In vitro evaluation of antitrypanosomal activity and molecular docking of benzoylthioureas

Article abstract of DOI:10.1016/j.parint.2020.102225

A series of sixteen benzoylthioureas derivatives were initially evaluated in vitro against the epimastigote form of Trypanosoma cruzi. All of the tested compounds inhibited the growth of this form of the parasite, and due to the promising anti-epimastigot

Full text of DOI:10.1016/j.parint.2020.102225

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In vitro evaluation of antitrypanosomal activity and molecular
docking of benzoylthioureas
Patricia M.L. Pereira, Priscila G. Camargo, Bruna T. Gonçalves,
Luiz A.P. Flores-Junior, Luiza R.S. Dias, Camilo H.S. Lima,
Phileno Pinge-Filho, Lucy M.Y. Lioni, Sueli F. Yamada-Ogatta,
Marcelle L.F. Bispo, Fernando Macedo
PII:
S1383-5769(20)30175-6
DOI:
https://doi.org/10.1016/j.parint.2020.102225
PARINT 102225
Reference:
To appear in:
Parasitology International
Received date:
Accepted date:
30 June 2020
12 October 2020
Please cite this article as: P.M.L. Pereira, P.G. Camargo, B.T. Gonçalves, et al., In vitro
evaluation of antitrypanosomal activity and molecular docking of benzoylthioureas,
Parasitology International (2019), https://doi.org/10.1016/j.parint.2020.102225
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IN VITRO EVALUATION OF ANTITRYPANOSOMAL ACTIVITY AND
MOLECULAR DOCKING OF BENZOYLTHIOUREAS
Patricia M.L. Pereiraa, Priscila G. Camargob, Bruna T. Gonçalvesa, Luiz A. P. Flores-
Juniorc, Luiza R. S. Diasc, Camilo H. S. Limac,d, Phileno Pinge-Filhoe, Lucy M. Y.
Lionia, Sueli F. Yamada-Ogattaa, Marcelle L. F. Bispob,*, Fernando Macedo Jrb.
aLaboratório de Biologia Molecular de Microrganismos, Departamento de Microbiologia, Centro de
Ciências Biológicas, Universidade Estadual de Londrina, Londrina, Paraná, Brazil.
bDepartamento de Química, Centro de Ciências Exatas, Universidade Estadual de Londrina, Londrina,
Paraná, Brazil.
cLaboratório de Química Medicinal, Faculdade de Farmácia, Universidade Federal Fluminense, Niterói,
Rio de Janeiro, Brazil.
dDepartamento de Química Orgânica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio
de Janeiro, Brazil.
eLaboratório de Imunopatologia Experimental, Departamento de Ciências Patológicas, Centro de Ciências
Biológicas, Universidade Estadual de Londrina.
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Abstract
A series of sixteen benzoylthioureas derivatives were initially evaluated in vitro
against the epimastigote form of Trypanosoma cruzi. All of the tested compounds
inhibited the growth of this form of the parasite, and due to the promising anti-
epimastigote activity from three of these compounds, they were also assayed against the
trypomastigote and amastigote forms. ADMET-Tox in silico predictions and molecular
docking studies with two main enzymatic targets (cruzain and CYP-51) were performed
for the three compounds with the highest activity. The docking studies showed that
these compounds can interact with the active site of cruzain by hydrogen bonds and can
be coordinated with Fe-heme through the carbonyl oxygen atom of the CYP51. These
findings can be considered an important starting point for the proposal of the
benzoylthioureas as potent, selective, and multi-target antitrypanosomal agents.
Keywords: Chagas disease, CYP51, cruzain, Trypanosoma cruzi, molecular docking.
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1. Introduction
American trypanosomiasis or Chagas disease is a neglected tropical illness
caused by the protozoan parasite Trypanosoma cruzi. Although still endemic in Latin
America, the presence of Chagas disease in other continents can be related to
immigration and the globalization events, which have changed its epidemiological
profile. According to the World Health Organization (WHO), about 8 million people are
infected with this protozoan worldwide, and more than 10000 deaths occur each year
[1,2].
The T. cruzi life cycle alternates between the insect vector and the host
mammalian. The natural human infection results from the bites of infected
hematophagous reduviid vectors which release the non-proliferative metacyclic
trypomastigotes into their excreta (feces and urine) close to the injured skin or intact
mucosal membranes [3,4]. In host cells, metacyclic trypomastigotes differentiate into
replicative amastigote forms [5], which multiply by binary fission. The intracellular
amastigote forms in high density transform into trypomastigotes, which can invade new
cells or are released into the bloodstream, reaching other organs [6]. The blood-
circulating parasites can serve to complete the life cycle when they are taken up by the
insect vector during its blood meal. In this invertebrate host, bloodstream
trypomastigotes differentiate into the proliferative epimastigotes, which multiply in the
midgut, also by binary fission. Finally, epimastigotes migrate and adhere to the rectal
epithelium and differentiate into metacyclic trypomastigotes [4]. T. cruzi can also be
transmitted by contaminated food, blood transfusion, organ transplantation, and
vertically from mother to newborn [7].
Overall, the clinical course of Chagas disease comprises acute and chronic
phases. The acute phase is generally asymptomatic or presents non-specific symptoms
[8]. This phase is characterized by high parasitemia and lasts 4-8 weeks; if untreated,
patients remain chronically infected and around 30-40% can develop organ involvement
such as cardiomyopathy and megaesophagus and/or megacolon after 10-30 years of
infection [7]. Progression of heart failure accounts for the most of Chagas disease
morbidity and related mortality [9]. The etiologic treatment for Chagas disease is
restricted to two nitroheterocyclic drugs introduced in the 1960-1970s: Benznidazole
and Nifurtimox [10]. Both are effective only in the acute or early chronic phases,
require prolonged treatment durations, have mutagenic potential [11] and exhibit a wide
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range of side effects, which lead to discontinuation of therapy by patients [12]. In
addition, there is no vaccine for the prevention of Chagas disease [13] and efforts are
focused on controlling transmission, which impulses to the search of new
antitrypanosomal agents with low toxicity and greater efficiency [14].
Due to its great synthetic and biological versatility, thiourea plays an important
role in drug discovery since thiourea-containing compounds have a wide range of
biological activities, such as antitumoral [15], anti-diabetes [16], anti-adipogenesis[17],
antibacterial [18], antifungal [19] and antiviral [20]. Furthermore, there are reports in
the literature indicating that thiourea scaffold could represent a promising
pharmacophore in the search for new therapies against protozoal diseases, such as
malaria [21], leishmaniasis [22] and Chagas disease [23]. Considering this huge
potential, our research group has been investigating the antimicrobial potential of a
series of benzoylthiourea, which showed interesting results against Mycobacterium
tuberculosis (minimal inhibitory concentration = 423.1 - 9.6 μM)[24] and against other
species of bacteria and fungi (unpublished data). Owing to these exciting results and
supported by the literature reports on antiprotozoal activity of thiourea derivatives, we
decided to investigate the antitrypanosomal activity of this class of compounds.
Therefore, in this work we described the in vitro evaluation of sixteen benzoylthiourea
derivatives (1a-p, Figure 1) against epimastigote forms of T. cruzi as well as the
cytotoxic evaluation in mammalian cells. Thereafter, a preliminary structure-activity
relationship (SAR) study was conducted to determine the important structural features
of this series of compounds for the biological activity and also to find out the hit
compounds. The most promising compounds were then selected to be assayed against
the infective trypomastigote and the intracellular replicative amastigote forms of T.
cruzi. Finally, molecular docking studies were carried out in two important molecular
targets of T. cruzi, cruzipain (CRZ) and C14α-sterol demethylase (CYP51) with the aim
to demonstrate possible molecular mechanisms of action of these compounds.
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Figure 1. Experimental approach to evaluate the antitrypanosomal potential of benzoylthioureas (1a-p).
2. Experimental section
2.1 Cytotoxicity to mammalian cells
LLCMK2 (kidney epithelial cells of Macaca mulatta, CCL-7, ATCC, USA) cell
line was maintained in Dulbecco′s Modified Eagle′s medium (DMEM) supplemented
with 10% fetal bovine serum (FBS, Invitrogen), 2 mM L-glutamine, 100 IU/mL
penicillin, 100 µg/mL streptomycin, 1% tylosin and 5% CO2 at 37°C. Peritoneal
macrophages were obtained from male BALB/c mice (6 to 8 weeks old). 5%
thioglycolate (1.0 mL) was injected intraperitoneally into the animals, and 4 days later,
the macrophages were harvested by washing the peritoneal cavities with sterile and cold
0.1 M phosphate-buffered saline (PBS), pH 7.2. The cytotoxicity of all benzoylthioureas
were evaluated on LLCMK2 cells; and the toxicity of lead compounds were also
evaluated on peritoneal macrophages. For both mammalian cells, 2.5 x 105 cells/mL
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were cultured into 96-well plate for 24 h [25]. Non-adherent cells were removed by
washing with sterile PBS. The medium containing different concentrations of
compounds (3.12 - 400 µg/mL) was added to each well and the plates were incubated
for further 48 h. Cells cultured in growth medium alone were used as controls. Cell
viability was determined by the reduction of the tetrazolium salt (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - MTT (Merck) method
according to the manufacturer’s recommendation. The 50% cytotoxic concentration
(CC50) that corresponds to the concentration of compounds needed to inhibit the viable
cells up to 50% was calculated by regression analysis. The selectivity index (SI) was
calculated using the equation: SI = CC50/IC50,where IC50 corresponds the minimal
concentration of the compounds that causes 50% of antitrypanosomal activity [25]. This
study protocol was approved by the Ethics in Animal Utilization Committee (CEUA) of
the Universidade Estadual de Londrina (UEL), Paraná, Brazil (CEUA/UEL number
15516.2018.20).
2.2 Trypanosoma cruzi
Trypanosoma cruzi Y strain was used in all assays. Epimatigotes were maintained
by weekly transfers in liver infusion tryptose (LIT) medium [26] supplemented with
10% of heat inactivated FBS at 28°C; and cells obtained from 4-day incubations were
used in all assays. Trypomastigotes were harvested by centrifugation (850 x g for 5 min)
from the supernatant of infected LLCMK2 cells cultured in 25 cm2 flasks at 37°C after
120 h post-infection.
2.3 Effect on growth of epimastigotes
Epimastigotes (1.0 x 106 cells/mL) in exponential growth phase were inoculated
into 24-well plate containing LIT-FBS medium with different concentrations of
compounds (6.25 - 100 µg/mL). The cultures were incubated at 28°C and cell growth
was estimated by direct counting in a hemocytometer (Improved Double Neubauer)
after 72 h. Wells containing growth medium alone and medium plus 1% DMSO were
used as controls. The results were expressed as the minimal concentration of the
compounds that causes 50% (IC50Epi) of growth inhibition compared to the untreated
control.
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2.4 Effect on viability of trypomastigotes
Trypomastigotes (1.0 x 107 cells) were incubated in DMEM supplemented with
10% FBS containing different concentrations of compounds (6.25 – 100 µg/mL) in 24-
well plate at 37°C and 5% CO2 for 24 h. Viable trypomastigotes were estimated by
motility and counted in a hemocytometer under a light microscope (Olympus CX31),
according to the Brener method [27]. The results were expressed as the minimal
concentration of the compounds that inhibits the viability of 50% (IC50Trypo) of parasites
compared to the untreated control.
2.5 Effect on intracellular amastigotes
Peritoneal macrophages (2.5 x 105 cells/mL) were inoculated into the 24-well
plates containing round glass coverslips. At confluence, non-adherent cells were
removed by gently washing with PBS. Trypomastigotes at a protozoan-cell-ratio of 5:1
was inoculated into the macrophages monolayer and incubated at 37°C in 5% CO2
atmosphere for 2 h. Non-interiorized trypomastigotes were removed by gently washing
with PBS. DMEM containing different concentration of benzoylthioureas (6.25 and 100
μg/mL) was added and the plates were incubated for 48 h. The coverslips were
subjected to fixation with methanol, stained with Giemsa for 20 min and permanently
prepared with Entellan (Merck). The number of infected cells and parasites per cell
were determined by direct counting randomly 200 cells under a light microscope. The
survival index (%) was determined by multiplying the percentage of infected cells by
the average number of amastigotes per infected cell. The survival index observed in the
control without treatment was considered as 100%. The results were expressed as the
minimal concentration of the compounds that causes 50% (IC50Ama) reduction in
survival index compared to the untreated control [28].
2.6 In silico predictions of pharmacokinetic and toxicity parameters (ADME-Tox)
In silico ADME and the parameters related to oral bioavailability of the
compounds 1c, 1g, and 1p were assessed by rule-based filters from Linpinski [29] and
Veber [30] using the SwissADME platform (http://www.swissadme.ch/) [31].
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Prediction of the in silico toxicity of these compounds were performed using
DataWarrior software (Version 5.2.1) [32].
2.7 Molecular docking
Molecular docking studies were performed using Autodock 4.2 [33] and the X-
ray crystal structures of CYP51 (PDB ID: 4C27, resolution: 1.95 Å) [34] and CRZ
(PDB ID: 4KLB, resolution 2.62 Å) [35] available in the Protein Data Bank (PDB)
server (rcsb.org) [36]. The protein preparation and the grid box size dimensions were
prepared according to the protocol previously described by our research group [37]. The
intermolecular interactions analyses and figures were generated by the PyMOL program
(DeLano Scientific).
3.
Results and Discussion
3.1. Anti-epimastigote activity and in vitro cytotoxicity of the Benzoylthioureas (1a-
p): a preliminary SAR study
Benzoylthioureas (1a-p, Scheme 1) were previously synthesized by our research
group by treatment of the corresponding benzoyl chloride with ammonium
isothiocyanate and different nucleophilic amines. The structure of the products was fully
characterized by assignments of IR, MS and 1H and 13C NMR spectra [24,38].
Scheme 1. Synthetic route for the preparation of Benzoylthioureas 1a-p, previously described by our
research group[24,38].
In the first step, the inhibitory effect of all benzoylthioureas was evaluated on
growth of epimastigote forms of T. cruzi Y strain, and on viability of LLCMK2 cells.
Except 1o compound, all benzoylthioureas (1a-n, 1p) inhibited the growth of
epimastigotes forms at non-toxic concentration to LLCMK2 cells in vitro. The IC50Epi
and CC50 values of the compounds ranged from 13.4 to 217.3 μM and 54.5 to 946.0 μM,
respectively (Table 1).
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Table 1. In vitro antitrypanosomal activity on epimastigotes forms (IC50Epi) of Trypanosoma cruzi,
toxicity (CC50) to LLCMK22 cells and selectivity index (SI) of Benzoylthioureas (1a-p).
IC50Epi
(µM)
N°
CC50 (µM)
SI
R1
R2
H
H
H
H
H
H
H
H
H
n-Bu
96.4
186.4
26.4
303.2
424.7
261.3
493.1
946.0
457.1
255.1
459.4
392.0
910.3
652.5
495.6
152.1
282.6
54.5
3.20
2.27
10.6
2.72
9.61
3.23
19.0
5.56
6.12
10.4
4.94
2.28
5.10
1.46
0.87
15.5
1a
1b
1c
1d
1e
1f
n-Hex
c-Hex
CH2Bn
180.8
98.3
4-OH-Ph
4-OMe-Ph
4-t-Bu-Ph
4-Cl-Ph
4-Br-Ph
3-NO2-Ph
4-OMe-Ph
4-Cl-Ph
3-NO2-Ph
4-OMe-Ph
4-Cl-Ph
3-NO2-Ph
141.2
13.4
1g
1h
1i
82.6
64.0
H
87.3
1j
Cl
132.0
217.3
29.8
1k
1l
Cl
Cl
Br
Br
1m
1n
1o
1p
193.7
62.3
Br
61.1
945.7
Based on the results presented in Table 1, a preliminary study of SAR was
conducted with the aim to indicate important structural features to the anti-epimastigote
activity of the benzoylthioureas series (1a-p). In general, considering only the R1-
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substituted derivatives (1a-j), the aromatic subset (1e-j) showed more relevant IC50
values than the aliphatic ones (1a-d) for the epimastigote forms. Among the aliphatic
subset (1a-d), the chain length could be considered a critical feature for the anti-
epimastigote effect since longer and bulky chains such as n-Hex (1b) and CH2Bn (1d)
have shown the lowest activities in comparison with n-Bu (1a) and c-Hex (1c). For the
aromatic subset (1e-j), the presence of electron-withdrawing groups attached to the
benzene ring, such as chlorine (1h), bromine (1i) and nitro (1j), were more effective
against epimastigote forms than strong electron releasing groups, such as hydroxyl (1e)
and methoxyl (1f). However, the best compound of this subset was 1g, which has a t-
butyl group attached at 4-position of the benzene ring, which is a weak electron
releasing group suggesting steric, conformational, or lipophilic effects also could be
important for the anti-epimastigote effect.
In regard to the subset of derivatives (1k-p), which have chloro or bromo at
para-position of benzoyl group (R2), the chlorinated derivatives (1k-m) were more
active than brominated ones (1n-o) against epimastigote forms, except for 1o. However,
the replacement of the hydrogen by a halogen seems not to contribute for the
improvement of IC50 values when 4-OMe-Ph (1k and 1n) and 4-Cl-Phe (1l and 1o) are
attached to R1 position. On the other hand, when derivatives have a 3-NO2-Ph at R1, the
presence of chloro (1m) or bromo (1p) increases the activity when compared to the
respective unsubstituted derivative (1j).
Generally, most benzoylthioureas (1a-n, 1p) were more relatively selective
towards the epimastigote forms of T. cruzi than to the mammalian cells, which can be
demonstrated by SI values that ranged from 1.46 to 19.0, except for 1o (SI = 0.87).
Noteworthy, the introduction of an electronegative atom at R2 position leads to the
rising of cytotoxicity, except for 1p (SI = 15.5). Among all benzoylthioureas, the most
promising compounds were 1c, 1g, 1j and 1p that showed SI values > 10 [39].
However, in combination with IC50 values, the derivatives 1c, 1g and 1p were selected
as the hit compounds of this series, since they also exhibited lower IC50 values, being
26.4, 13.4 µM and 61.1 µM, respectively. Therefore, these three derivatives were
selected to be subjected to more specific assays, in order to also verify their effects
against trypomastigote and amastigote forms of the parasite.
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3.2. Anti-trypomastigote and anti-amastigote activity of the Benzoylthioureas 1c,
1g and 1p
The antitrypanosomal activity of lead compounds 1c, 1g and 1p were also
evaluated in clinically significant developmental forms in vitro, using benznidazole
(BZD) as a reference drug. All derivatives selectively inhibited the viability of
trypomastigote forms (Table 2). Compound 1g was 3 times more active than the
reference drug, which is also 12.2 and 6.7 times more active than 1c and 1p. The
inhibitory effect on trypomastigotes is particularly of note, since in general these non-
proliferative cells exhibit a higher inherent capacity to resist the action of
antitrypanosomal compounds [40] including BZD [41].
During the acute phase of Chagas disease, macrophages play an important role
in controlling Trypanosoma cruzi infection. Besides acting as effector cells in the initial
immune defense response, these cells can serve as host for the parasite replication [42].
In this sense, we investigate the effect of the lead compounds on replication of
amastigotes in murine peritoneal macrophages. The benzoylthiourea 1c, 1g and 1p
displayed relatively low toxicity to macrophage cells, exhibiting CC50 values of 261.3,
255.1 and 94.57 µg/mL, respectively. The derivative 1p showed the most promising
antiamastigote effect, exhibiting both the lowest IC50 and highest SIama values,
indicating that this compound could be tolerable and safer.
Table 2. In vitro antitrypanosomal activity in trypomastigote (IC50Trypo) and amastigote (IC50Ama) forms of
T. cruzi and selective index (SI) of Benzoylthioureas (1a-p) and benznidazole (BZD).
IC50Trypo
(µM)
IC50Ama
(µM)
N°
CC50
(µM)
SITrypo
SIAma
R1
R2
H
c-Hex
54.9
4.5
24.8
21.9
261.3
255.1
945.7
768.5
4.76
59.9
31.2
56.9
10.5
11.6
1c
1g
4-t-Bu-Ph
3-NO2-Ph
-----
H
Br
-----
30.3
13.5
16.8
56.2
1p
5.28[43]
145.5
BZD
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3.3 In silico pharmacokinetic and toxicity parameters (ADME-Tox) predictions
In order to predict the oral bioavailability, permeability profile and drug-likeness
of the selected compounds (1c, 1g and 1p), we conducted an in silico study of their
physicochemical properties (Table 3).
Table 3. In silico prediction of physicochemical and pharmacokinetic properties and toxicological risks
of the lead compounds 1c, 1g and 1p.
ADMET-Tox
Properties
1c
1g
R1 = 4-t-Bu-Ph R2
= H
1p
R1 = 3-NO2-Ph R2
= Br
R1 = c-Hex
R2 = H
MW (g.mol-1)
cLogP
H-bond acceptor
H-bond donor
TPSA (Ų)
262.3
2.96
2
312.4
4.20
1
365.2
3.41
2
2
2
2
73.22
5
0
73.22
6
0
82.45
6
0
Rotatable bonds
Violations
GI absorption
BBB permeant
P-gp substrate
Mutagenic
Tumorigenic
Reproductive Effective
Irritant
High
Yes
No
None
None
High
None
High
Yes
No
None
None
High
None
High
No
No
None
None
High
None
MW: molecular weight; cLog P: partition coefficient log; TPSA: topological polar surface area; GI:
gastrointestinal; BBB: blood-brain barrier; P-gp: P-glycoprotein-; Calculated on: SwissADME platform
(http://www.swissadme.ch/). Toxicological risks are calculated using DataWarrior software
(Version 5.2.1)
The three compounds (1c, 1g, and 1p) do not present violations according to
Lipinski's [29] and Veber's rules [30] (Table 3), following the requirements that indicate
that these substances have drug-likeness features, they are likely to be membrane-
permeable and have high bioavailability following oral administration.
The ability to permeate membranes can be reinforced by the parameters of
gastrointestinal absorption and permeation of the blood-brain barrier. Except for 1p,
these compounds showed a high probability to exhibit absorption through the
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gastrointestinal tract, although they may also access the central nervous system, which
could lead to possible adverse effects [44]. Due to the P-glycoprotein affects the
pharmacokinetics of several structurally and pharmacologically distinct drugs [45],
inhibition or induction of this protein can result in toxicity or under treatment caused by
decrease absorption at the intracellular level [46]. However, the in silico results indicate
that these compounds are not P-glycoprotein substrates. The toxicity profile of 1c, 1g,
and 1p compounds was performed using DataWarrior software [32]. The results
indicated that the compounds have no mutagenic, tumorigenic, and irritant effects, but
showed reproductive effective risk. Moreover, it is important to be mentioned that
experimental data should be obtained aiming to corroborate with these predictions.
3.2. Molecular docking
Acyl urea and thioureas can be considered lead scaffolds for the development of
non-covalent inhibitors of cruzain (CRZ) [23], and thioureas derivatives could interact
with fungal C14α-sterol demethylase (CYP51) [19,47], as reported in the literature.
Since the inhibitory activity of benzoylthioureas derivatives could be associated with
multi-target in the T. cruzi [48,49], we carried out molecular docking simulations of the
highest activity compounds (1c, 1g, and 1p) in two of the main enzymatic targets of T.
cruzi, CRZ [50,51] and CYP51 [52,53] to obtain more information about the
antitrypanosomal activity.
The validation of the docking protocol was carried out through a re-docking
simulation, considering the protocol described in the literature [37], from the co-
crystallized structure of each enzyme deposited in Protein Data Bank (PDB): CRZ
(PDB ID: 4KLB; resolution: 2.62 Å) [34] and CYP51 (PDB ID: 4C27; resolution: 1.95
Å) [35]. The protocol selected from the AutoDock program was able to reproduce the
crystal conformation with showed a root mean square deviations (RMSD) values below
2 Å (1.01 Å and 0.68 Å for CYP51 and CRZ, respectively).
The docking complexes between the ligands (1c, 1g, and 1p) and CRZ showed
that these compounds could interact with this enzyme by hydrogen bonds, but the
simulation showed different orientations for these compounds (Figure 2). The
compound 1c can interact by a hydrogen bond with Leu160 in the S3 subsite (Figure 2-
A). Although 1p (R1= 3-NO2-Ph) can also interact in the S3 subsite, this would happen
with the Ser64 residue (Figure 2-C), while the 4-bromophenyl group would be inserted
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into the cavity formed by S1 and S2. However, the R1 replacement by a 4-(tert-butyl)-
phenyl group allows the interaction in Gly66 in the S2 subsite (1g, Figure 2-B), which is
a residue already described for CRZ inhibition [34].
The molecular docking simulations of 1c, 1g, and 1p as CYP51-ligands revealed
that the three compounds can coordinate with Fe-heme through the carbonyl oxygen
atom. Compound 1c can also interact by hydrogen bonding with the NH group from
Ala291 and by hydrophobic interactions with cyclohexane group and residues Phe291,
Met460, and Val461 (Figure 2-D), while compound 1p can interact by hydrogen
bonding with –CONH- group and Tyr103 residue (Figure 2-F). On the other hand, the
compound 1g showed that the pose underwent rotation, leading to loss of the hydrogen
bonding interaction and allowing the tert-butyl phenyl group to be conducted to a
hydrophobic area near the Tyr103 and Tyr116 residues (Figure 2-E). The interactions
by coordination with ferryl heme, hydrogen bonds, and/or hydrophobic interactions
indicate that these compounds are capable of reducing the enzyme’s reactivation time as
described in the literature [35,54–56].
Figure 2. Main intermolecular interactions of the top docking pose of 1c (A and D), 1g (B and E) and 1p
(C and F) in the active site of CRZ (top) and CYP51 (down), respectively.
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4.
Conclusions
In summary, we observed that the series of benzoylthiourea derivatives inhibited
in vitro the growth of epimastigote forms of T. cruzi. A preliminary SAR study
indicated that aromatic substituents attached at N2 of the thiourea moiety improve the
antiepimastigote activity, and among these aromatic derivatives, the steric effects are
more prominent to biological activity than electronic effects. Moreover, it was possible
to identify the three most promising compounds (1c, 1g, and 1p), due to their highest
activity and SI > 10. These hit compounds also inhibited the viability and replication of
trypomastigote and amastigote forms, respectively, in non-toxic concentrations for
mammalian cells.
The results of the molecular docking study showed that these compounds may
interact with the active site of CRZ and CYP51 enzymes indicating that the 1c, 1g, and
1p compounds can inhibit these enzymes. Furthermore, ADME-Tox in silico
predictions indicated that these compounds may have high bioavailability following oral
administration, and no mutagenic, tumorogenic and irritant toxicological risks. Our
results reinforce the highlight of the benzoylthioureas as a scaffold for trypanosomal
activity.
Funding sources
This study was supported by Coordenadoria de Aperfeiçoamento Pessoal de
Nível Superior (CAPES, Brazil).
Declarations of Competing Interest
The authors declare no conflict of interest.
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Highlights
 Antitrypanosomal activity evaluation in vitro of benzoylthioureas.
 All tested compounds presented anti-epimastigote activity.
 Three promising compounds were evaluated against trypomastigote and amastigote
forms of T. cruzi.
 ADME-Tox in silico predictions was performed.
 Docking studies with two main enzymatic targets (cruzain and CYP-51) were
investigated.
22