4-Chlorophenylthioacetone-derived thiosemicarbazones as potent antitrypanosomal drug candidates: Investigations on the mode of action

Article abstract of DOI:10.1016/j.bioorg.2021.105018

Chagas disease (ChD), caused by Trypanosoma cruzi, remains a challenge for the medical and scientific fields due to the inefficiency of the therapeutic approaches available for its treatment. Thiosemicarbazones and hydrazones present a wide spectrum of bi

Full text of DOI:10.1016/j.bioorg.2021.105018

Bioorganic Chemistry 113 (2021) 105018
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
4-Chlorophenylthioacetone-derived thiosemicarbazones as potent
antitrypanosomal drug candidates: Investigations on the mode of action
Diego Rodney Rodrigues de Assisa, Alexandre Almeida Oliveirab, Samuel Luiz Portoa,
a
c
a
ˆ
Rayane Aparecida Nonato Rabelo , Eduardo Burgarelli Lages , Viviane Correa Santos ,
d
e
a
˜
Matheus Marques Milagre , Stenio Perdigao Fragoso , Mauro Martins Teixeira ,
a
a
c
ˆ
Rafaela Salgado Ferreira , Carlos Renato Machado , Lucas Antonio Miranda Ferreira ,
Nivaldo Lucio Spezialif, Heloisa Beraldob , Fabiana Simao Machado
,
*,
1
a,g,*,1
˜
a
ˆ
´
Departamento de Bioquímica e Imunologia, Instituto de Ciencias Biologicas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
b
c
d
e
f
ˆ
Departamento de Química, Instituto de Ciencias Exatas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
ˆ
´
Departamento de Produtos Farmaceuticos, Faculdade de Farmacia, Universidade Federal de Minas Gerais 31270-901 Belo Horizonte, MG, Brazil
´
Escola de Farmacia, Universidade Federal de Ouro Preto, 35400-000 Ouro Preto, MG, Brazil
˜
Fundaçao Oswaldo Cruz, Instituto Carlos Chagas, 81350-010 Curitiba, PR, Brazil
ˆ
Departamento de Física, Instituto de Ciencias Exatas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
g
ˆ
Faculdade de Medicina, Programa em Ciencias da Saúde: Infectologia e Medicina Tropical, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Chagas disease (ChD), caused by Trypanosoma cruzi, remains a challenge for the medical and scientific fields due
to the inefficiency of the therapeutic approaches available for its treatment. Thiosemicarbazones and hydrazones
present a wide spectrum of bioactivities and are considered a platform for the design of new anti-T. cruzi drug
candidates. Herein, the potential antichagasic activities of [(E)-2-(1-(4-chlorophenylthio)propan-2-ylidene)-
hydrazinecarbothioamides] (C1, C3), [(E)-N’-(1-((4-chlorophenyl)thio)propan-2-ylidene)benzohydrazide] (C2),
[(E)-2-(1-(4-, and [(E)-2-(1-((4-chlorophenyl)thio)propan-2-ylidene)hydrazinecarboxamide] (C4) were investi-
gated. Macrophages (MOs) from C57BL/6 mice stimulated with C1 and C3, but not with C2 and C4, reduced
amastigote replication and trypomastigote release, independent of nitric oxide (NO) and reactive oxygen species
production and indoleamine 2,3-dioxygenase activity. C3, but not C1, reduced parasite uptake by MOs and
potentiated TNF production. In cardiomyocytes, C3 reduced trypomastigote release independently of NO, TNF,
and IL-6 production. C1 and C3 were non-toxic to the host cells. A reduction of parasite release was found during
infection of MOs with trypomastigotes pre-incubated with C1 or C3 and MOs pre-stimulated with compounds
before infection. Moreover, C1 and C3 acted directly on trypomastigotes, killing them faster than Benznidazole,
and inhibited T. cruzi proliferation at various stages of its intracellular cycle. Mechanistically, C1 and C3 inhibit
parasite duplication, and this process cannot be reversed by inhibiting the DNA damage response. In vivo, C1 and
C3 attenuated parasitemia in T. cruzi-infected mice. Moreover, C3 loaded in a lipid nanocarrier system (nano-
emulsion) maintained anti-T. cruzi activity in vivo. Collectively, these data suggest that C1 and C3 are candidates
for the treatment of ChD and present activity in both the host and parasite cells.
Trypanosoma cruzi
Chagas disease
Hydrazones
Thiosemicarbazones
Treatment
Lipid nanoparticles
Abbreviations: MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; Bz, benznidazole; CMs, cardiomyocytes; ChD, Chagas disease; DMSO,
Dimethyl Sulfoxide; IDO, indoleamine 2,3-dioxygenase; iNOS, inducible nitric oxide synthase; IFN-γ, interferon gamma; IL, interleukin; LDH, lactate dehydrogenase;
MOs, macrophages; NE, nanoemulsion; Nfx, nifurtimox; NO, nitric oxide; ROS, reactive oxygen species; TNF, tumor necrosis factor.
ˆ
´
* Corresponding authors at: Departamento de Bioquímica e Imunologia, Instituto de Ciencias Biologicas (F. Machado) and Departamento de Química, Instituto de
ˆ
Ciencias Exatas (H. Beraldo), Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
˜
E-mail address: machadofs@icb.ufmg.br (F. Simao Machado).
1
These authors contributed equally to this manuscript.
https://doi.org/10.1016/j.bioorg.2021.105018
Received 16 March 2021; Received in revised form 19 May 2021; Accepted 20 May 2021
Available online 25 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
1. Introduction
metal complexes have been extensively reported [10-12]. Thiophenol-2-
ylidene-thiosemicarbazones and thiazolylhydrazones, which have a
spacer group (-S-CH2) between the phenyl ring and the hydrazone/thi-
osemicarbazone backbone (see Fig. 1), have shown promising anti-
T. cruzi activities [13,14].
More than 100 years after its discovery, ChD, which is caused by
Trypanosoma cruzi, remains a serious problem due to its high morbidity.
It is estimated that eight million people are infected with T. cruzi
worldwide, mainly in Latin America, where this disease remains a public
health problem, causing disability in infected individuals and more than
10,000 deaths per year [1]. ChD is characterized mainly by two distinct
phases: acute and chronic. The chronic phase is considered to be of
greater relevance, in which approximately 30% of patients present with
pathogenesis, including chronic cardiomyopathy (myocarditis, heart
failure, and eventually sudden death) and digestive alterations (mega-
esophagus and megacolon) [2].
Currently, targets including cruzain protease [15], DNA damage
response (DDR) [16], and topoisomerase enzymes from T. cruzi [17]
have been investigated in drug development due to their capacity to
interfere with pathways that are crucial for T. cruzi survival. Thiazo-
lylhydrazones have been shown to act as inhibitors of cruzain [18].
In the present work, a family of 4-chlorophenylthioacetone-derived
hydrazones and thiosemicarbazones (Fig. 1) were obtained, and their
anti-trypanosomal activities were evaluated. Our investigation aimed to
elucidate their mechanism of action and their molecular targets, in both
host and parasite cells.
During parasite-host interaction, cells of the mononuclear phago-
cytic system, mainly macrophages (MOs), play a fundamental role in
decreasing parasitemia by controlling T. cruzi proliferation. These cells
respond to pathogen-associated molecular patterns (e.g., lipids, carbo-
hydrates, nucleic acids, and various proteins derived from the parasite)
and orchestrate a Th1-type response (activation of natural killer cells
and TCD4+ lymphocytes), primarily through the production of IFN-γ, IL-
12, and TNF cytokines [3]. These cytokines are crucial for activating
oxygen-dependent mechanisms such as production of reactive oxygen
species (ROS), and oxygen-independent (such as nitric oxide – NO
production) [4,5] trypanocidal mechanisms. In addition, other trypa-
nocidal mechanisms that involve nutrient deprivation (such as ideol-
amine 2,3-dioxygenase enzyme – IDO – activation) are also important
for limiting the intracellular growth of T. cruzi, because IDO activity
reduces the levels of tryptophan, an amino acid involved in T. cruzi
multiplication. Moreover, IDO activation results in release of sub-
products that are toxic to the parasite [6]. Cardiomyocytes are also
involved in the development of ChD pathogenesis because these cells
actively participate in the control of parasite replication by producing
and responding to inflammatory molecules [7].
The critical steps in developing an effective drug include effective
absorption by the host, protection against chemical and/or enzymatic
degradation, and half-life improvement. Several research groups have
resorted to the use of drug delivery systems based on lipid nanocarriers
[19], mainly for compounds with reduced pharmacological activity due
to low solubility in aqueous media, such as certain hydrophobic
hydrazones and thiosemicarbazones [19]. Hence, lipid nanocarrier for-
mulations of the compounds were also prepared, and their antiparasitic
activities were tested. Thus, the compounds under study and their
nanocarrier formulations were investigated as a platform for the
development of new drug candidates for the treatment of ChD.
2. Materials and methods
2.1. Ethics statement
This research study was carried out in strict accordance with the
Brazilian guidelines on animal work and the Guide for the Care and Use
of Laboratory Animals of the National Institute of Health (NIH). The
animal ethics committee (CEUA) of Universidade Federal de Minas Gerais
(UFMG) – Brazil (BR), approved all experiments and procedures (Permit
Number 414/2018).
Benznidazole (Bz) and nifurtimox (Nfx) are the only drugs consid-
ered effective for the treatment of the acute phase of human Chagas
infection. However, these therapeutics present very low cure rates in the
chronic phase of the disease. Furthermore, when used for long-term
regimens (30 to 60 days), both drugs cause undesirable side effects in
30% to 87% of patients, resulting in treatment interruptions [8,9]. Thus,
the search for new antichagasic drug candidates with high efficacy and
minimal toxicity is extremely relevant.
2.2. Synthesis and characterization of C1-C4.
The reagents were purchased from Aldrich and used as received.
Partial elemental analyses were performed on a Perkin Elmer CHN 2400
analyzer. Melting points were determined with a Mettler MQAPF-302
apparatus. Infrared spectra were recorded on a Perkin Elmer Spectrum
One equipment employing the attenuated total reflectance (ATR)
Thiosemicarbazones and hydrazones are Schiff base compounds,
which exhibit a wide range of pharmacological applications as anti-
neoplastic, antimicrobial, and antiparasitic agents. The anti-
trypanosomal activities of thiosemicarbazones, hydrazones, and their
Fig. 1. (a) Compounds studied by other authors [13,14]; (b) 4-chlorophenylthioacetone-derived thiosemicarbazones (C1, C3) and hydrazones (C2, C4) studied in
the present work.
2

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
method (4000–400 cmꢀ 1). NMR spectra were obtained at 25 ◦C with a
Bruker DPX-400 Advance (400 MHz) spectrometer using DMSO‑d6 as
solvent. Mass spectra were recorded with a Shimadzu LCMS-IT-TOF
instrument working at high-resolution.
2.3.1. [(E)-2-(1-(4-chlorophenylthio)propan-2-ylidene)-N-
methylhydrazinecarbothioamide] (C1)
White-yellowish solid. Yield: 64%. Melting point: 106.4 ◦C ꢀ
108.1 ◦C. Anal. Calc. for C11H14ClN3S2: C, 45.90; H, 4.90; N, 14.60.
Single crystal X-ray diffraction measurements were carried out on an
Oxford-Diffraction GEMINI-Ultra diffractometer (LabCri-UFMG) using
Found: C, 45.94; H, 4.88; N, 14.44. FW: 287.82 g molꢀ 1. IR (ATR, cmꢀ 1):
–
3188
ν
(N2-H), 1520
ν
(C = N1), 848
ν
(C S). 1H NMR [400.13 MHz,
–
graphite-Enhance Source Mo K
α
radiation (λ = 0.71073 Å) at 293(2) K.
DMSO‑d6, δ (ppm)]: 10.12 [s, 1H, N2H], 8.10 [d, 1H, N3H], 7.42 [d, 2H,
H3, H5], 7.33 [d, 2H, H2, H6], 3.82 [s, 2H, H7], 2.96 [d, 3H, H11], 1.96
[s, 3H, H9]. 13C NMR [100.61 MHz, DMSO‑d6, δ (ppm)]: 178.6 [C10],
148.0 [C8], 134.1 [C4], 130.8 [C1], 130.6 [C3, C5], 128.8 [C2, C6],
40.1 [C7], 30.8 [C11], 15.1 [C9]. HRMS [ESI(+), IT-TOF] calculated for
Data collection, cell refinements and data reduction were performed
using the CrysAlisPro software package [20]. An absorption correction
based on a multi-scan method was applied. The structures were solved
by direct methods using SHELXS-2013/1 [21]. Full-matrix least-squares
refinement procedure on F2 with anisotropic thermal parameters was
carried out using SHELXL-2014/7 [22]. Positional and anisotropic
atomic displacement parameters were refined for all non-hydrogen
atoms. Hydrogen atoms were placed geometrically and the positional
parameters were refined using a riding model. Suitable single crystals of
C1 and C3 were obtained at room temperature from slow evaporation of
a 9:1 methanol: DMSO solution of the compounds. A summary of the
crystal data, data collection details and refinement results is listed in
Table S1 (Supplementary Material). Molecular graphics were plotted
using PLATON [23].
C
11H15ClN3S2, [M + H]+: 288.0396, found 288.0389.
2.3.2. [(E)-N’-(1-((4-chlorophenyl)thio)propan-2-ylidene)
benzohydrazide] (C2)
White solid. Yield: 57%. Melting point: 126.7 ◦C ꢀ 128.2 ◦C. Anal.
Calc. for C16H15ClN2OS: C, 60.28; H, 4.74; N, 8.79. Found: C, 60.13; H,
4.58; N, 8.87. FW: 318.82 g molꢀ 1. IR (ATR, cmꢀ 1): 3243
ν
(N2-H), 1520
1
–
ν
(C = N1), 1650
ν
(C O). H NMR [400.13 MHz, DMSO‑d , δ (ppm)]:
–
6
10.55 [s, 1H, N2H], 7.82 [sl, 2H, H12, H16], 7.60–7.50 [m, 1H, H14],
7.50–7.40 [m, 4H, H3, H5, H13, H15], 7.35 [d, 2H, H2, H6], 3.90 [s, 2H,
H7], 2.05 [s, 3H, H9]. 13CNMR [100.61 MHz, DMSO‑d6, δ (ppm)]: 163.5
[C10], 157.1 [C8], 134.6 [C4], 133.9 [C11], 131.4 [C14], 130.8 [C1],
130.4 [C3, C5], 128.8 [C2, C6], 128.2 [C13, C15], 127.8 [C12, C16],
40.6 [C7], 15.7 [C9]. HRMS [ESI(+), IT-TOF] calculated for
2.3. Syntheses of the 4-chlorophenylthioacetone-derived
thiosemicarbazones and hydrazones
The thiosemicarbazones and hydrazones were prepared as previ-
ously described [24]. Briefly, in a 50 mL flask containing 10 mL of
methanol, 207 mg (1 mmol) of (4-chlorophenylthio) acetone were sol-
ubilized. Three drops of acetic acid were added and, after 10 min with
stirring, 1 mmol of the appropriate thiosemicarbazide or hydrazide was
added. In all cases, the reaction mixture was kept under magnetic stir-
ring and reflux for 4 h (see Scheme 1). After cooling at room tempera-
ture, the mixture was kept in the freezer for 24 h, then it was filtered,
washed with a minimum of methanol and diethyl ether, and the solids
were subsequently dried in a desiccator under reduced pressure.
The compounds were characterized by elemental analysis and by
means of their infrared, NMR and HRMS spectra. The carbon atom
numbering scheme and all spectra of C1 - C4 are shown in the Supple-
mentary Material.
C
16H16ClN2OS, [M + H]+: 319.0672, found 319.0690.
2.3.3. [(E)-2-(1-(4-chlorophenylthio)propan-2-ylidene)
hydrazinecarbothioamide] (C3)
White solid. Yield: 83%. Melting point: 136.3 ◦C ꢀ 137.0 ◦C. Anal.
Calc. for C10H12ClN3S2: C, 43.87; H, 4.42; N, 15.35. Found: C, 43.85; H,
4.40; N, 15.14. FW: 273.80 g molꢀ 1. IR (ATR, cmꢀ 1): 3150
ν(N2-H),
1591
ν(C = N1), 864
ν
(C S). 1H NMR [400.13 MHz, DMSO‑d6, δ
–
–
(ppm)]: 10.10 [s, 1H, N2H], 8.13 [s, 1H, N3H], 7.54 [s, 1H, N3H], 7.41
[d, 2H, H3, H5], 7.32 [d, 2H, H2, H6], 3.82 [s, 2H, H7], 1.96 [s, 3H, H9].
13C NMR [100.61 MHz, DMSO‑d6, δ (ppm)]: 178.8 [C10], 148.2 [C8],
134.0 [C4], 130.7 [C1], 130.3 [C3, C5], 128.7 [C2, C6], 39.8 [C7], 15.1
[C9]. HRMS [ESI(+), IT-TOF] calculated for C10H12ClN3NaS2, [M +
Na]+: 296.0059, found 296.0065.
Scheme 1. Syntheses of (4-chlorophenylthio)acetone-derived thiosemicarbazones (C1, C3) and hydrazones (C2, C4). Reagents and conditions: (a) thio-
semicarbazide, MeOH, acetic acid (three drops), reflux, 4 h; (b) hydrazide, MeOH, acetic acid (three drops), reflux, 4 h.
3

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
2.3.4. [(E)-2-(1-((4-chlorophenyl)thio)propan-2-ylidene)
hydrazinecarboxamide] (C4)
calculated using the following formula:
[Total] ꢀ [Filtered] ꢀ Ultrafiltrate]
White-yellowish solid. Yield: 75%. Melting point: 158.2 ◦C ꢀ
161.1 ◦C. Anal. Calc. for C10H12ClN3OS: C, 46.60; H, 4.69; N, 16.30.
Found: C, 46.09; H, 4.53; N, 16.24. FW: 257.74 g molꢀ 1. IR (ATR, cmꢀ 1):
EE(%) =
x100
[Total]
Ultrafiltrate: represents the fraction of the compound soluble in the
aqueous phase of the formulation. Filtered: represents the fraction of the
compound insoluble in the aqueous phase of the formulation; Total: the
sum of the drug fraction retained in the lipid matrix (encapsulated), the
fraction soluble in the aqueous phase, and the insoluble fraction in the
aqueous phase.
1
–
3164
ν
(N2-H), 1584
ν
(C = N1), 1685
ν
(C O). H NMR [400.13 MHz,
–
DMSO‑d6, δ (ppm)]: 9.12 [s, 1H, N2H], 7.40 [d, 2H, H3, H5], 7.32 [d,
2H, H2, H6], 6.24 [s, 2H, N3H], 3.78 [s, 2H, H7], 1.85 [s, 3H, H9]. 13
C
NMR [100.61 MHz, DMSO‑d6, δ (ppm)]: 157.1 [C10], 143.8 [C8], 134.4
[C4], 130.5 [C1], 130.3 [C3, C5], 128.7 [C2, C6], 40.1 [C7], 14.5 [C9].
HRMS [ESI(+), IT-TOF] calculated for C10H13ClN3OS, [M + H]+:
258.0468, found 258.0478.
2.5. Animals
Wild-type (WT) C57BL/6 mice, female, aged 8–10 weeks, were ob-
tained from the Animal Care Facilities of Universidade Federal de Minas
Gerais. Inducible nitric oxide synthase knockout (iNOS KO), gp91Phox
knockout (Phox KO), and indoleamine 2,3 dioxygenase knockout (IDO
KO) female mice were bred on a C57BL/6 genetic background under
2.4. Preparation of a C3 lipid nanocarrier
C3 was encapsulated in a lipid nanocarrier (nanoemulsion - NE),
according to the composition described in the Table 1. The oily and
aqueous phases were weighed separately and heated to 80 ◦C. After
fusion of the lipids, the aqueous phase was slowly poured over the oil
phase under constant agitation at 8000 rpm using the Ultra Turrax T-25
homogenizer (Ika Labortechnik, Germany). After 2 min, the emulsion
was sonicated (21% amplitude, 10 min) using a high-intensity ultrasonic
processor (model CPX 500, Cole-Palmer Instruments, USA). The ob-
tained emulsion was cooled to room temperature under magnetic stir-
ring. The pH was adjusted to 7.0 and the final volume was completed to
10 mL with water. The formulation was stored at 4 ◦C, protected from
light and in a nitrogen atmosphere.
ˆ
´
pathogen-free conditions at the Instituto de Ciencias Biologicas, Uni-
versidade Federal de Minas Gerais, Brazil.
2.6. Infection of mice and cells
The T. cruzi Y strain was used for both in vivo and in vitro experiments.
C57BL/6 mice were infected intraperitoneally (i.p.) with 1x103 trypo-
mastigotes, and parasitemia levels were periodically measured in 5 μL of
blood from the tail vein. For in vitro infection, trypomastigotes were
grown in cultures of a monkey kidney epithelial cell line (LLC-MK2) and
then used for infection at a 5:1 parasite/host cell ratio [7].
2.4.1. Nanoemulsion characterization
The nanoemulsion (NE) formulation was characterized by its average
particle diameter, polydispersity index (PI), zeta potential (Zp), and
encapsulation efficiency (EE). The average particle diameter and PI were
measured by means of dynamic light scattering (DLS) using a Zetasizer
Nano ZS90 (Malvern Instruments, United Kingdom) at a fixed angle of
90◦ and temperature of 25 ◦C. Measurements of Zp were performed by
dynamic light scattering (DLS) determinations of electrophoretic
mobility at 25 ◦C. To carry out the analyses, 10 µL of the formulation was
diluted in 1 mL of distilled water.
2.7. Macrophage cultures, treatment, and NO-inhibition
MOs were harvested from peritoneal cavities as previously described
[7], from C57BL/6 (WT), iNOS KO, Phox KO, or IDO KO mice, and
plated (2x105 or 1x106 cell/well – 96 or 24 well, respectively) onto
culture plates (Nunc, Rochester, NY, USA). Infections were performed as
described above. Cells were treated with C1 - C4 at concentrations in the
10 μM ꢀ 50 μM range. IFN-γ at 100 ng/mL (Sigma) was used as a pos-
itive control for T. cruzi replication in MOs. In addition, WT and Phox KO
MOs were infected with T. cruzi (as described above) and posteriorly
To determine the EE of the compound, filtration and ultrafiltration
methods were used. Thus, the procedure was divided into three stages:
total, filtered, and ultrafiltered. In the first (total concentration) stage,
500 µL of the formulation was transferred to a 5 mL volumetric flask.
Tetrahydrofuran (THF, 2 mL) was added. Then, 300 µL were transferred
to a 5 mL volumetric flask, and the volume was completed with DMSO.
In the second step (filtration), 1 mL of the formulation was filtered
through a 0.45 µm membrane. Then, 500 µL of the filtrate were trans-
ferred to a 5 mL volumetric flask, 2 mL of THF were added, and the
volume was completed with DMSO. Then, 300 µL were transferred to a 5
mL volumetric flask, and the volume was again completed with DMSO.
In the third stage (ultrafiltration), 300 µL of the formulation were
transferred to an Amicon 100 kDa filtration device (Millipore, USA),
which was subjected to centrifugation at 2400 × g for 10 min. Then, 50
µL of the filtrate were transferred to an eppendorf, and 450 µL of DMSO
were added. The concentration of C3 in all analyses was evaluated by
UV–VIS spectrometry, using the absorbance at 280 nm. EE was
stimulated with C1 (20
μM), C3 (30 μM), or IFN-γ (100 ng/mL).
Simultaneously, the arginine amino acid analog NG-monomethyl-L-
arginine (L-NMMA) (Sigma-Aldrich Co.) was added at 400 μM, to inhibit
NO production [7]. MOs were treated with compounds at 2, 6, 12, and
24 h after infection to evaluate the impact of the compounds on amas-
tigote replication and differentiation.
2.8. Cardiomyocyte H9c2-line culture, infection, and treatment
Adherent H9c2 rat embryonic cardiomyocytes (CMs), H9c2(2–1)
(ATCC® CRL-1446™) were grown in Dulbecco’s Modified Eagle Me-
dium (DMEM – Cultilab, Campinas, SP/BR), pH 7.6, supplemented with
10% fetal bovine serum (Cultilab) and 1% penicillin/streptomycin
(Gibco, BLR Life Technologies). Cells were cultured at 37 ◦C in 5% CO2.
Before reaching confluence, the cells were removed from the bottle
using trypsin/EDTA (Sigma) and centrifuged for 10 min at 500×g. The
pellet was homogenized in DMEM, and cells were counted in a Neu-
bauer’s chamber with the aid of trypan blue stain (Gibco) and seeded at
a density of 4x104 cells/well. These cells were infected as described
Table 1
Composition of the nanoemulsion (NE) containing C3.
Formulation (NE)
above, and after 2 h of infection, C3 or IFN-⋎ was added at 0.62
M or 100 ng/ml, respectively.
μM ꢀ 5
μ
Oily phase
Aqueous phase
MCT
5.0%
0.5%
1.5%
0.1%
Glycerol
2.25%
100%
2.9. Parasite count
The number of intracellular amastigotes was determined in macro-
Cholesterol
Tween 80
C3
Water qsp
MCT: medium-chain triglycerides
´
´
phages fixed and stained with Panotico Rapido (LB Laborclin, Pinhais,
4

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
PR/BR) at four and 24/48 h post-infection, to evaluate parasite uptake
and intracellular growth, respectively. The number of released trypo-
mastigotes from MOs and CMs was quantified in the supernatants from
three to seven days post-infection (dpi), as previously described [25].
2.15. MTT assay
Parasites were seeded (1x106/well) in 96-well plates in the presence
or absence of compound. After treatment, 10 µL of MTT (Sigma) solution
(5 mg/mL) was added to the wells, and immediately afterwards, the
plates were incubated at 37 ◦C for 2 h, for the trypomastigotes assay, or
28 ◦C for 2 h, for the epimastigotes assay. After this period, the parasites
were removed from the plates, placed in 1.5 mL tubes, and centrifuged
(2438 × g, 10 min). The supernatants were discarded, and the pellet was
2.10. Nitric oxide and cytokines determinations
Nitric oxide determination was performed in supernatants by the
Griess colorimetric method as previously described [26]. Optical den-
sities were measured at 540 nm in a plate reader (96 wells) (Elx800 - Bio
Tek). TNF and IL-6 cytokines were assayed in the supernatants of the
macrophage cultures by the sandwich enzyme-linked immunosorbent
assay (ELISA) method, according to the manufacturer’s instructions (R &
D Systems Inc., Minneapolis, Minnesota). Optical densities were
measured at 450 nm in a plate reader (96 wells) (Elx800 - Bio Tek).
resuspended in 200 µL of DMSO to dissolve formazan crystals. A 200 μL
aliquot was pipetted into a 96-well plate for reading on a spectropho-
tometer at a wavelength of 490 nm. The results were expressed as optical
density (OD) and compared with the OD values obtained from the
control group.
2.16. Cruzain inhibition assay
2.11. LDH assay
The catalytic activity of cruzain was monitored by the cleavage of the
fluorogenic substrate Z-Phe-Arg-amidomethylcoumarin (Z-FR-AMC) in
a Synergy 2 plate reader (Biotek), as previously described [29]. For this
purpose, 4 nM cruzain was incubated in the presence of C1, C3, or Bz at
100 µM for 10 min at room temperature. Then, 2.5 µM of the substrate
was added to start the reaction. The positive control for inhibition (E64)
was used. The assays were performed in triplicate using 0.1 M sodium
acetate (pH 5.5) in the presence of 5 mM beta-mercaptoethanol and
0.01% Triton X-100 in a 96-well plate (flat black background). Aggre-
gation assays were performed as previously described [30], with the
following modification: the inhibitory activity of the compounds was
evaluated in the presence of 0.001%, 0.01%, and 0.1% Triton X-100. For
IC50 determination, the activity of C3 was evaluated in eleven concen-
trations, ranging from 0.0015 µM to 500 µM. Two independent experi-
ments were performed in triplicate, and the data were analyzed with
GraphPad Prism 5.0, employing a nonlinear regression analysis of log
[C3] versus response with a variable slope and four parameters.
At 24 or 48 h after treatment with C1 and C3, the culture superna-
tants (from MOs and CMs) were collected for cytotoxicity assay (LDH -
lactate dehydrogenase). The LDH test is a marker of plasma membrane
integrity [27]. The assay consists of removing the supernatant from cells
and measuring the activity of the released enzyme by offering its sub-
strate (Bioclin® Kit, Belo Horizonte, MG/BR).
2.12. Pre-incubation of macrophages and T. cruzi-trypomastigotes with
C1 or C3
MOs were plated as above; however, before infection with the
parasite, the cells were stimulated with C1, C3, or IFN-γ for 1 h. After
this interval, the cells were washed twice with RPMI and then infected
(as above). Trypomastigotes were obtained as described above, counted,
and incubated with RPMI in the presence of C1 (30
μM) or C3 (20
μ
M).
◦
Incubation occurred in microtubes for 1 h (37 C, 5% CO2). After in-
cubations, the “mix” parasite “C1 + T. cruzi” or “C3 + T. cruzi” was
centrifuged at 2438 × g for 10 min. The parasites were resuspended in
RPMI medium for subsequent MO infection (as described above).
2.17. T. cruzi-infected mice treatments
Animals were infected (as described above) and treated 8 h after
infection, up to the ninth dpi, twice a day (12/12 h). Mice received the
compounds (C1 or C3) at a dose of 1 mg/Kg by gavage (orally) or
intraperitoneal routes. The control group received a vehicle solution.
The parasitemia level was determined as described above. In the second
experiment, infected mice were treated 8 h after infection, up to the
ninth dpi with C3 loaded into the NE system or with Bz, both at doses of
10 mg/kg by gavage (orally), once a day. The control group received an
empty NE solution. Parasitemia level and survival were monitored.
2.13. Trypomastigotes incubation with C1, C3 and benznidazole
Trypomastigotes (Y strain) from LLC-MK2 cells were cultured in the
presence of RPMI alone, C1 (30 μM), C3 (20 μM), or Benznidazole (30
μ
M) for 1 h. After incubation, the parasites were centrifuged (2438 × g,
10 min) to remove the compounds and resuspended in RPMI culture
medium. A second experimental group remained in the presence of the
compounds throughout the analysis. Viable parasites (flagellar move-
ment and elongated morphology characteristic of trypomastigote forms)
were observed under an optical microscope and counted in a Neubauer’s
chamber at two, four, and six h after initial treatment.
3. Results
3.1. Characterization of the 4-chlorophenylthioacetone-derived
thiosemicarbazones and hydrazones
2.14. Epimastigotes culture, treatment, and caffeine-pretreatment
Microanalyses and high-resolution mass spectra were compatible
with formation of C1-C4. In the infrared spectra of C1 and C3, absorp-
tions at 1520–1591 cmꢀ 1 were attributed to
ν
(C N). The vibrations
–
Epimastigotes (Y strain – WT, DM28 strain – WT, topoisomerase 3
α
–
(C S) were observed at 848 cmꢀ 1 and 864 cmꢀ 1 (com-
–
–
KO DM28 strain) were grown in liver infusion tryptose (LIT) medium
(pH 7.4) supplemented with 10% fetal bovine serum (FBS, Gibco BRL,
Invitrogen, Carlsbad, CA, USA) and 1% streptomycin/penicillin (Gibco)
attributed to
ν
pounds C1 and C3, respectively). For the hydrazones, the vibrations
ꢀ 1
(C O) were observed at 1650 cm and 1685 cmꢀ 1
–
attributed to
ν
–
at 28 ◦C. Parasite transfection (Topo-3
α
KO background DM28) was
(compounds C2 and C4, respectively).
1
performed using electroporation, as previously described [28]. Cells
were cultured until the logarithmic growth phase before all experiments.
For the pretreatment assay, epimastigotes were incubated, or not, with
caffeine (Sigma) at doses of 1 mM to 4 mM for 1 h. After caffeine in-
cubation, parasites were centrifuged (2438 × g, 10 min), seeded in
NMR spectra were recorded in DMSO‑d6. The H resonances were
assigned on the basis of chemical shifts, multiplicities and by using 2D
homonuclear 1H–1H correlation spectroscopy (COSY). The carbon type
(C, CH) was determined by using distortionless enhancement by polar-
ization transfer (DEPT-135) experiments and the assignments were
made by 2D heteronuclear multiple quantum coherence (HMQC) and
heteronuclear multiple bond coherence (HMBC) experiments.
culture plates, and stimulated, or not, with 30 μM of C1, C3, or Bz. The
parasite growth profile was followed from three to 48 h after treatment.
5

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
1
Formation of C1-C4 was evidenced in the H NMR spectra by the
presence of a signal corresponding to N2H in the 9.12–10.55 ppm range.
In the 13CNMR spectra a signal close to 150 ppm was assigned to the
iminic carbon.
cytotoxicity assay demonstrated that even at the highest concentration
used (50 μM), the thiosemicarbazones C1 or C3 did not induce toxicity
to the host cells (Fig. 3D). Similar to the results found with infected-MOs
stimulated with the compounds, only C1 and C3 had direct activity
against the epimastigote form of the parasite (Fig. 3E).
1
Duplicated signals were observed in the H NMR spectra (see Sup-
plementary Material), which corresponded to a mixture of E (89–96%)
and Z stereoisomers (11–4%) in solution, as confirmed by Two-
dimensional Nuclear Overhauser Effect Spectroscopy (NOESY) spectra
which showed intramolecular 1H–1H coupling involving C8CH3 and
N2H. In the 13C NMR spectra, the signals did not appear to be duplicates
because of the low concentration of the Z isomer in solution.
3.3. C1 and C3 attenuate T. Cruzi growth in macrophage cultures
independently of nitric oxide production
After initial screening, we started a more detailed analysis of the
trypanocidal activity of C1 and C3 compared to stimulation with the
IFN-γ cytokine (a potent inducer of MO trypanocidal activity). As shown
in Fig. 4A, infected cells stimulated with C1 presented a lower parasitic
load compared to unstimulated infected cells at both 24 and 48 h after
treatment, and this effect was similar to the IFN-γ activity. Regarding
parasite uptake, the presence of C1 did not alter the capacity of trypo-
mastigote invasion in MOs (Fig. 4B). Moreover, C1 treatment also
reduced the release of trypomastigotes when compared to unstimulated
infected cells (Fig. 4C). Of note, treatment with C3 presented the highest
capacity to control amastigote replication, even higher than IFN-γ
(Fig. 4D), and decreased parasite uptake/invasion (Fig. 4E) and trypo-
mastigotes release by infected-MOs (Fig. 4F).
X-ray crystallography analyses showed that the thiosemicarbazones
C1 and C3 crystallized in the monoclinic and triclinic systems, respec-
tively. The compounds crystallized in the EE conformation in relation to
the C8-N1 and C10-N2 bonds. The bond lengths (Å) and angles of the
thiosemicarbazones were similar. The C10-S2 and C8-N1 bond distances
–
were close to 1.685 and 1.272 Å, respectively, as expected for C S and
–
–
–
C
N double bonds [31]. Crystal data and refinement results are listed in
Table S1 (Supplementary Material). The atom arrangements and atom
numbering scheme for C1 and C3 are shown in Fig. 2. Selected intra-
molecular bond distances and angles are in Table S2 (Supplementary
Material).
Next, the involvement of NO, IL-6, and TNF cytokines in the anti-
T. cruzi activity induced by C1 or C3 in infected cells was investigated.
Moreover, to exclude the requirement of NO in macrophage activity by
compounds, iNOS deficient macrophages (iNOS KO) were used. Fig. 4G
and 4I demonstrated that at the time of analysis, only IFN-γ stimulus
was able to induce NO production by infected MOs. In fact, NO pro-
duction triggered by iNOS was not necessary for the trypanocidal ac-
tivity of the compounds under study here, because WT or iNOS KO MOs
showed similar trypanocidal capacity when treated with C1 (Fig. 4H) or
C3 (Fig. 4J). C1 or C3 induced the uninfected MOs to secrete TNF
(Fig. 4K), but not IL-6 (Fig. 4K). When these cells were infected, treat-
ment with C3, but not with C1, increased TNF production, compared
with the unstimulated infected group. On the other hand, treatment with
C1, but not with C3, decreased IL-6 levels when compared to the
unstimulated infected group (Fig. 4K). Moreover, ROS production, the
enzyme IDO, and NO were not necessary for maintaining the micro-
bicidal activities induced by C1 or C3 in T. cruzi-infected MOs. Phox KO
MOs, in the presence or absence of LNMMA (NO production inhibitor),
and IDO KO macrophages reduced the parasite number in macrophages
when stimulated with C1 or C3 (Fig. S18, Supplementary Material).
3.2. C1 and C3, but not C2 and C4, induce trypanocidal activity without
presenting host cell toxicity
The anti-T cruzi activities of C1–C4 was evaluated in MO cultures.
Dose-response data were obtained for C1. Infected cells treated with
doses of 30, 40, or 50 µM, presented a reduced number of amastigotes
48 h after infection and treatment, when compared with unstimulated
infected cells (Fig. 3A). At a dose of 20 μM, C1 failed to induce anti-
T. cruzi activity (Fig. 3A). Infected-MOs in the presence of DMSO (0.5%
v/v), the maximum percentage used in our assays, and maximum con-
centration allowed for cell cultures [32], had similar amounts of
amastigotes at 48 h compared to unstimulated infected-cells, which
shows that this low DMSO content did not influence the results (Fig. 3A).
As no significant difference was observed among the numbers of
intracellular parasites found in stimulated macrophages incubated with
30, 40, or 50 µM of C1 (Fig. 3A), the lowest concentration was chosen for
subsequent experiments. The other compounds (C2, C3, and C4), which
are structurally similar to C1, were tested in infected-MO cultures, and
only C3 was able to reduce amastigote replication when compared to
unstimulated infected cells (Fig. 3B). In the assay to test decreasing
doses of C3, it was observed that at all tested concentrations, C3 effi-
ciently controlled intracellular T. cruzi replication at 48 h (Fig. 3C). At
3.4. C3 controls trypomastigote release by cardiomyocytes without
inducing nitric oxide, TNF, and IL-6 production
20
activity of C3 was similar at 20
chosen for the subsequent experiments. Additionally, the LDH-
μ
M, C3 showed higher anti-T. cruzi activity than at 10
μM, and since
μ
M and 30 M, the 20 M condition was
μ
μ
The microbicidal activity of C3 was also evaluated in non-phagocytic
H9c2 cardiomyocytes cells. Infected cardiomyocytes stimulated with
Fig. 2. Molecular plots of C1 and C3 showing the labeling scheme of the non-H atoms and their displacement ellipsoids at the 50% probability level.
6

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
Fig. 3. C1 and C3, but not C2 and C4, attenuate T. cruzi replication in macrophages and on epimastigote axenic cultures. MOs from mice (C57BL/6) were
cultured, infected (5:1 - T. cruzi:cell ratio), and stimulated, or not, with C1 (50 to 20 M) or in the presence of 0.5% DMSO (A) or stimulated by C2 (30 M), C3 (30 to
10 M), or C4 (30 M) (B). Next, intracellular parasite counts were performed on fixed and stained cells at 48 h (C). MOs were cultured in the presence of C1 (30 M),
C3 (20 M), or culture medium only, and after 48 h, the supernatants were collected for toxicity assessment (LDH). For the positive lysis control, 2% triton was used
(D). Epimastigotes (Y strain) were incubated, or not, in the presence of C1–C4 (30 M) or DMSO 0.5%, and 48 h after incubation, parasite replication analysis was
performed (E). Each point represents the mean ± SEM of one of the two independent experiments, * = p ≤ 0.05; ** = p < 0.01; *** = p < 0.001. (ns = not significant).
μ
μ
μ
μ
μ
μ
μ
C3, in all tested concentrations, reduced trypomastigote release when
infection. As shown in Fig. 6A, when MOs pre-treated with C1 or C3
were infected, there was a lower parasite release at the seventh dpi,
without uptake impairment (Fig. 6B). In order to investigate the possible
direct activity of compounds on T. cruzi, trypomastigotes were incubated
with or without C1 or C3 for 1 h before infection. MOs that were infected
with parasites pre incubated with C1 or C3 showed decreased number of
released trypomastigotes from the fourth to seventh dpi, when
compared with parasites pre-incubated with medium alone (Fig. 6C).
Moreover, pre-incubation with C1 or C3 reduced capacity of parasites to
invade the host cells (Fig. 6D). Next, we investigated whether C1 or C3
could act at different stages of T. cruzi infection, mainly by directly
inhibiting proliferation and differentiation of amastigotes in trypomas-
tigotes. C1 reduced the number of trypomastigotes released when added
compared to unstimulated infected cells (Fig. 5A). The dose of 2.5 μM
was chosen for the next tests, because it sufficiently controlled parasite
release and was not toxic to the host cell (Fig. 5B). Treatment with C3
and IFN-γ did not induce NO (Fig. 5C), TNF (Fig. 5D), or IL-6 (Fig. 5E)
production by cardiomyocytes at 48 h after treatment.
3.5. C1 and C3 act directly on trypomastigote forms of the parasite and in
different stages of macrophage infection to decrease parasite release
First, to determine whether the compounds could pre-activate the
host cell microbicidal response against T. cruzi before infection, MOs
were pre-treated with C1 or C3 for 1 h, and the cells were washed before
7

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
Fig. 4. C1 and C3 control amastigote replication and trypomastigote release in macrophages, independent of NO production. WT or iNOS KO peritoneal
MOs were plated and/or infected with trypomastigote forms and stimulated, or not, with compounds. Intracellular amastigote count was performed at 24 h and 48
h after infection and treatment with C1 (30 μM) (A) or C3 (20 μM) (D). For a positive control of T. cruzi intracellular replication, IFN-γ 100 ng/mL was used.
Parasite uptake was evaluated 4 h after simultaneous infection and treatment with C1 (30
third to seventh dpi after C1 treatment (30 M) (C), or with C3 (20 M) (F), and after C1 (30
MOs (H) or C3 (20
μ
M) (B) or C3 (20
μM) (E). Trypomastigote release was followed from the
μ
μ
μ
M) treatment in WT (empty symbols) × iNOS KO (filled symbols)
μ
M) treatment in WT (empty symbols) × iNOS KO (filled symbols) MOs (J). At 24 h and 48 h intervals, supernatants were collected for NO
dosing (G–C1 treatment; I–C3 treatment) and cytokine measurements after treatment with C1 or C3 (K): TNF – top panel; IL-6 – bottom panel. “CT” (control) =
uninfected and unstimulated cells. Each point represents the mean ± SEM of one of two independent experiments, * = p ≤ 0.05; ** = p < 0.01; *** = p < 0.001.
to the culture at different times after infection (a.i.): 4 h (Fig. 6E), 12 h
(Fig. 6F), 24 h (Fig. 6G), and 48 h (Fig. 6H). C1 also reduced the number
of parasites when added to cultures at 4, 12, and 24 h a.i. Of note, C3
displayed a potent trypanocidal capacity, as did IFN-γ, and drastically
reduced the number of amastigotes in cells and trypomastigotes in the
culture supernatant when added to culture after all analyzed infection
times (Fig. 6E-H). These results strongly suggest that compounds C1 and
C3 have direct activity on T. cruzi.
C1 and C3 reduced the number of parasites, when compared to the
control (parasite incubated with medium alone) and, of great relevance,
to the group incubated with Bz. At 12 h of incubation, C1 and C3
continued to decrease the number of parasites; and only at this time after
incubation was it possible to observe the trypanocidal activity of Bz,
which took place 10 h later than that of C1 and 6 h after that of C3
(Fig. 6J). Through the MTT assay, we observed that the incubation of
epimastigotes with C1 or C3, but not with Bz, decreased parasite
viability within 4 h of treatment (Fig. 6K).
To confirm the direct action of compounds on the parasite, free
trypomastigotes were pre-incubated with C1, C3 or benznidazole (Bz,
used as positive control) for 1 h, or incubated with the compounds for all
times analyzed. As shown in Fig. 6I, a decreased parasite number was
observed 12 and 24 h after the initial treatment for 1 h with C1 or C3,
respectively. In the assay where the parasites remained in contact with
the compounds during all analysis times, after 2 h of incubation, C1
decreased the number of parasites (Fig. 6J). After 6 h of incubation, both
3.6. C1 and C3 decrease the number of circulating parasites in the blood
of mice
The potential therapeutic capacities of C1 and C3 were investigated
in an acute model of T. cruzi infection. Our results showed that the
treatment of infected animals with C1 reduced the number of parasites
8

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
Fig. 5. C3 protects H9c2 cardiomyocytes from T. cruzi lytic activity, independently of cytokine (TNF and IL-6) and NO production. H9c2 CMs were cultured,
infected (5:1 - T. cruzi:cell ratio), and stimulated, or not, with C3 (5 to 0.6 M). Subsequently, trypomastigotes release was followed from the third to fifth dpi (A).
Infected-CMs were cultured in the presence of C3 (2.5
μ
μ
M), IFN-γ (100 ng/mL), or culture medium only. After 24 and 48 h, the supernatants were collected for toxicity
assessment (LDH) (B). At the fifth dpi, the supernatants were collected for NO measurement (C). The supernatants were also collected at 48 h to evaluate cytokine
production; TNF (D) and IL-6 (E). Each point represents the mean ± SEM of one of two independent experiments, * = p ≤ 0.05; ** = p < 0.01; *** = p < 0.001.
in the blood of animals at both the seventh and ninth dpi when
administered orally and at the seventh dpi when administered intra-
peritoneally (Fig. 7A). C3 reduced the level of parasitemia at both the
seventh and ninth dpi by both routes of administration (Fig. 7B).
experiments were carried out in which cruzain was incubated in the
presence of the compounds at 100 M and later, the fluorescent sub-
strate was added, and its cleavage was measured by fluorescence
emission. Initially, C3 was able to inhibit cruzain, with IC50 = 41 ± 5
μ
μM
(Fig. 8D). An additional assay was carried out to verify whether the
observed enzyme inhibition by C3 was due to nonspecific inhibition. It is
known that small molecules can aggregate and inhibit enzyme activity
in a non-specific manner. A feature of inhibitory aggregates is that non-
ionic detergents can disrupt their formation and reduce their influence
on enzyme inhibition [33,34]. In detergent tests, a 31% change in C3
activity was observed when comparing 0.1% with 0.001% for Triton X-
100. Taken together, the sensitivity of C3 to detergent, its low potency
against cruzain, and the absence of cruzain inhibition by the structurally
related C1 indicates that the trypanocidal activity of C3 is not due to
cruzain inhibition.
3.7. T. cruzi-topoisomerase-3
not to Bz and C3
α is involved in parasite resistance to C1, but
First, we evaluated whether the activities of C1 and C3 were trypa-
nocidal or trypanostatic against epimastigote forms. As seen in the
growth curves of the parasites (Fig. 8A), the presence of C1 and C3
inhibited parasite growth.
To check whether the growth inhibition was due to immediate DNA
damage caused by C1 and C3 that led to the activation of the DNA
damage response, we analyzed the effect of caffeine on the activities of
C1 and C3. For this, epimastigotes were pre-incubated with caffeine,
which is an ATM/ATR (kinases that function as the main damage sensor
during DNA Damage Response; DDR) pathway inhibitor. To choose an
inhibitor concentration, the parasites were incubated with increasing
doses of caffeine, and the growth curve was monitored. We found that
the concentrations of 1, 2, or 4 mM of caffeine did not change the
parasite growth pattern at all times observed, when compared to the
control group (Fig. S19, Supplementary Material), with an interme-
diate dose of 2 mM. Next, epimastigotes were incubated with caffeine at
2 mM for 1 h, then “washed” to remove the compound, and immediately
afterwards, seeded in culture plates and incubated with Bz, C1, or C3.
Inhibition of the ATM/ATR pathway by caffeine did not change the
capacity of C1 or C3 to control parasite growth (Fig. 8B).
3.8. C3 loaded in lipid NE maintains anti-Trypanosoma cruzi activity in
vitro and in vivo
In order to improve its pharmacological properties, C3 was subjected
to encapsulation in nanoemulsion (NE). The characterization results
showed particles with an average diameter close to 180 nm and a narrow
size distribution (PI < 0.30), indicating a monodisperse system. The zeta
potential was slightly negative, probably due to the free fatty acids
present on the NE surface. The EE (%) was close to 100%, which dem-
onstrates that practically all of the added compound was properly
encapsulated inside the nanostructures (Fig. 9A).
Our biological results demonstrated that encapsulated C3 main-
tained anti-T. cruzi activity, similar to that of C3 in its free form, as
demonstrated by the reduction of trypomastigote release by MOs, from
the third to fifth dpi (Fig. 9B), without inducing toxicity to the host cells
(Fig. 9C). In an animal model of acute chagasic infection, our results
showed that infected animals treated with the formulation containing
C3 reduced the parasitemia at the peak of infection (Fig. 9D). The same
phenotype was observed with Bz treatment (Fig. 9D). Of note, treatment
with the C3 formulation, but not with Bz, increased the survival
In another approach, we investigated whether the absence of T. cruzi
topoisomerase-3α could alter the control pattern of parasitic replication
exhibited in the presence of C1 and C3, since this enzyme plays an
important role during DNA duplication and repair. In Fig. 8C we can see
that Topo-3
α knockout parasites were more susceptible to C1 anti-
T. cruzi activity. Notably, both WT and KO parasites were highly sus-
ceptible to the anti-T. cruzi action induced by C3 (Fig. 8C). The ability of
C1 and C3 to inhibit the cruzain enzyme was also evaluated. For this,
9

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
Fig. 6. C1 and C3 act directly on trypomastigotes, faster than benznidazole, and infected-cells by reducing replicative capacity and amastigote differ-
entiation. Peritoneal MOs were plated and stimulated, or not, with C1 (30 μM), C3 (20 μM), or IFN-γ 100 ng/mL for a period of 1 h. After this interval, cells were
washed with RPMI medium and infected, and trypomastigotes release was followed from the third to seventh dpi (A). Cells were in simultaneous contact (parasite +
compounds [C1 or C3 or IFN-γ], or parasite only) for a period of 4 h, to evaluate the parasite entry profile (uptake) in the host cells (B). Trypomastigotes were incubated,
or not, in the presence of C1 (30 μM) or C3 (20 μM) for a period of 1 h. After this time, parasites were centrifuged to remove the compounds, and plated MOs were
posteriorly infected with pre-incubated trypomastigotes, and trypomastigote release was followed from the third to seventh dpi (C). Additionally, parasites went through
the same process described before and were in contact with MOs for 4 h (pre-incubated trypomastigote uptake) (D). MOs were plated and infected with trypomastigotes,
and after 4 h (E), 6 h (F), 12 h (G) or 24 h (H) post infection, compounds were added, and trypomastigote release (panel above) and amastigote replication/differ-
entiation (panel below) were analyzed. Trypomastigotes were cultured in the presence of RPMI alone, C1 (30 μM), C3 (20 μM), or benznidazole (30 μM) for 1 h. After
this time, the parasites were centrifuged to remove the compounds and plated (96 wells-plate) (I), or the trypomastigotes remained throughout the analysis in the
presence of the compounds (J). Quantitation of the viable parasites was performed at 2, 6, 12 and 24 h after previous treatment (optical microscope) and at 2 and 4 h
after incubation, by the MTT assay (K). Each point represents the mean ± SEM of one of the two independent experiments, * = p ≤ 0.05; ** = p < 0.01; *** = p < 0.001.
Fig. 7. C1 and C3 decrease parasitemia in T. cruzi-infected mice. Female C57BL/6 mice were infected (1x103 trypomastigotes – Y strain) and treated with C1 (A) or
C3 (B) at 1 mg/Kg by gavage (ORAL) or intraperitoneal (IP) routes for 12/12 h. From the third to fifteenth dpi, blood was collected through the caudal vein every two days
for parasite quantification. “Tc” = infected animals. “Tc þ C1” = infected animals treated with C1. “Tc þ C3” = infected animals treated with C3. * = p ≤ 0.05; ** = p <
0.01; *** = p < 0.001 in relation to the infected group treated with the infection control. Each point represents the mean ± SEM of one of the two independent experiments.
10

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
Fig. 8. Evaluation of the involvement of the ATM/ATR pathway, the Topo-3
α
enzyme, and cruzain inhibition in the anti-T. cruzi activity of C1 or C3.
Epimastigotes (Y strain) were pre-treated, or not (A), with caffeine (2 mM) for 1 h (B), and subsequently incubated, or not, in the presence of the compounds (A, B).
Parasite growth was followed at 3, 6, 12, 24, and 48 h after treatment with C1, C3, or Bz molecules (A, B). *** = p < 0.001 (in comparison to the treated group with
the untreated control). WT (DM28 strain; empty symbols) or Topo-3α KO (filled symbols) epimastigotes were cultured and treated, or not, with 30 μM C1, C3, or Bz.
Parasite growth was evaluated at 24 and 48 h after treatment (C). ** = p < 0.01; *** = p < 0.001 (in comparison to the treated group with the untreated control).
Each point represents the mean ± SEM of one of the two independent experiments. To determine the compound IC50, cruzain (4 nM) was incubated in the presence of
100 μM C1, C3, or Bz for 10 min. Then, 2.5 µM of the substrate, Z-Phe-Arg-aminomethylcoumarine (Z-FR-AMC), was added to start the reaction. To assess the
sensitivity to detergent inhibition (triton X-100), the compounds [IC50 ꢀ 0.01% triton X-100] were pre-incubated with a solution containing cruzain and 0.001%,
0.01%, or 0.1% triton X-100 for 10 min. Then, a substrate solution with the same concentration of triton X-100 was added to the plates, and the fluorescence was
monitored. Reported values refer to the average and standard deviation obtained from two independent experiments, in triplicate. “N.D.” = not determined (D).
percentage during the time investigated (Fig. 9E).
exposed simultaneously to the compounds and the parasite). The results
revealed that in the presence of C3, but not C1, parasite invasion into the
uninfected cells was decreased. These results are extremely important
and indicate that C3 interferes with the parasite-cell interaction to
reduce parasite proliferation and dissemination of the infection.
Studies with humans and experimental animal models suggest the
involvement of the immune system in the effectiveness of many drug
treatments [36], including the dependence of “immunological factors”
for Bz therapeutic activity [37]. Thus, we investigated whether immu-
nological mechanisms were involved in the microbicidal activity of C1
or C3 in macrophages. In this context, the investigation of NO produc-
tion is relevant, since this compound can act directly on the parasite to
induce its death [38]. However, our results showed that the trypanocidal
activities of C1 and C3 in infected MOs were independent of NO pro-
duction and/or the presence of the iNOS enzyme.
4. Discussion
Chemotherapies with benznidazole and nifurtimox have been used
as the gold standard for treating ChD for more than half a century.
However, Bz and Nfx therapies require long-term treatment that is
associated with potential side effects, and the effectiveness of using
these drugs for complete parasite elimination remains unclear. In this
context, we investigated the trypanocidal activity of new thio-
semicarbazones (C1 and C3) and hydrazones (C2 and C4) derived from
4-chlorophenylthioacetone.
Our results showed that among the studied compounds, only C1 and
C3 controlled the intracellular replication of T. cruzi in MOs. In fact, C2
and C4 were not able to control T. cruzi proliferation in MOs. The same
phenomenon was observed in the axenic culture of epimastigotes, where
C1 and C3 limited the replication of the parasites, while C2 and C4 did
not show any anti-T. cruzi activity. Hence, we may infer that replace-
ment of the sulfur atom by oxygen completely abolishes the anti-
trypanosomal activity of the 4-chlorophenylthioacetone-derived Schiff
bases.
TNF and IL-6 cytokines are also important mediators of T. cruzi
infection and exert protective effects against the parasite [39,40]. Thi-
osemicarbazone C3 increased TNF production and maintained IL-6
secretion levels by infected-MOs, which may contribute to the greater
microbicidal activity of C3 in relation to C1.
ROS production has been reported as a mechanism of resistance
against various infections [41]. In the context of T. cruzi infection, low
ROS concentrations promote signaling for parasite proliferation. How-
ever, in high “doses,” ROS behaves as toxic agents against the parasite
[42].
The presence of extracellular LDH (in the culture supernatant) is a
fundamental characteristic of cells undergoing apoptosis, necrosis, and
other forms of cell damage [27]. Our results showed that even at higher
doses, C1 and C3 did not induce toxicity (increased extracellular LDH) in
the host cells.
Our results showed that the inhibition of NADPH oxidase activity by
the absence of the catalytic subunit gp91phox did not cause any inter-
ruption of the anti-T. cruzi activity triggered by C1 or C3 in the MOs.
Other pathways, such as those that induce nutrient deprivation, are also
important for limiting intracellular pathogen growth [43]. Hence, we
investigated whether the activity of the IDO enzyme (resistance can
correlate with Trp deprivation and/or the generation of metabolites
produced by IDO activity that are toxic to the parasite) could be involved
In the context of ChD, the IFN-γ production or administration cor-
relates with resistance to T. cruzi infection, mainly in the acute phase
[35]. Our data showed that C1 has similar trypanocidal activity to IFN-γ,
while C3 has a higher activity than IFN-γ in vitro. Our experiments were
designed to mimic a scenario where parasites complete their intracel-
lular cycle and are released into the bloodstream, at a point at which
patients would receive treatment with C1 or C3 (where MOs were
11

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
Fig. 9. The effect of NE containing C3 on T. cruzi infection in vitro and in vivo. Parameters including particle diameter, polydispersity index (PI), Zeta
potential (Zp), and encapsulation efficiency (EE) were evaluated after the construction of the nanosystem (A). MOs from mice (C57BL/6 – female) were
cultured, infected (5:1 - T. cruzi:cell ratio), and stimulated, or not, with free or encapsulated C3 at 20 μM. Trypomastigote release was followed from the third
to eights dpi after C1 treatment (B). After 48 h, the supernatants were collected for toxicity assessment (LDH). For the positive lysis control, 2% triton (T) was
used (C). C57BL/6 mice were infected (1x103 trypomastigotes – Y strain) and treated, or not, with C3-NE (nanoemulsion) or Bz (Benznidazole) at 10 mg/Kg
by oral route, once a day. From the third to fifteenth dpi, blood was collected every two days for parasite (D) and survival (E) evaluations. “CT” = non-
infected cells/animals. “Tc” = infected cells/animals. “Tc þ C3” = infected cells treated with C3 (free). “Tc þ NE” = infected cell/animals treated with
empty NE. “Tc þ C3-NE” = infected cells/animals treated with NE containing C3. “Tc þ Bz” = infected animals treated with Benznidazole. *** = p < 0.001
(Tc + NE × Tc + Bz), # # # = p < 0.001 (Tc + NE × Tc + C3-NE). Each point represents the mean ± SEM of one of the two independent experiments.
in the trypanocidal mechanism of C1 or C3 in MOs. Our data demon-
strated that IDO deficiency did not cause a loss in the trypanocidal ac-
tivity triggered by C1 or C3 in MOs, which indicates that this pathway
does not participate in the microbicidal activity of these molecules.
The investigation of the anti-T. cruzi activity of C3 in cardiomyocytes
(a cell type of extreme importance due to its involvement in the cardiac
pathophysiology of ChD) revealed that, unlike IFN-γ, the treatment of
infected cardiomyocytes with C3 decreased the release of trypomasti-
gotes, independent of NO, TNF, and IL-6 production. Furthermore, the
treatment of infected-CMs with C3 protected against the lithic activity of
T. cruzi (maintenance of LDH levels similar to that of uninfected cells).
Collectively, the results obtained with MOs and CMs strongly suggest
that the thiosemicarbazones C1 and C3 act mainly by mechanisms that
induce direct death of the parasite, independently of the activation of
the main “classical” host cell microbicidal mechanisms described so far
(i.e., NO and ROS production).
To understand the role of host cells in the trypanocidal mechanisms
of C1 and C3, host cells were pre-incubated with C1 or C3 before
infection. The compounds were still able to control the release of try-
pomastigotes by MOs without changing the parasite invasion capacity in
pre-stimulated cells.
Here, the direct activity of C1 and C3 on parasites was demonstrated.
Pre-incubation of the trypomastigotes with the compounds led to a
reduced number of parasites released in the supernatant correlated with
the reduced invasion capacity of pre-incubated parasites. This indicates
that the compounds may act either indirectly or directly on the parasites
to control their replication in MOs.
Our data obtained through the treatment of MOs, beginning after
different times of infection, suggest that C1 and C3 effectively inhibited
amastigote replication and differentiation. Of great relevance, our
12

D. Rodney Rodrigues de Assis et al.
Bioorganic Chemistry 113 (2021) 105018
results from the direct incubation of trypomastigotes with C1 and C3
strongly suggest that these compounds act to directly induce the parasite
death, faster than Bz, without the need for the host cell microbicidal
machinery. Validating our findings in vitro, we found that, importantly,
C1 or C3 maintained their anti-T. cruzi activity in an animal model of
acute chagasic infection, decreasing the parasitemia level of the infected
animals. However, further studies in vivo are required to compare the
efficacy of the treatments with C1, C3 and BZ, and to demonstrate the
molecular mechanisms responsible for the observed effects.
Institute for Science and Technology in Dengue and Host-microbial in-
teractions (Programa INCT; APQ-03606–17), INCT-INOFAR (CNPq
˜
`
465.249/2014–0) and Fundaçao de Amparo a Pesquisa do Estado de
´
Minas Gerais, Rede Mineira de Imunobiologicos (FAPEMIG; REDE-
00140–16) and FAPEMIG APQ-00628–16.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Based on the results of the compounds’ direct activity on the para-
sites, we investigated certain signaling pathways and molecular targets
on which the compounds could act to lead to parasite death. First, we
investigated whether C1 and C3 induce the parasite death through a
signaled response, more precisely, via ATM/ATR signal transducer ki-
nases. Our results suggest that the ATM/ATR pathway does not play a
role in the maintenance of anti-T. cruzi activity exerted by C1, C3, or Bz,
since pre-incubation of parasites with an inhibitor of this pathway,
caffeine (cells treated with caffeine exhibit various phenotypic abnor-
malities reported in cells with ATM or ATR function deficiency) [44],
did not impair the anti-T. cruzi action of the compounds. This suggests
that the action of C1 and C3 was not immediate, but that it causes
delayed damage that blocks parasite duplication without immediate
DNA damage response (DDR) activation.
Acknowledgements
The authors would like to thank Dr. M. A. Ribeiro, Departamento de
Química (Universidade Federal do Espírito Santo, Brazil) and Jacqueline
Barbosa de Oliveira Viana (Universidade Federal de Minas Gerais,
Brazil) for technical assistance. We also thank Allison Doak and Dr.
Brian Shoichet, from the University of California San Francisco, for
gently providing recombinant cruzain.
Appendix A. Supplementary data
Contrary to the results obtained with the inhibition of ATM/ATR
enzymes, the absence of topoisomerase-3α in the parasite resulted in a
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.105018.
greater trypanocidal capacity of C1, which demonstrates that the pres-
ence of this enzyme is important for parasite resistance against the ac-
tion of C1. This compound probably induces damage to the DNA of the
parasite, in which topoisomerase and other enzymes are involved in its
repair.
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