Theoretical study of new LmDHODH and

LmTXNPx complexes: structure-based

relationships

Plinio Cantero-López, Sara M. Robledo

Restrepo, Osvaldo Yañez, César Zúñiga
& Gilmar G. Santafé-Patiño

Structural Chemistry
Computational and Experimental
Studies of Chemical and Biological
Systems

ISSN 1040-0400

Struct Chem
DOI 10.1007/s11224-020-01624-7

1 23
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 Author's personal copy
Structural Chemistry
https://doi.org/10.1007/s11224-020-01624-7

ORIGINAL RESEARCH

Theoretical study of new LmDHODH and LmTXNPx

complexes: structure-based relationships
Plinio Cantero-López 1,2 & Sara M. Robledo Restrepo 3 & Osvaldo Yañez 4,5,6 & César Zúñiga 7,8 &
Gilmar G. Santafé-Patiño 9

Received: 14 July 2020 / Accepted: 20 August 2020

# Springer Science+Business Media, LLC, part of Springer Nature 2020

Abstract
In this work, a series of eight novel ring-substituted styrylquinolines were synthesized, and in silico physicochemical properties
were estimated. The inhibitory activity of these compounds was evaluated in intracellular amastigotes of Leishmania (Viannia)
panamensis, and their affinity for L. major dihydroorotate dehydrogenase DHODH (LmDHODH) and L. major tryparedoxin
peroxidase TXNPx (LmTXNPx) was calculated by molecular docking, NCI index, and the recently developed IGM analysis,
providing us useful insights about the forces governing the ligand-protein coupling. The eight synthesized molecules do not break
the Lipinski, Ghose, Veber, Egan, and Muegge rules. Therefore, the bioavailability and absorption will not be poor.

Keywords Leishmania (Viannia) panamensis . NCI index . ADMET properties . Molecular modeling . Ring-substituted
styrylquinolines

Introduction Leishmania (Leishmania spp.), which are transferred to mam-

During the last decades, the parasitic disease leishmaniasis has
become one of the biggest health problems worldwide, caus-
ing approximately 50,000 deaths every year and exposing
over 20 billion people to contagium [1, 2]. Leishmaniasis is
generated by intracellular protozoan parasites of the genus
mals, including men, by the bite of females infected with
phlebotomous insects of the genus Lutzomya, Phlebotumus,
and Psychodopygus. The main forms of clinical manifesta-
tions are cutaneous, mucous, and visceral. The symptons can
include several skin and mucosal ulcers, anemia, fever, and
death [3]. The trypanosomatid protozoa Leishmania is

Electronic supplementary material The online version of this article

(https://doi.org/10.1007/s11224-020-01624-7) contains supplementary
material, which is available to authorized users.

* Plinio Cantero-López
pliniocantero@gmail.com; p.canterolpez@uandresbello.edu
1
Relativistic Molecular Physics Group (ReMoPh), PhD program in
Molecular Physical Chemistry, Facultad de Ciencias Exactas,
Universidad Andres Bello, República 275, Santiago, Chile
2
Center of Applied Nanoscience (CANS), Facultad de Ciencias
Exactas, Universidad Andres Bello, Av. República 330,
Santiago, Chile
3 8
PECET-Programa de Estudio y Control de Enfermedades Tropicales.
Facultad de Medicina, Universidad de Antioquia, Calle 70 No. 52–
21, Medellín A 1226, Colombia
4 9
Computational and Theoretical Chemistry Group, Departamento de
Ciencias Químicas, Facultad de Ciencias Exactas, Universidad
Andrés Bello, República 498, Santiago, Chile
5
Center for Bioinformatics and Integrative Biology (CBIB), Facultad
de Ciencias de la Vida, Universidad Andres Bello, Av. Republica ́
330, 8370146 Santiago, Chile
6
Center of New Drugs for Hypertension (CENDHY), Santiago, Chile
7
Instituto de Ciencias Naturales, Facultad de Medicina Veterinaria y
Agronomía, Universidad de Las Américas, Sede Providencia,
Manuel Montt 948, 7500972 Santiago, Chile
Facultad de Ciencias de la Salud, Universidad Central de Chile, Lord
Cochrane 417, Santiago, Chile
Grupo de Investigación Química de los Productos Naturales,
Departamento de Química, Universidad de Córdoba, Carrera6
Núm.76-103, Montería, Córdoba, Colombia
 Author's personal copy
endemic in approximately 100 tropical and sub-tropical coun-
tries enclosing various continents around the world [1, 3].
The treatment of leishmaniasis in any of its clinical forms is
aimed at the eradication of amastigotes, the reduction and
healing of lesions, and to prevent the spread of the parasite.
Pentavalent antimony compounds such as sodium
stibogluconate (Pentostam®) or meglubin antimoniate
(Glucan-time®) are the first-line treatment against this disease
since 1959 [4, 5]. This type of compound has drawbacks as-
sociated to its high toxicity, the persistence of side effects,
prolonged treatments that favor the emergence of resistant
strains, and parenteral administration. For these reasons, other
therapeutic alternatives such as pentamidine or amphotericin B
have been used, which have serious and irreversible toxic effects
[6]. Additionally, accessible pharmacological treatments for the
majority of neglected tropical diseases (NTDs) similar to leish-
maniasis are restricted in terms of cost, and resistance phenom-
ena frequently increase [1, 2, 7–9]. Therefore, development of
new drugs against Leishmania is an open area of investigation
and remains critically important to reduce most human deaths
attributable to these diseases [10]. Molecules with quinolinic or
hydroxyquinoline nuclei from natural sources or also obtained
by synthetic methods have been shown to have important
leishmanicidal activity [11]. In the same way, styrylquinolines
have shown a broad spectrum of biological activities, among
which are their ability to inhibit the replication of the HIV virus,
antiproliferative, antifungal, and anti-protozoal activity [12, 13].
For their part, polyhydroxylated styrylquinolinines have shown
a great capacity to inhibit transcriptase and integrase, enzymes
necessary for the replication of viral cycles.
In this work, we report the synthesis of 8 new
styrylquinolines obtained by a condensation reaction type
Perkin between quinaldine (1–4) or 8-hydroxyquinoline (5–
8) with the appropriate aromatic aldehydes [14, 15]: 2 - [(E) -
2- (2 -acetyloxy-4-methoxyphenyl) ethenyl] quinoline (1), 2 -
[(E) -2- (2-acetyloxy-5-nitrophenyl) ethenyl] quinoline (2), 2 -
[(E) -2- (3 -methoxyphenyl) ethenyl] quinoline, (3) 2 - [(E) -2-
(4-methoxyphenyl) ethenyl] quinoline (4), (E) -2- [2- (8-
hydroxyquinolin-2-yl) ethenyl] -5-methoxyphenylacetate (5),
(E) -2-ethoxy-4- [2- (8-hydroxyquinolin-2-yl) ethenyl]
phenylacetate (6), 2 - [(E) -2- (4- methoxyphenyl) ethenyl]
quinolin-8-ol (7), and 2 - [(E) -2- (3-methoxyphenyl) ethenyl]
quinolin-8-ol (8); yields were 34 to 62%. The compounds
obtained were isolated, purified, and characterized by chro-
matographic and nuclear magnetic resonance techniques, re-
spectively. Synthesized compounds were evaluated for
leishmanicidal activity on intracellular amastigotes of
Leishmania (Viannia) panamensis by flow cytometry. The
amastigotes of this protozoon were used as a model to test
leishmanicidal activity. Under this outline, the use of compu-
tational tools has allowed offering useful information to inter-
pret trends and state structure-activity relationships and con-
duct a systematic study of protein-drug interactions,
Struct Chem
identifying meaningful intermolecular characteristics. For this
purpose, following the protocols previously published by
Andricopulo, Muskus, Brindisi, and coworkers [1, 3, 16],
we select Leishmania protein targets, broadly used to strongly
bind the most promising molecules synthetized in this work.
Experimental section
Synthesize of styrylquinolines
The styrylquinolines were obtained from 8-hydroxyquinoline
through Perkin-type condensation reaction by using high-
purity reagents in acetic anhydride (Ac2O). The general syn-
thetic procedure is the following: A solution of 8-
hydroxyquininoline was added to 6 mL of acetic anhydride.
The mixture was stirred for 1 h without heating, and then a
stoichiometric excess of 3-ethoxy-4-hydroxybenzaldehyde
was added. The reaction mixture was refluxed for 8 to 12 h
at 180 °C, monitoring periodically the progress of the reaction
by the countercurrent distribution method (CCD) to verify
precursor consumption and formation of the product. Upon
completing the reaction, the mixture was left to dry at room
temperature, and then enough amounts were added of a satu-
rated solution of NaHCO3 until hydrolyzing the remnant
Ac 2 O. The mixture was extracted with three portions
(60 mL) of benzine mixture: ethyl acetate (1: 2), the organic
phase was dried using anhydrous Na2SO4, filtered, and con-
centrated with reduced pressure. Finally, the raw product was
purified by column chromatography using PB:EtOAc as elu-
ent with increasing polarity gradient and silica gel 60 F254
(Merck) as stationary phase. In Scheme 1, it is possible to see
an example of the synthesis reaction.
Biological activity
Cells and culture conditions
The U-937 promonocytes (CRL-1593.2™) (American Type
Culture Collection, Manassas, VA, USA) were cultivated in
complete medium containing RPMI-1640 (Sigma) enriched
with 10% fetal bovine serum (FBS) (Gibco, Life
Technologies, Grand Island, NY, USA) and 1% antibiotics
(100 U/ml of penicillin and 0.1 mg/ml of streptomycin)
(Sigma). The cells were under standard culture conditions at
37 °C, 5% CO2 with medium change every 72 h until use.
Cultivation of L. (V) panamensis
A virulent L. (V) panamensis (UA140-pIReGFP), previously
obtained from a hamster lesion aspirate, was used. Parasite
was maintained as promastigotes in biphasic culture NNN
(Novy-MacNeal-Nicholle) medium and PBS enriched with
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Struct Chem
Scheme 1 Illustration of synthesis of compound 6 presented in this work
glucose, pH 6.9. The cultures were incubated at 26 °C. The
environment was modified every week at maximum for
2 months. Parasites in the stationary phase of growth were left
by 6-day in culture.
In-vitro antileishmanial activity on amastigotes of L. (V)
panamensis
To evaluate the effectiveness on intracellular amastigotes, the
strain of Leishmania (Viannia) panamensis UA140-pIReGFP
was used. U937 cells at a concentration of 300.00 cells/mL
maintained in RPMI 1640 at 10% SFB medium with 0.1 μg/
mL of forbolmeristate acetate (PMA) in 24-well cell culture
dishes were infected with promastigotes in the stationary
phase of growth in one 15:1 parasite ratio: cell, for 3 hours.
Infected cells were incubated with the corresponding concen-
tration of the compounds to be evaluated as well as
amphotericin B, a substance used as a control of effectiveness.
For all compounds and control, 4 concentrations were evalu-
ated from the previously obtained LC50. After 72 h of incu-
bation in the presence of the compounds and the control, the
effect of these substances on the viability of intracellular
amastigotes will be determined by means of flow cytometry
reading at 488 nm excitation and 525 nm emission with a laser
of Argon. The tests were performed twice with 3 replicates for
each concentration evaluated. Leishmanicidal activity is
expressed as effective concentration 50 (EC50) which was
calculated using the Probit method [17].
The cytotoxicity was evaluated on the U-937 cell lines
which were used in the exponential phase of growth and were
adjusted to a concentration of 1 × 106 cells/mL in 96-well cell
culture dishes in RPMI-1640 medium with 10% SFB and the
corresponding concentration of the compounds to be evaluat-
ed as well as amphotericin B, which was used as a cytotoxicity
control [18, 19]. For all compounds and the control, 6 double
serial concentrations from 200 μg/mL were evaluated. After
72 h of incubation in the presence of the compounds and the
control, their effect on the viability of the cells was determined
using the MTT method on a 570-mn spectrophotometer. The
tests will be performed twice with 3 replicates for each con-
centration evaluated. Cytotoxic activity was expressed as
Lethal Concentration 50 (LC50) which was calculated by
the Probit method. Values are expressed as mean ± SD. Data
were tested by a two-way ANOVA, considered significant
differences when P < 0.05. A rigorous statistical analysis
was to carry with Prism 6.0 (GraphPad Prism, San Diego,
CA, USA) [20].
Computational studies
ADMET prediction
ADMET properties of a compound deal with its absorption,
distribution, metabolism, excretion, and toxicity in and
through the human body. ADMET, which constitutes the
pharmacokinetic profile of a drug molecule, is very essential
in evaluating its pharmacodynamic activities. In this study, we
have used the SwissADME prediction tool [21], for in silico
physicochemical properties such as molecular hydrogen bond
acceptor (HBA), hydrogen bond donor (HBD), weight (MW),
topological polar surface area (TPSA), rotatable bond count
(RB), octanol/water partition coefficient (LogP), water solu-
bility (LogS), and skin permeation (logKp).
Molecular modeling and quantum chemistry
calculations
Molecular docking
A computational protein-ligand docking approach was used to
analyze structural complexes of the L. major dihydroorotate
dehydrogenase (LmDHODH) [22, 23] and L. major
tryparedoxin peroxidase I (LmTXNPx) [16] with selected mol-
ecules as potential inhibitors. We used the binding energies as
a selection criterion. Thus, compounds that exhibited the most
negative values were selected, and this agrees with the litera-
ture [3]. In this way, two L. major crystals were used for
validation.
The binding sites of LmDHODH and LmTXNPx inhibitors
have been characterized based on structural information de-
rived from ligand position in proteins co-crystallized with a
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Struct Chem

Table 1 Structures of 2-styrylquinolines (1–8) synthetized in this work*

Styrylquinolines

*Compounds 1, 2, 3, 4: R1 = H; compounds 5, 6, 7, 8: R1 = − OH. The appropriate aromatic aldehydes used can be seen in the supporting information
(Table S1)
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Struct Chem

bound ligand. Literature information was useful to determine
active site residues for these structures.
The binding sites were characterized using the co-
crystallized protein with a bound ligand, and these sites
were compared with the available literature [1, 3, 16,
22–24], showing a great correspondence with them.
Simulations were performed with AutoDock (v 4.2.1)
a n d A u t o D o c k V i n a ( v 1 . 0 . 2 ) [ 2 5 ] . T h e th r e e -
dimensional coordinates used have been obtained from
the PubChem database (http://pubchem.ncbi.nlm.nih.gov)
and energy optimized using MOPAC2016 [26] software
by PM6 semiempirical method [27] with RMS gradient of
0.001 kcal/mol. When the structures of ligands is not
available from database, then they are drawn using
Discovery Studio 3.1 (Accelrys, CA) [28]. The ligand
files were prepared using the AutoDock-Tools package
[29] provided by AutoDock by accepting all rotatable
bonds; moreover, the atomic charges are computed toward
the Gasteiger [30] procedure, and nonpolar hydrogen
atoms are merged. The crystal structure of LmDHODH
(PDB Code: 3MJY) and LmTXNPx (PDB Code: 4K1F)
was downloaded from the Protein Data Bank [31].
Schrödinger’s Protein Preparation Wizard [32] was used to
adding polar hydrogen atoms, where nonpolar hydrogen
atoms were merged and charges assigned. The proteins during
the docking procedure was treated as rigid bodies in order to
reduce the search space for optimal structures. This protocol
was implemented with Schrödinger’s Maestro suite [33].
Noncovalent interactions
Noncovalent interaction index (NCI) [34, 35] is an appro-
priate model for the visualization of areas where weak
interactions predominate, the origin of which may be
dispersive in nature, hydrogen bridges, dipole-dipole in-
teractions, or repulsive steric effects, based on the reduced
density gradient (s), which comes from the electronic den-
sity and its first derivative and is expressed as:
1 ∇ρ
s¼ ð1Þ
2ð3π2 Þ1=3 ρ4=3
This parameter can be described qualitatively using
color representations, which are based on the sign of the
second eigenvalue (λ2) of the electron density matrix
(Hessian matrix). Thus, the red color represents strong
repulsive interactions (steric repulsion, λ > 0), the blue
color represents a strong attractive interaction (Hydrogen
bonds, λ < 0), and the green color represents weak attrac-
tive interactions (van der Waals interactions, λ = 0). The
NCI was evaluated using the NCIPLOT program [34].
Molecular visualization of the systems was carried out
with the VMD software package [36].
A complementary approach was performed using the
Independent Gradient Model (IGM) analysis conducted by
using the IGMPlot code [37] with promolecular electronic
density. Within the IGM approach, the descriptor δg identifies
the net ED gradient attenuation due to interactions resulting
from the electron sharing between fragments [38]. Plotting
δginter isosurfaces in real space enables a visual understanding
of the noncovalent forces between these fragments. In addi-
tion to identifying the interactions, the δg index can be inte-
grated to quantify the electron sharing interaction through a
Δg score, which measures the ligand-pocket binding at a
moderate computational cost. The δginter isosurfaces are gen-
erally colored on a Blue Green Red (BGR) color scale accord-
ing to the density value oriented with the sign of λ2 (second
eigenvalue of the electronic density hessian previously
mentioned.)

Table 2 In silico predicted physicochemical properties of all compounds 1–8

1 2 3 4 5 6 7 8

Log P 4.38 4.28 4.46 4.37 4.09 4.48 4.16 4.16

MW (g/mol) 319.35 335.35 306.32 261.32 335.35 349.38 277.32 277.32
TPSA (Å2) 48.42 68.65 42.35 42.35 22.12 68.65 88.85 22.12
HBA 4 6 5 2 5 5 3 3
HBD 1 1 1 1 2 2 2 2
RB 5 5 3 3 3 5 5 3
Log S − 4.95 −5 − 4.81 − 4.71 − 4.65 −5 − 5.16 − 4.65
log Kp (cm/s) − 5.25 − 5.61 −5 − 5.07 − 4.72 − 5.61 − 5.78 − 4.72
Molar refractivity(MR) 94.87 94.98 83.56 90.22 96.54 101.16 85.23 85.23
N° Violation 0 0 0 0 0 0 0 0

MW = 150–500 g/mol; TPSA = 20 Å2 –130 Å2 ; HBA = N° of H-bond acceptors ≤ 10; HBD = N° of H-bond donor ≤ 5; RB = 0–9; Log S = Insoluble < −
10 < Poorly < − 6 < Moderately < − 4 < Soluble < − 2; Log P ≤ 5; log Kp ≥ −2.5 considered to be permeable; N° Violations of Lipinski, Ghose, Veber,
Egan and Muegge rules
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Struct Chem

Table 3 In vitro activity of tested compounds against intracellular amastigotes of Leishmania (Viannia) panamensis*

Compound CL50 (μg/mL) U-937 % inhibition EC50 (μg/mL) SI

Amastigotes intracellular

1 6.1 ± 0.4 61.8 ± 0.6 1.8 ± 0.2 3.4

2 5.9 ± 0.5 74.9 ± 4.1 2.5 ± 0.4 2.4
3 0.8 ± 0.2 5.5 ± 0.9 4.7 ± 0.2 0.1
4 54.1 ± 3.1 40.9 ± 5.9 4.2 ± 0.2 12.8
5 > 200 16.9 ± 3.5* >6 –
6 2.8 ± 0.3 65.7 ± 6.5** 0.2 ± 0.0 14
7 2.1 ± 0.4 19.4 ± 2.3** 5.1 ± 0.3 3.8
8 3.4 ± 0.3 49.5 ± 5.5*** 4.1 ± 0.2 12
Amphotericin B 52.5 ± 3.6 62.7 ± 1.4**** 0.05 ± 0.1 1050

*EC50, effective concentrations; CL50, median lethal concentration; IS, selectivity index (SI = CL50/CE50)

Results and discussion inhibition percent on intracellular amastigotes of

Chemistry and biological evaluation
All synthesized compounds and their respective precursors are
show in Table 1. All the compounds were isolated in good
manner and spectroscopically characterized (section 1 in the
Supporting Information).
In order to have a preliminary idea about the drug-likeness
behavior of potential molecular targets studied, Gelovani’s
and Lipinski’s predictions were performed [39, 40]. The re-
sults can be seen in Table 2. These rules predict the probability
of success or failure of a certain drug, for example, according
to Lipinski’s rules molecules that complying at least two of the
following aspects [41]: (1) number of H-bond donors ≤ 5, (2)
molecular mass (MW) ≤ 500, (3) number H-bond acceptors ≤
10, (4) high lipophilicity (CLogP < 5), and (5) molar
refractivity(MR) between 40 and 130. These characteristics
are of great help in preclinical development, saving time and
money. According to Table 2, it is observable that the poten-
tial candidates are within the range of expected values for the
Lipinski’s and Gelovani’s parameters. Therefore, in the case
of passing the corresponding clinical phases, the bioavailabil-
ity and absorption will not be poor.
The next step of this work was to evaluate the cytotoxic
activity and in vitro activity. In vitro analysis was evaluated in
intracellular amastigotes of L. panamensis. While the cytotox-
ic activity was tested in human macrophages U-937, this was
expressed as median lethal concentration (CL50, mean ±
SEM), SEM means standard error of the mean. Data were
represented as mean ± SD, and statistical differences were
significant at P < 0.05. The results obtained are shown in
Table 3.
From Table 3, it is clear that compounds 1 (EC50 1.8 μg/
mL), 2 (EC50 2.5 μg/mL), and 6 (EC50 0.2 μg/mL) exhibited
the highest in vitro activity against the tested cell lines, with
CL50 (μg/mL) U-937 over 6.1, 5.9, and 2.8 and higher
L. panamensis. Moreover, the compounds 3, 4, 5, 7, and 8
exhibited strong cytotoxic effects against cell lines evaluated.
However, according to Lipinski, Ghose, Veber, Egan, and
Muegge parameters, these compounds could be useful in an-
other kind of target. At this point, we will proceed with the rest
of the discussion with the potential candidates for Leishmania
panamensis. Indeed, for the mentioned compounds, there is a
great correlation between % inhibition amastigotes intracellu-
lar and CL50 (μg/mL) U-937(R2 = 0.97), for the most prom-
ising compounds. Therefore, we select Leishmania protein
targets to strongly bind the most promising molecules previ-
ously mentioned.
Molecular docking analysis
In the literature, there are interesting studies of molecular
docking of quinoline derivatives that include studies of
leishmanicidal activity [42]. Based on this evidence, calcula-
tions were performed to estimate the interactions of two in-
hibitors quite described in the literature as constituting the
L. major dihydroorotate dehydrogenase DHODH
(LmDHODH) and L. major tryparedoxin peroxidase TXNPx
(LmTXNPx) [1].
It is well-known in the scientific community that
flavoenzyme dihydroorotate dehydrogenase act efficiently as
a good target for parasites responsible for many neglected
diseases (ND), especially leishmaniasis because most of its
components are essential to parasite survival. Additionally,
this enzyme is absent in the host. In this sense, the defense
of these parasites against reactive oxygen species (ROS) con-
stitute the enzymes involved in metabolism such as
trypanothione peroxidase and trypanothione reductase, where
the latter is essential for the survival of the parasite [16]. In
addition, dihydroorotate dehydrogenase catalyzes the fourth
reaction of the de novo pyrimidine, which in turn plays an
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Struct Chem
Fig. 1 Docking analysis for ligands (a) molecule 1, (b) molecule 2, and
(c) molecule 6 bound to LmDHODH. (I) The surrounding amino acid
residues in the binding pocket of LmDHODH within 3 Å from the com-
pounds. (II) Two-dimensional interaction map of the molecules and
LmDHODH. The arrows indicate potential interactions between amino
acid residues and compounds under study. (III) NCI plot graphs of
important function in the cells, especially within DNA and
RNA biosynthesis [43].
Leishmania TXNPx is an excellent pharmacological target,
since trypanothione-dependent metabolism has allowed the
identification of several compounds as potential enzyme in-
hibitors; this is because it is a homodimer whose active site is
formed by the N-proximal peroxidative cysteine (Cp), which
is well described by Brindisi and coworkers [16, 44].
The analysis of the regions S1–S5 is useful to design new
specific inhibitors of the parasite. The folding and activity of
LmDHODH depend on the binding of the flavin cofactor;
thus, the S1 subsite interacts with orotate substrate and is
conserved within the inhibitor (DHODH), next to S1 is the
cavity of S2, which contains active amino acids within the
noncovalent interactions on the structure of LmDHODH. The red color
represents strong repulsive interactions (steric repulsion, λ > 0), the blue
color represents a strong attractive interaction (Hydrogen bonds, λ < 0),
and the green color represents weak attractive interactions (van der Waals
interactions, λ = 0)
loop (a4-bA) [22, 23]. It was corroborated that the S2 subsite
is essential for the activity of the LmDHODH enzyme, but this
could be improved by exploiting the S3 to S5 subsites that are
more internal to the protein and show some conformational
Table 4 ΔG° of binding for styrylquinolines, obtained from docking
analysis with DHODH and TXNPx
Compound/ 1 2 3 4 5 6 7 8
target
DHODH − 7.2 − 7.9 − 7.4 − 7.0 − 7.6 − 7.5 − 8.1 − 7.7
TXNPx − 6.7 − 7.5 − 7.2 − 6.5 − 6.6 − 6.8 − 7.5 − 6.8
*All energies are in kcal/mol
 Author's personal copy
Fig. 2 Docking analysis for ligands (a) molecule 1, (b) molecule 2, and
(c) molecule 6, bound to LmTXNPx. (I) The surrounding amino acid
residues in the binding pocket of LmTXNPx within 3 Å from the com-
pounds under study. (II) Two-dimensional interaction map of molecules
and LmTXNPx. The arrows indicate potential interactions between amino
acid residues and styrylquinolines. (III) NCI plot graphs of noncovalent
differences [3]. The coupling modes were obtained for
LmDHODH. The binding sites are illustrated in Fig. 1.
These coincide with those previously reported by the scientific
community, which are the S1 and S2 sites, this finish validat-
ing the protocol used in this work [3, 22, 23, 45]. Additionally,
the potential inhibitors evaluated here interact with the before
mentioned pockets, through electrostatic and hydrophobic in-
teractions with the following S1 residues (Lys44, Asn68,
Met70, Gly71, Leu72, Asn128, Asn195, Ser196), S2
(Leu72, Ser100, Leu102, Asn107, Asn128, Ser130, Gln139,
Val140), and other residues (Ser103, Met104, Cys150) not
present at sites S3 to S5. In the case of compound 1, it presents
hydrophobic interactions. In the case of molecules 2 and 6
have hydrogen-bonding interactions, respectively. For com-
pound 2, (i) it happens between the oxygen atom at position
1 of the nitro group with Lys44 (site S1) and (ii) oxygen atom
at position 1, =O of nitro group with Leu72 (site S1-S2), and
(iii) between the oxygen atom at position 2, -O- of nitro group
with Asn68 (site S1). For compound 6, (i) it occurs between
Struct Chem
interactions on the structure of LmTXNPx. The red color represents
strong repulsive interactions (steric repulsion, λ > 0), the blue color rep-
resents a strong attractive interaction (Hydrogen bonds, λ < 0), and the
green color represents weak attractive interactions (van der Waals inter-
actions, λ = 0)
the carbonyl group with Lys44 (site S1) and (ii) carbonyl
group with Leu72 (site S1–S2). At this point, it is possible
to visualize in principle that the interactions that play a pre-
dominant role are hydrophobic forces as well as hydrogen
bonds. Finally, the calculated interaction Gibbs free energies
(ΔGbind) are − 7.2, − 7.9, and − 7.5 kcal·mol-1, for the 1-
LmDHODH, 2-LmDHODH, and 6-LmDHODH complexes,
respectively (Table 3).
As mentioned above, LmTXNPx has been considered a
molecular target in ligand-based drug design studies since it
reduces hydroperoxides originated by contaminated macro-
phages [1, 16]. LmTXNPx is known to catalyze TS2-
dependent peroxide detoxification. This type of mechanisms
is useful for the design of new drugs since they are unique to
the parasite and necessary for its survival [24]. Furthermore,
the C-terminal tail (residues 169–199), which does not have
LmTXNPx (PDB Code: 3TUE) [24], is visible in LmTXNPx
(PDB Code: 4K1F). This allows the active site of the protein
to be fully explored.
 Author's personal copy
Struct Chem
Fig. 3 IGM analysis of
noncovalent interactions with a
0.3 a.u. isosurface and a BGR
color code in the range − 0.1 < ρ
sign (λ2) < 0.1 a.u. for
LmDHODH and LmTXNPx
The results obtained for the coupling modes at the
LmTXNPx active site can be seen in Fig. 2 and Table 4. ΔG°
of binding for styrylquinolines does not have meaningful dif-
ferences between them. However, as previously set, the cyto-
toxicity assays confirm that the better molecules are the 1, 2,
and 6. Thus, the binding site of 1, 2, and 6 in the LmTXNPx
coincides with those previously reported by another works
[16, 46]. Analogous results were obtained with LmDHODH,
where the three inhibitors are perfectly inserted into the pock-
et. The trend continues. Therefore, these interactions are of the
hydrophobic and electrostatic type with the following resi-
dues: Pro45(A), Leu46(A), Thr49(A), Phe50(A), Val51(A),
Cys52(A), Glu120(A), Ser122(A), Gln123(A), Val125(A),
Arg128(A), Met147(A), Pro148(A), Asn9(B), Val172(B),
and Pro186(B) residues, where A and B are protein chains.
In the case of 1, it presents hydrogen-bonding interaction,
between the carbonyl group with Asn9(B). It is noteworthy
again that the molecules 2 and 6 have hydrogen-bonding in-
teractions, respectively. For 2, (i) it happens between the ox-
ygen atom at position 1, =O of nitro group with Val51(A), and
(ii) oxygen atom at position 2, -O- of nitro group with
Arg128(A). For 6, (i) it occurs between the carbonyl group
with Phe50(A), (ii) between the carbonyl group with
Val51(A), and (iii) e carbonyl group with Arg128(A). The
calculated interaction Gibbs free energies (ΔG) are − 6.7, −
7.5 and − 6.8 kcal·mol−1, for the 1-LmTXNPx, 2-LmTXNPx,
and 6-LmTXNPx complexes (see Table 3). In general, all the
molecules evaluated were promising in both targets according
to the experimental and theoretical results. Remarkably, the
best potential inhibitors interaction energy is the molecule 2,
which was identified as the most effective in leishmanicidal
activity experiments.
 Author's personal copy
The NCI results are presented in Figs. 1 and 2, where it is
possible to see the preferential interaction between
styrylquinolines with the surrounding amino acid residues of
LmDHODH and LmTXNPx targets. Undoubtedly, the interac-
tions are predominantly dominated by weak interactions such
as London dispersion interactions that is part of the Van der
Waals potential (attractive parameter), as well as hydrogen
bonding; all this kind of interactions represent an important
function in the stabilization of protein-drug system.
IGM analysis
IGM analysis provides us a more quantitative identification
about the main interactions responsible of the ligand accom-
modation inside the protein pocket (see Figure 3). In the case
of LmDHODH complexes, compound 1, it presents London
interactions through its methoxy group and quinolone moie-
ties. In the case of molecule 2, it exhibits three hydrogen
bonds involving the nitro group, whereas molecule 6 estab-
lishes two hydrogen bonds through its acetoxy group. More in
detail, the nitro group of compound 2 interacts with Lys44
(site S1), Leu72 (site S1–S2), and Asn68 (site S1). For com-
pound 6, the hydrogen bonds involve Lys44 (site S1) and
Leu72 (site S1–S2). The Δg score for the three systems are
4.10, 3.83, and 5.25 for 1, 2, and 6, respectively, suggesting a
more favorable binding situation in the last case. A similar
situation was evidenced for the LmTXNPx complexes, with
the corresponding residues described in the docking analysis.
In these systems, Δg scores of 4.10, 5.63, and 5.77 obtained
for 1, 2, and 6, respectively, suggest that the best pocket-
ligand pair corresponds to compound 6. In general, all the
molecules evaluated were promising in both targets according
to the experimental and theoretical results.
Therefore, the better inhibitors character is the result of
direct mechanisms.
Conclusion
The synthesis, cytotoxicity, and activity against L. (V)
panamensis of 8 new ring-substituted 8 ring-substituted
styrylquinolines are reported. The compounds were obtained
by Perkin-type condensation reaction. Three of them were
really promising against L. (V) panamensis. The possible
binding modes of potential inhibitors in the L. major
dihydroorotate dehydrogenase DHODH (LmDHODH) and
L. major tryparedoxin peroxidase TXNPx (LmTXNPx) were
calculated. The potential Leishmania candidates are within the
range of expected values for the Lipinski’s and Gelovani’s
parameters. Noncovalent interaction index (NCI) and IGM
analysis were used to evaluate intermolecular interactions. It
is clear from these analyses that ligand encapsulation is ruled
by London interactions as well as hydrogen bonding. Our
Struct Chem
theoretical results are in full agreement with the experimental
data. Therefore, the better inhibitors character is the result of
direct mechanisms, like hydrogen bonds, steric repulsion, and
van der Waals interactions. Finally, all the results suggest that
these compounds have potential as templates for drug devel-
opment against Leishmania (Viannia) panamensis. The eval-
uation in another kind of protozoan parasites of the genus
Leishmania will be the target of further studies.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
interest.
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