycle (lights on at 06:00) in a controlled room
temperature (22 °C), and free access to food and water. Groups
of two animals were used in each test group. The experiments
were performed after the protocol was approved by the local
Institutional Ethics Committee (protocol number 53/2012), which
follows the Legislation for the protection of animals as described
by the Directives from the European Commission.
7
0 Å in all dimensions taking the coordinates for the center as 29,
2
5 and 22 for x, y and z respectively. Rigid docking was carried
out, using Lamarckian genetic algorithm as search parameter,
keeping the other default options. After, we performed a
structural comparison through a superposition of 1N1E with
docked DHC and 1X0X to identify differences between the
binding sites for useful subpockets to be explored for selectivity.
L. amazonensis promastigotes (MPRO/BR/1972/M1841-LV-
9) freshly isolated from mice were kept at 28°C in liver infusion
tryptose (LIT) supplemented with 10% fetal bovine serum (FBS),
penicillin and streptomycin (Sigma-Aldrich®).
The best pose out of 10 conformations in terms of energy
scoring was used as the initial complex to set up the system for
molecular dynamics simulation with Desmond v3.8[24] with
graphical user interface from Maestro.[22] The system was
neutralized with adequate counter ions, NaCl in a concentration
of 150 mM was added, and the complex of G3PDH-DHC was
surrounded by single point charge (SPC) model of water
molecules. The force field assigned for the system was OPLS-
AA[25]in orthorhombic box shape with the distance of 10 Å
from the complex to the box wall in all directions. The SHAKE
algorithm was used for geometrical constrains of the molecules
and bond lengths with hydrogens, and long-range electrostatic
interactions treated by Smooth Particle Mesh Ewald while a
cutoff radius of 9 Å was used to calculate short-range coulombic
interactions. Minimization was carried out using steepest descent
method until 1.0 kcal/mol/Å of convergence and equilibration
following a multistep protocol. Basically, these were the steps: i)
system relaxation with Brownian dynamics using NVT ensemble
at 10 K with position restraints for protein and ligand, except for
hydrogens; ii) simulation with NVT ensemble and Berendsen
thermostat with temperature kept at 10 K; iii) simulation with
NPT ensemble and Berendsen for both thermostat and barostat in
7
Flat-bottom plates (TPP®) were seeded with cultured
promastigotes from L. amazonensis at the end of exponential
growth phase (7 days) to achieve 8 x 106 parasites/mL per well.
DHC was dissolved in DMSO, added to each well in dilution
series ranging from 0.195 µg/mL to 100.0 µg/mL, and incubated
at 28 °C for 72 h. The assays were carried out in triplicate.
Leishmanicidal effect was assessed by 3-[4,5-dimethylthiazol-2-
yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) method
modified accordingly.[16] Absorbance was read at 490 nm
(
Robonik®). The drug concentration corresponding to 50% of
parasite growth inhibition was expressed as the inhibitory
concentration (IC50).
To evaluate the cytotoxicity, thioglycolate-stimulated mice
were used to collect peritoneal macrophages. Murine peritoneal
macrophages were seeded in 96 well flat-bottom plates (TPP®)
at a density of 1 x 105 cells/well (100 µL/well) in RPMI-1640
medium supplemented with 10% heat-inactivated FBS, 25 mM

the same temperature with 1 atm of pressure, with position
restraints excepting for hydrogens; iv) Solvation of the
interaction site; v) simulation in NPT ensemble with Berendsen
thermostat and barostat, at 300 K and pressure of 1 atm, keeping
solution restraints, a fast relaxing constant of temperature, and a
slow relaxing of pressure; vi) simulation in NPT ensemble, with
the same thermostat and barostat at 300 K, pressure of 1 atm, a
fast relaxing constant of temperature, and a normal relaxing
constant of pressure. Then, simulation was run during 10 ns at
dashed lines) identified are highlighted, and also the pocket surface
of DHC. BE: binding energy; LE: ligand efficiency; nH-bonds:
number of H-bonds. Figure generated with Maestro.[22]
In the context of the drug design process the fragment-based
approach has proved to be a very consistent and reward strategy
to discover new leads, and Rule of Three (Ro3) is used largely as
a guidance to enrich fragment libraries for this purpose.[28] As it
can be seen from figure 2, important H-bonds contacts between
DHC and the residues Val92, Lys125 and Arg274 were identified
by docking, and the BE and LE indexes highlight that DHC is a
promising fragment for the process of chemical optimization to
generate new lead candidates.[29] Furthermore, the superposition
of reconstructed 1N1E and 1X0X revealed differences in the
residues surrounding the binding pocket used as receptor target
for docking, which can be chemically explored for selectivity
(figure 3).
3
00 K and afterwards several analyses of the trajectory were
performed to evaluate the time-dependent structural
modifications and molecular interactions.
DHC was assayed in a culture of promastigote cells from L.
amazonensis and its cytotoxic activity was evaluated with
peritoneal macrophages. The half of the maximal inhibitory
concentration (IC50) and the cytotoxic concentration (CC50)
values are given in table 1. The leishmanicidal activity of DHC
was nearly 16 times higher than pentamidine, a drug that is used
in the treatment of Leishmaniasis in South America[26], and it
was about 200 times more selective for the parasite in
comparison to the same control.
Table 1. Biological activity of DHC and Pentamidine against
promastigotes and amastigotes of L. amazonensis (IC50),
macrophages (CC50) and selectivity index (SI).
IC50 (µM)
promastigotes
CC50 (µM)
macrophages
Compound
SI
DHC
Pentamidine
0.4 ± 0.1
6.7 ±0.5
416.7 ± 2.1
35.7±6.8
1041.7
5.3
Selectivity index represented by the ratio between CC50 and IC50 for
promastigotes.
Based on the good results from these biological assays and
data previous reported we proceeded our investigation by
applying docking and molecular dynamics simulation. As cells
from L. mexicana and L. amazonensis show high morphological
and a very similar mode of biochemical interaction with the
host[27] we used the crystal structure of G3PDH from
L.mexicana (PDB ID 1N1E) as the biochemical target for
computational studies. As this is an essential enzyme in
glycolysis pathway from which the parasite is highly dependent
in a specific form of its life cycle, it is reasonable to propose the
interaction between DHC and this biological target to explain the
leishmanicidal effect observed.
Figure 3. Superposition of the active sites from 1N1E and 1X0X.
DHC is showed positioned in the center with respect to its docking
onto 1N1E. Surface denotes the binding pocket of DHC and
differences in terms of the residues from the binding sites are
highlighted. The residues Val40, His95, Ile119 and Gln298 in 1X0X
are substituted by His45, Thr95, Thr124 and Glu300 in 1N1E
respectively. Hip: protonated Histidine. Residues colored by
properties. Gray: hydrophobic; cyan: hydrophilic; red: anionic; blue:
protonated. Figure generated with Maestro.[22]
Investigating the dynamics between DHC and G3PDH is
fundamental for revealing the structural pattern and chemical
groups that respond for the intermolecular recognition. Then, we
investigated such complex by running 10 ns of MD simulation in
an attempt to identify the time-dependent events that can dictate
the formation of the DHC-G3PDH complex identified by
docking. The results are shown in figure 4.
The poses from the docking of DHC into the binding pocket
of the 1N1E resulted in an estimative of the binding energy
-
1
ranging from -6.68 to-8.39 kcal.mol (figure 2).
BE = -8.39
LE = 0.47
nH-bonds = 3
Protein
Ligand
Figure 2. Pose that retrieved best scoring from the DHC docked in
the binding site of 1N1E. The residues and the H-bonds (yellow
A

shows compatible flexibility to follow the dynamics of the
binding site as simulation proceeds. MD uncovered the formation
of additional H-bonds with other residues, in particular between
the 4´-hydroxy group and Glu300, providing a clue to emphasize
the need of an H-bond donor in this position to assure high
affinity for the binding site of G3PDH, which was not identified
by docking. This is a relevant structural pattern and distinguished
molecular behavior of DHC as its structure can follow the
conformations adopted by the binding site of G3PDH, thus
allowing complementary of the interactions with other residues
also buried in the binding pocket. DHC structure shows both
flexibility and strategic groups to switch interactions with various
residues in additional hot spots as the structure of the protein
moves ahead and that can operate as a paramount feature of the
good DHC affinity for G3PDH. Then, a combination of factors
like strategic positions of groups for H-bond interactions,
compatible volume to a buried binding pocket and reasonable
flexibility of DHC collaborate for promoting the complex
formation with G3PDH. The pattern of DHC interaction with
G3PDH by the MD simulation indicated a prevalence of H-bond
with the residues Val92, Lys125, Arg274, and Glu300 equal or
more than 40% of the simulation time. The more important
hydrophobic contact was with Phe63 located at the entrance of
the binding site, and together with others hydrophobic residues in
this region could reinforce the DHC interaction by decreasing the
solvent accessibility and avoiding the return of DHC to the
aqueous phase. Ionic interaction was identified with Arg274,
which persisted about 30% of the trajectory. The H-bonds with
Glu300 (100% of the trajectory), Lys125 (more than 80%),
Val92 (more than 40%) together with the multiple interactions
with Arg274 were the prevalent hydrophilic forces. Table 2 lists
the residues and the type of interactions they kept with DHC.
α-helice
ß-sheet
turn
B
water bridges
H-bond
C
ionic
hydrophobic
Table 2. Main residues that interacted with DHC and type of
interactions observed during the MD simulation.
Residue
Type of
interaction
Chemical group in
DHC
Occupancy
(%)
Ser23
Hb
Phenyl ring
C=O
2
2
Gly24
Ala25
Phe26
Phe63
Val92
Pro94
Hb (m, d)
Hb (m, d)
Hb (m, d)
h (s)
C=O
2
C=O, -OH (2´)
Phenyl ring
-OH (2´)
1
54
43
21
27
84
72
23
100
Hb (m, a)
h
D
dihydroxyphenyl ring
-OH (2´)
Figure 4. Dynamics of interactions between DHC and G3PDH.
A) Root Mean Square Deviation (RMSD) from DHC-G3PDH
complex observed for the trajectory during 10 ns of simulation; B)
Root Mean Square Fluctuation (RMSF) from the G3PDH C-alphas
and numbered residues interacting with DHC (green lines); D) Bar
plot of main interactions between DHC and G3PDH binding pocket
during the simulation; D) Representative interaction of the main H-
bonds detected with an interactions cutoff of 35%. Figures generated
with Maestro.[22]
Cys123
Lys125
Arg274
Val298
Glu300
Hb (m, a)
Hb (m, d)
Hb (s, d), i
Hb (m, d)
Hb (s, a)
-OH (4´)
C=O
C=O
-OH (4´)
wb: water-bridge; Hb: H-bond; i: ionic; h: hydrophobic; m: main
chain; s: side chain; d: donor; a: acceptor: C=O: carbonyl oxygen;
-
OH: hydroxyl group numbered accordingly.
The RMSD graph of the backbone (figure 4.A) highlights a
fluctuation of about 3 Å of the protein backbone in comparison to
the initial complex until a more stable conformation of this
system is reached, which occurs at around 3 ns, thus reflecting
that G3PDH exhibits small changes in its conformation with
exception of the random N- and C-terminal portions. DHC
showed conformational changing (RMSD) up to 1 Å as the
simulation proceeds, indicating that DHC fills in the pocket and
The MD simulation provided a substantial evidence of the hot
spots in the binding pocket of G3PDH with respect the DHC
compound. The side chains of Arg274 and Glu300 as well as the
NH of main chain from Lys125 provided the stronger hydrophilic
interactions, and they can be considered as key residues for H-
bond interactions with DHC. On the other hand, the residues

Val92, Cys123, and Val298 can provide additional H-bonds to
also explore for more potent DHC ligands. The carbonyl group
attached to and the dihydroxyphenyl ring is likely to hold the
moiety that keeps the effective interaction with the binding site,
and thus it represents the lowest structural fragment that should
be retained in the structure of new derivatives to assure its
inhibitory activity against G3PDH. Low occupancy observed
with other residues also contributes transiently to enforce the
complex, and can be important to the design of new inhibitors.
These findings open up a new route to address the development
of even other classes of G3PDH inhibitors starting from a
carbonyl dihydroxyphenyl ring moiety. Additionally, the side
chains of the residues His45, Thr95, Thr124 and Glu300 that are
equivalent to Val40, His95, Ile119 and Gln298 respectively in
the human G3PDH provide important subpockets for the
exploration of more selective inhibitors.
[4] N. Singh, M. Kumar, R.K. Singh, Leishmaniasis: Current status
of available drugs and new potential drug targets, Asian Pacific
Journal of Tropical Medicine, 5 (2012) 485-497.
[
5] P. Bhargava, R. Singh, Developments in Diagnosis and
Antileishmanial Drugs, Interdisciplinary Perspectives on Infectious
Diseases, 2012 (2012) 13.
[
6] P.A. Bates, Transmission of Leishmania metacyclic
promastigotes by phlebotomine sand flies, International Journal for
Parasitology, 37 (2007) 1097-1106.
[
7] L.H. Freitas-Junior, E. Chatelain, H.A. Kim, J.L. Siqueira-Neto,
Visceral leishmaniasis treatment: What do we have, what do we need
and how to deliver it?, International Journal for Parasitology: Drugs
and Drug Resistance, 2 (2012) 11-19.
[8] F. Chappuis, S. Sundar, A. Hailu, H. Ghalib, S. Rijal, R.W.
Peeling, J. Alvar, M. Boelaert, Visceral leishmaniasis: what are the
needs for diagnosis, treatment and control?, Nat Rev Micro, 5 (2007)
873-882.
[
9] G. Kaur, B. Rajput, Comparative Analysis of the Omics
In summary, we have identified a 2´,4´-dihydroxychalcone as
an important leishmanicidal compound against. Previous studies
indicated high affinity of chalcones for G3PDH and results from
biological assays evidenced the potency and selectivity of DHC
against promastigote cells from L. amazonenesis, which
supported the investigation of the mechanism by which DHC
could inhibit G3PDH. Docking studies retrieved good values of
binding estimation which corroborates that this essential
glycolytic enzyme could be a target of such compound. MD
simulation highlighted the interactions that could dictate the
molecular behavior from G3PDH-DHC complex, providing the
molecular knowledge for the design of new analogue inhibitors
with increased potency and selectivity for G3PDH of Leishmania
species. Additionally, the simulation data revealed that DHC is
very suitable to employ for chemical optimization to generate a
new antileishmanial lead. The better IC50, CC50, and selectivity
profile in comparison to the drug pentamidine – which is in
clinical use – is also a substantial evidence of its features that
establishes the DHC structure as a very useful ligand with
increased chances to provide a new chemotherapeutic agent to
treat Leishmaniasis. Furthermore, the carbonyl dihydroxyphenyl
ring can be considered as a very relevant substructure to be
included in a fragment-based pipeline for discovering new
inhibitor classes of G3PDH from Leishmania species.
Technologies Used to Study Antimonial, Amphotericin B, and
Pentamidine Resistance in Leishmania, Journal of Parasitology
Research, 2014 (2014) 11.
[10] M. Ouellette, J. Drummelsmith, B. Papadopoulou,
Leishmaniasis: drugs in the clinic, resistance and new developments,
Drug Resistance Updates, 7 (2004) 257-266.
[11] I.V. Ogungbe, W.R. Erwin, W.N. Setzer, Antileishmanial
phytochemical phenolics: Molecular docking to potential protein
targets, Journal of Molecular Graphics and Modelling, 48 (2014)
1
05-117.
[
12] R. Romagnoli, P.G. Baraldi, M.D. Carrion, O. Cruz-Lopez, C.L.
Cara, J. Balzarini, E. Hamel, A. Canella, E. Fabbri, R. Gambari, G.
Basso, G. Viola, Hybrid α-bromoacryloylamido chalcones. Design,
synthesis and biological evaluation, Bioorganic
Chemistry Letters, 19 (2009) 2022-2028.
& Medicinal
[
13] Z. Nowakowska,
inflammatory chalcones, European Journal of Medicinal Chemistry,
2 (2007) 125-137.
A review of anti-infective and anti-
4
[
[
14] Marvin 5.7.1, ChemAxon, www.chemaxon.com.
15] V.F.X. M.L. Zeraik, L.O. Regasini, L.A. Dutra, D.H.S. Silva,
L.M. Fonseca, D. Coelho, S.A.S. Machado, V.S. Bolzani, 4´-
Aminochalcones as novel inhibitors of the chlorinating activity of
myeloperoxidase., Current Medicinal Chemistry, 19 (2012) 5405-
5413.
[16] V.A.F.F.M. Santos, L.O. Regasini, C.R. Nogueira, G.D.
Passerini, I. Martinez, V.S. Bolzani, M.A.S. Graminha, R.M.B.
Cicarelli, M. Furlan, Antiprotozoal Sesquiterpene Pyridine Alkaloids
from Maytenus ilicifolia, Journal of Natural Products, 75 (2012) 991-
Acknowledgements
9
95.
The authors are very grateful to São Paulo Research
Foundation (FAPESP) and the PROPe-UNESP from São Paulo
State University, for providing the scholarships and the funding
sources for the researches as well.
[17] F.C. Bernstein, T.F. Koetzle, G.J.B. Williams, E.F. Meyer,
M.D. Brice, J.R. Rodgers, O. Kennard, T. Shimanouchi, M. Tasumi,
The Protein Data Bank, European Journal of Biochemistry, 80
(
[
1977) 319-324.
18] T.U. Consortium, Reorganizing the protein space at the
Universal Protein Resource (UniProt), Nucleic Acids Research, 40
(
[
2012) D71-D75.
Conflict of Interest
19] J. Choe, D. Guerra, P.A.M. Michels, W.G.J. Hol, Leishmania
The authors declare that there are no conflicts of interest.
mexicana
Glycerol-3-phosphate
Dehydrogenase
Showed
Conformational Changes Upon Binding a Bi-substrate Adduct,
Journal of Molecular Biology, 329 (2003) 335-349.
[
20] X. Ou, C. Ji, X. Han, X. Zhao, X. Li, Y. Mao, L.-L. Wong, M.
References and Notes
Bartlam, Z. Rao, Crystal Structures of Human Glycerol 3-phosphate
Dehydrogenase 1 (GPD1), Journal of Molecular Biology, 357 (2006)
858-869.
[
1] A.K. Haldar, P. Sen, S. Roy, Use of Antimony in the Treatment
of Leishmaniasis: Current Status and Future Directions, Molecular
Biology International, 2011 (2011) 23.
[21] J.P.G.L.M. Rodrigues, M. Levitt, G. Chopra, KoBaMIN: a
knowledge-based minimization web server for protein structure
refinement, Nucleic Acids Research, 40 (2012) W323-W328.
[22] v. Schrödinger Release 2014-2: Maestro, Schrödinger, LLC,
New York, NY, 2014.
[
2] J.N. Burrows, R.L. Elliott, T. Kaneko, C.E. Mowbray, D.
Waterson, The role of modern drug discovery in the fight against
neglected and tropical diseases, MedChemComm, 5 (2014) 688-700.
[
3] C.A. Philips, C.R. Kalal, K.N.C. Kumar, C. Bihari, S.K. Sarin,
Simultaneous Occurrence of Ocular, Disseminated Mucocutaneous,
and Multivisceral Involvement of Leishmaniasis, Case Reports in
Infectious Diseases, 2014 (2014) 4.
[23] G.M. Morris, R. Huey, W. Lindstrom, M.F. Sanner, R.K.
Belew, D.S. Goodsell, A.J. Olson, AutoDock4 and AutoDockTools4:

Automated docking with selective receptor flexibility, Journal of
Computational Chemistry, 30 (2009) 2785-2791.
[26] S.L. Croft, P. Olliaro, Leishmaniasis chemotherapy—challenges
and opportunities, Clinical Microbiology and Infection, 17 (2011)
1478-1483.
[
24] v. Schrödinger Release 2014-2: Desmond Molecular Dynamics
System, D. E. Shaw Research, New York, NY, 2014. Maestro-
Desmond Interoperability Tools, version 3.8, Schrödinger, New
York, NY, 2014.
[27] L. Soong, Subversion and utilization of host innate defense by
Leishmania amazonensis, Frontiers in Immunology, 3 (2012).
[28] H. Jhoti, G. Williams, D.C. Rees, C.W. Murray, The 'rule of
three' for fragment-based drug discovery: where are we now?, Nat
Rev Drug Discov, 12 (2013) 644-644.
[29] R.A.E. Carr, M. Congreve, C.W. Murray, D.C. Rees, Fragment-
based lead discovery: leads by design, Drug Discovery Today, 10
(2005) 987-992.
[
25] G.A. Kaminski, R.A. Friesner, J. Tirado-Rives, W.L. Jorgensen,
Evaluation and Reparametrization of the OPLS-AA Force Field for
Proteins via Comparison with Accurate Quantum Chemical
Calculations on Peptides†, The Journal of Physical Chemistry B, 105
(
2001) 6474-6487.