Antiparasitic activity of furanyl N-acylhydrazone derivatives against Trichomonas vaginalis: In vitro and in silico analyses

Article abstract of DOI:10.1186/s13071-020-3923-8

Background: Trichomonas vaginalis is the causative agent of trichomoniasis, which is one of the most common sexually transmitted diseases worldwide. Trichomoniasis has a high incidence and prevalence and is associated with serious complications such as HI

Full text of DOI:10.1186/s13071-020-3923-8

et al. Parasites Vectors
https://doi.org/10.1186/s13071-020-3923-8
(2020) 13:59
Alves
Parasites & Vectors
Open Access
RESEARCH
Antiparasitic activity of furanyl
N-acylhydrazone derivatives
against Trichomonas vaginalis: in vitro and in
silico analyses
Mirna Samara Dié Alves1 , Raquel Nascimento das Neves1, Ângela Sena‑Lopes1, Micaela Domingues2,
Angela Maria Casaril2, Natália Vieira Segatto3, Thaís Cristina Mendonça Nogueira4,
Marcus Vinicius Nora de Souza4,5, Lucielli Savegnago2, Fabiana Kömmling Seixas3, Tiago Collares3
and Sibele Borsuk1*
Abstract
Background: Trichomonas vaginalis is the causative agent of trichomoniasis, which is one of the most common sexu‑
ally transmitted diseases worldwide. Trichomoniasis has a high incidence and prevalence and is associated with seri‑
ous complications such as HIV transmission and acquisition, pelvic infammatory disease and preterm birth. Although
trichomoniasis is treated with oral metronidazole (MTZ), the number of strains resistant to this drug is increasing
(2.5–9.6%), leading to treatment failure. Therefore, there is an urgent need to fnd alternative drugs to combat this
disease.
Methods: Herein, we report the in vitro and in silico analysis of 12 furanyl N‑acylhydrazone derivatives (PFUR 4, a‑k)
against Trichomonas vaginalis. Trichomonas vaginalis ATCC 30236 isolate was treated with seven concentrations of
these compounds to determine the minimum inhibitory concentration (MIC) and 50% inhibitory concentration (IC50).
In addition, compounds that displayed anti‑T. vaginalis activity were analyzed using thiobarbituric acid reactive sub‑
stances (TBARS) assay and molecular docking. Cytotoxicity analysis was also performed in CHO‑K1 cells.
Results: The compounds PFUR 4a and 4b, at 6.25 µM, induced complete parasite death after 24 h of exposure with
IC50 of 1.69 µM and 1.98 µM, respectively. The results showed that lipid peroxidation is not involved in parasite death.
Molecular docking studies predicted strong interactions of PFUR 4a and 4b with T. vaginalis enzymes, purine nucleo‑
side phosphorylase, and lactate dehydrogenase, while only PFUR 4b interacted in silico with thioredoxin reductase
and methionine gamma‑lyase. PFUR 4a and 4b led to a growth inhibition (<20%) in CHO‑K1 cells that was compara‑
ble to the drug of choice, with a promising selectivity index (>7.4).
Conclusions: Our results showed that PFUR 4a and 4b are promising molecules that can be used for the develop‑
ment of new trichomonacidal agents for T. vaginalis.
Keywords: Antiparasitic, Lipid peroxidation, Molecular docking, Trichomoniasis, Trichomonacidal
*Correspondence: sibeleborsuk@gmail.com
1 Laboratório de Biotecnologia Infecto‑parasitária, Centro de
Desenvolvimento Tecnológico, Biotecnologia, UFPel, Pelotas, RS
96010‑900, Brazil
Full list of author information is available at the end of the article
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adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and
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Alves et al. Parasites Vectors
(2020) 13:59
Page 2 of 13
Background
Methods
Trichomonas vaginalis is an anaerobic, fagellated pro- Chemicals
tozoan parasite, which infects the urogenital tract of Te synthesis of furanyl N-acylhydrazone derivatives was
humans causing trichomoniasis, the most prevalent carried out as described by Cardoso et al. [26] and the
non-viral sexually transmitted infection worldwide [1]. synthesized samples were kindly provided by Dr Marcus
Trichomoniasis has an estimated incidence rate of 276 Vinicius Nora de Souza from the Instituto de Tecnologia
million cases per year [1]. In Brazil, the prevalence var- em Fármacos-Far-Manguinhos, Fiocruz, Brazil. Briefy,
ies between 2.5% and 20% in women [1–6]. Trichomonas the synthesis was carried out by the reaction between
vaginalis has a greater impact on public health as it is 2-(H2NNHCO-furan), which were generated from
associated with increased risk of preterm delivery, pelvic methyl furan-2-carboxylate, and arylaldehydes result-
infammatory disease [7] and HIV acquisition and trans- ing in the formation of 12 derivatives, which are as fol-
mission [8, 9]. It is important to note that non-adherence lows: PFUR 4 (precursor), PFUR 4a, PFUR 4b, PFUR 4c,
and/or noncompliance to treatment for trichomoniasis, PFUR 4d, PFUR 4e, PFUR 4f, PFUR 4g, PFUR 4h, PFUR
which happens frequently, helps in maintaining the chain 4i, PFUR 4j and PFUR 4k (Fig. 1).
of infection [8].
For more than fve decades, in most countries, the
treatment for T. vaginalis has been restricted to 5-nitro- Trichomonas vaginalis culture
imidazoles, with metronidazole (MTZ) and tinidazole Te isolate T. vaginalis Donne ATCC 30236 that is sus-
(TNZ) being the only drugs approved by the U.S. Food ceptible to MTZ was axenically grown in pre-warmed
and Drug Administration Agency [9, 10]. Te activ- trypticase-yeast extract-maltose (TYM) medium with-
ity of MTZ depends on the reduction of its nitro group out agar (pH 6.0), supplemented with 10% sterile bovine
by hydrogenosome metabolism and by favin enzyme serum (SBS) (previously inactivated at 56 °C for 30 min)
thioredoxin reductase (TrxR) [11]; however, the increas- with 3% streptomycin (5 mg/ml) and incubated at 37 °C
ing number of T. vaginalis isolates resistant to 5-nitro- under microaerobic conditions [27]. Te motility and
imidazoles is a major concern [12–14] representing morphology of the culture were analyzed under light
2.5–9.6% of clinical cases [12, 13, 15]. Nonetheless, inhi- microscopy at 400× magnifcation. A trypan blue (0.4%)
bition of TrxR and other enzymes such as lactate dehy- exclusion assay was performed to ensure that minimum
drogenase (LDH), methionine gamma-lyase (MGL), viability of 95% and logarithmic growth phase had been
and purine nucleoside phosphorylase (PNP) also seem achieved before proceeding to antiparasitic and bio-
to be a rational approach for chemotherapy against T. chemical assays.
vaginalis due to their importance on protozoan survival
[16–18].
Anti‑T. vaginalis assay
In this context, there is a need to identify new mole- Drug susceptibility assays were performed to analyze the
cules with potential trichomonacidal activity as an alter- antiparasitic potential of 12 PFUR compounds against T.
native to 5-nitroimidazoles. N-acylhydrazone derivatives vaginalis as previously described [28, 29]. All compounds
have been found to have several biological properties, were diluted in dimethylsulfoxide (DMSO). For the assay,
such as antimalarial [19], antitumor [20], antifungal [21] 96-well microtiter plates were seeded with 2.6×105
and antibacterial [22] activities, exhibiting good phar- trophozoites/ml of TYM (initial density), then PFUR
macological potential. In addition, furan and related drugs were added into the wells at a concentration of 100
derivatives represent an important class of heterocyclic µM, and the plates were incubated at 37 °C with 5% CO2
compounds that have been reported to exhibit a wide for 24 h. Following incubation, an aliquot of trophozo-
range of biological activities, such as antibacterial, anti- ites and trypan blue (0.4%) (1:1, v/v) was counted in the
viral, anti-infammatory, antifungal, antitumor, etc. Neubauer chamber to determine trophozoite motility,
[23–25].
morphology and viability through trypan blue dye exclu-
Considering the need for new trichomonacidal com- sion. Tree controls were used in each assay: a negative
pounds, a series of furanyl N-acylhydrazone derivatives control containing only trophozoites; a 0.6% DMSO con-
(PFUR) was designed by molecular hybridization tech- trol (vehicle for solubilization); and a positive control
nique combining both pharmacophoric groups, i.e. the containing MTZ at 100 µM (Sigma-Aldrich, St. Louis,
N-acylhydrazone and furan moieties in the molecules. Missouri, USA). Only the compounds that reduced the
Terefore, this study was aimed at e the in vitro activ- viability of parasites by 100% were used in the subsequent
ity and selectivity of 12 synthesized PFUR derivatives experiments.
against T. vaginalis and at identifying their likely binding
to T. vaginalis proteins in silico.
Te minimum inhibitory concentration (MIC) and 50%
inhibitory concentration (IC50) values against T. vaginalis

Alves et al. Parasites Vectors
(2020) 13:59
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Thiobarbituric acid reactive substances (TBARS) assay
were established in the same conditions described previ-
ously with trophozoites exposed for 24 h to PFUR con-
centrations ranging from 1.5625 µM to 100 µM. Te
MIC was confrmed by transferring the culture solutions
contained in 96-well plates from MIC and from concen-
trations directly below and above, as well as controls, to
tubes containing 1.5 ml of fresh TYM medium, with SBS
and antibiotics. Tubes were then re-incubated at 37 °C
with 5% CO2 for 96 h and trophozoites were counted in
the Neubauer chamber every 24 h to confrm MIC. Te
IC50 was calculated using GraphPad Prism 7.0 software.
A kinetic growth curve was established to understand
the time required for the antiparasitic activity of com-
pounds on T. vaginalis cultures. 96-well plates were pre-
pared as previously described, compounds were added at
MIC, and the plates were incubated at 37 °C with 5% CO2
for 96 h. Te viability of trophozoites was observed under
light microscopy and the growth analysis was performed
at 1, 6, 12, 24, 48, 72 and 96 h using trypan blue (0.4%)
dye exclusion and motility and morphology analysis. All
assays were performed in three independent set-ups in
triplicate and the results were expressed as a percentage
of viable trophozoites compared with the negative control.
Te treatment and control groups were evaluated for
the formation of TBARS in an acid-heating reaction,
and malondialdehyde (MDA) was chosen as the bio-
marker for lipid peroxidation. Te assay was performed
as described by Ohkawa et al. [30] with modifcations.
For this, 96-well plates were seeded with T. vaginalis
(2.6×105 trophozoites/ml), the compounds (at MIC) and
controls were added, and plates were incubated at 37 °C
with 5% CO2 for 24 h. After incubation, aliquots of T.
vaginalis control and treatment groups were incubated
with 0.8% TBA, acetic acid/HCl (pH 3.4), and 8.1% SDS
(sodium dodecyl sulfate) at 95 °C for 2 h. Te absorbance
of the samples was measured at 532 nm and the results
were expressed as nmol MDA/105 trophozoites.
Molecular docking
Molecular docking of PFUR compounds was performed
using crystal structure of T. vaginalis purine nucleoside
phosphorylase (TvPNP; PDB 1Z36), lactate dehydroge-
nase (TvLDH; PDB 5A1T), and methionine gamma-lyase
(TvMGL; PDB 1E5E), which were retrieved from RSCB
protein data bank (http://www.rcsb.org/). Te structure
Fig. 1 Chemical structures of PFUR 4 and 4a‑k synthesized by the reaction of 2‑(H2NNHCO‑furan) and aryl aldehydes (EtOH, RCHO, r.t., 1–72 h,
40–97%)

Alves et al. Parasites Vectors
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Page 4 of 13
of T. vaginalis thioredoxin reductase (TvTrxR) was con- concentration (CC50) was calculated using GraphPad
structed using the server SWISS-MODEL [31] based on Prism 7.0 software. Te assay was performed at least
the UniProt entry A2DSU2. Target protein structures three independent times in triplicate. Te selectivity
were prepared by removing ligands, ions, and water index (SI) was calculated through the ratio CC50/IC50.
with CHIMERA 1.5.3 [32] software. Non-polar hydro-
gen atoms were merged, Gasteiger atomic charges were Statistical analysis
assigned, the atom type of protein structures was speci- Statistical analysis was carried out by two-way analysis of
fed, and the molecular docking simulation was per- variance (ANOVA) for the kinetic growth curve and for
formed using Autodock Vina 1.1.2 software [33]. Te all other assays by one-way ANOVA, both followed by
binding sites were defned by the co-complexed ligands in Tukey’s post-hoc test for multiple comparisons between
the crystal structure and the grid box was set up accord- groups. All tests were performed using GraphPad Prism
ing to the corresponding residues. PFUR 4a, PFUR 4b, 7.03 software, considering a probability value of P<0.05.
and MTZ were built and optimized in the software Avog- Data are expressed as the mean standard deviation
adro 1.1.1 [34]. Followed by structural optimization, the (SD).
ligands were prepared for docking by merging non-polar
hydrogen atoms, defning rotatable bonds and assigning
partial atomic charge using AutoDock Tools version 1.5.6
[35]. Docking poses of the investigated compounds were
visualized using Accelrys Discovery Studio 3.5.
Results
Anti‑T. vaginalis assay
Te screening for trichomonacidal activity showed the
potential of two compounds from the PFUR group, 4a
and 4b, as both reduced the trophozoites viability by
100% at 100 µM after 24 h (ANOVA: F(14, 30) =57.32,
Cell culture
Chinese Hamster Ovary (CHO-K1) cells obtained from P<0.0001). Moreover, PFUR 4h had no efect on tropho-
the Rio de Janeiro Cell Bank (PABCAM, Federal Univer- zoites growth, PFUR 4d and 4f were able to reduce
sity of Rio de Janeiro, RJ, Brazil) were grown as a mon- growth by 26.3% and 43.1%, respectively; however, such
olayer in Dulbecco’s Modifed Eagle’s Medium (DMEM) reduction was not signifcant when compared with MTZ,
(Vitrocell Embriolife, São Paulo, Brazil), supplemented as for other compounds (PFUR 4, 4c, 4e, 4g, 4i-k) viabil-
with 10% fetal bovine serum (Vitrocell Embriolife), 1% ity reduction rates varied from 5.5% to 18% and were not
l-glutamine, and 1% penicillin/streptomycin at 37 °C in statistically diferent from the negative control group
an atmosphere of 95% humidifed air and 5% CO2.
(ANOVA: F(14, 30)=57.32, P<0.0001) (Fig. 2). Unsur-
prisingly, 0.6% DMSO and negative controls induced
no reduction in viability, exhibited negative trypan blue
(0.4%) staining, and showed good motility and morphol-
ogy, whereas treatment with MTZ completely dimin-
ished viability and exhibited positive trypan blue staining
and negative motility.
Cytotoxicity assay
Te cytotoxicity assay was performed following a
method previously described [28, 36]. CHO-K1 cell
line viability and proliferation were determined by
measuring the formation of water-insoluble formazan
as a result of 3-(4,5-dimethythiazol-2-yl)–2,5-diphe-
nyl tetrazolium bromide (MTT) reduction. In 96-well
plates, 2 × 104 cells were seeded per well and grown at
37 °C, 5% CO2 for 24 h prior to the cell viability assay.
After that, the cells were treated with compounds
at 1.5625, 3.125, 6.25 and 12.5 μM for another 24 h
at 37 °C, 5% CO2. In addition, 0.6% DMSO and MTZ
at 100 μM were added as controls. After incubation,
MTT (Sigma-Aldrich) at 5 mg/ml was added per well
and the plates were re-incubated for 3 h at 37 °C, 5%
CO2. Subsequently, DMSO was added to solubilize
formazan crystals. Te absorbance was measured on a
microplate reader at a wavelength of 492 nm. Te inhi-
bition of cell growth was determined according to the
formula: Cell proliferation inhibition (%) = (1 − Abs492
Considering the full viability reduction caused by PFUR
4a and 4b, these compounds were chosen for antipara-
sitic and biochemical tests and molecular docking. Te
tests showed that PFUR 4a (ANOVA: F(9, 20)=317.1,
P<0.0001) and PFUR 4b (ANOVA: F(9, 20)=799.5,
P<0.0001) were able to completely reduce trophozoites
viability at 6.25 µM (Fig. 3a, b), which was determined as
MIC. IC50 was statistically found to be 1.69 µM and 1.98
µM for PFUR 4a and PFUR 4b, respectively, after 24 h
exposure (Table 1).
Te data obtained from kinetic growth curve showed
that PFUR 4a at 6.25 µM was able to reduce trophozoite
growth by 36.8% at 12 h and 98.7% at 24 h; conversely,
the negative control maintained full growth at all expo-
sure times and MTZ completely inhibited growth at 12 h
exposure (Two-way ANOVA: F(12, 36)=299.2, P<0.0001)
(Fig. 3c). As for PFUR 4b at 6.25 µM, growth reduction
cells/Abs492
cells) × 100. Te 50% cytotoxic
treated
control

Alves et al. Parasites Vectors
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Page 5 of 13
was 79.9% at 12 h and 99.7% at 24 h, whereas for the TvTrxR and TvMGL; however, no favorable binding
negative control and MTZ, the profle was the same as mode was found (data not shown).
described above (Two-way ANOVA: F(12, 36)=401.1,
In the case of PFUR 4b, Fig. 6a and b show that it
P<0.0001) (Fig. 3d). For both compounds, there were no may form four conventional hydrogen bonds and one
viable trophozoites after 24 h.
carbon-hydrogen bond (GLY89) with the active site
residues of the enzyme TvPNP, alongside other non-
covalent interactions that yield a docking score of − 6.8
kcal/mol (Table 2). PFUR 4b has a signifcant bind-
ing score of − 6.0 kcal/mol with TvTrxR as shown in
Fig. 6c and d and it could be involved in six hydrogen
bonds that might help in maintaining the binding mode
(Table 2).
TBARS assay
Data analysis demonstrated that treatment with 0.6%
DMSO did not increase lipid peroxidation levels when
compared with the control group (untreated tropho-
zoites). Te results also showed that MTZ, PFUR 4a,
and PFUR 4b treatments were unable to signifcantly
induce lipid peroxidation levels than those of the control
(ANOVA: F(4, 25)=4.901, P=0.0047) (Fig. 4).
It is worth mentioning that PFUR 4b might poten-
tially form seven hydrogen bonds with residues in the
active site of TvLDH (Fig. 6e, f, and Table 2). Tis bind-
ing mode is complemented with hydrophobic interac-
tions and Van der Waals forces, generating a ΔGbind of
−5.9 kcal/mol (Table 2). Furthermore, a likely binding
mode of PFUR 4b in the active site of TvMGL is shown
in Fig. 6g and h (docking score of −5.7 kcal/mol). Hydro-
gen bonds might be formed between PFUR 4b and three
residues of TvMGL, and other chemical interactions may
help in maintaining the binding mode of the compound
(Table 2).
Molecular docking
Next, a molecular docking study was carried out with the
two most active compounds, PFUR 4a and 4b, to investi-
gate their possible mechanism of action through their in
silico predicted interaction with proteins that play a role
in T. vaginalis survival.
We noticed that PFUR 4a interacts in silico with TvPNP
with binding free energy (ΔGbind) of −6.4 kcal/mol and
four residues may be involved in hydrogen bonds with
PFUR 4a (Fig. 5a, b, Table 2). As shown in Fig. 5c and d,
PFUR 4a could possibly form eight conventional hydro-
gen bonds and one carbon-hydrogen bond (SER240:
2.29 Å) with active site residues of the enzyme TvLDH,
alongside other non-covalent interaction that yield a
docking score of −5.8 kcal/mol (Table 2). We also per-
formed docking studies of PFUR 4a with enzymes
Considering that MTZ is the reference drug for T. vagi-
nalis treatment, we also investigated its possible interac-
tion with the abovementioned enzymes. In silico analysis
showed MTZ possibly binds to the active site of TvPNP
(ΔGbind of −5.0 kcal/mol; Fig. 7a, b), TvLDH (ΔGbind of
−5.2 kcal/mol; Fig. 7c, d), and TvMGL (ΔGbind of −4.2
kcal/mol; Fig. 7e, f). Te possible hydrogen bonds and
other interactions of MTZ with the enzymes can be seen
in Table 2.
Cytotoxic assay
Te MTT assay showed that PFUR 4a induced 18.6%
of growth inhibition at 6.25 µM and only 4.4% at 3.125
µM, none of the concentrations tested for this com-
pound were signifcantly diferent from MTZ (ANOVA:
F(5, 12)=15.98, P<0.0001), which caused 8.5% of inhibi-
tion (Fig. 8a). For PFUR 4b, the results demonstrated
8.8% of growth inhibition at 6.25 µM and 4.9% inhibi-
tion at 3.125 µM, once again none of the concentrations
tested were signifcantly diferent from MTZ (ANOVA:
F(5, 12)=9.427, P=0.0008) (Fig. 8b). As expected, 0.6%
DMSO did not alter cell growth and 1.5625 µM concen-
trations of both compounds also had no efect on cells.
Te CC50 for both compounds was higher than the high-
est concentration tested (12.5 µM) (Table 1) because less
than 50% of cell growth was inhibited at this concentra-
tion (Fig. 8a, b).
Fig. 2 In vitro trichomonacidal activity of PFUR 4 and 4a‑k at 100
µM against Trichomonas vaginalis ATCC 30236 isolate, confrmed
by the trypan blue (0.4%) assay after 24 h of exposure: Control
(untreated trophozoites), 0.6% DMSO (vehicle for solubilization), MTZ
(metronidazole at 100 µM). Viability of 100% in control corresponds to
2.6×105 trophozoites/ml. Data are presented as the mean standard
deviation of at least three independent experiments. Diferent letters
show a signifcant diference at P <0.05

Alves et al. Parasites Vectors
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Fig. 3 Anti‑Trichomonas vaginalis assay. MIC and IC50 for the antiparasitic activity of PFUR 4a (a) and PFUR 4b (b) against Trichomonas vaginalis
ATCC 30236 after exposure to 1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 µM concentrations for 24 h. Kinetic growth curves of Trichomonas vaginalis
ATCC 30236 after 1, 6, 12, 24, 48, 72 and 96 h of treatment with PFUR 4a (c) and PFUR 4b (d) at 6.25 µM. Growth was completely inhibited after 24 h.
Control (untreated trophozoites), 0.6% DMSO (vehicle for solubilization), MTZ (metronidazole at 100 µM). Viability of 100% in control corresponds to
2.6×105 trophozoites/ml. Data are presented as the mean standard deviation of at least three independent experiments. Diferent letters show a
signifcant diference at P <0.05
Table 1 Anti‑Trichomonas vaginalis activity and cytoxicity efects
of furanyl N‑acylhydrazone (PFUR) derivatives 4a and 4b
Compound
Trichomonas vaginalis
CHO‑K1 cells
CC50
MIC
IC50 SEa
SI
PFUR 4a
PFUR 4b
a
6.25
6.25
1.69 0.208
1.98 0.121
>12.5
>2.5
>7.4
>6.3
Standard error (SE) as calculated by GraphPad Prism 7.03 software
Notes: All results are expressed in μM, except for SI values. Trophozoites and cells
were exposed to PFUR 4a and 4b for 24h for all in vitro assays
Fig. 4 Lipid peroxidation levels measured through thiobarbituric
acid reactive substances assay with malondialdehyde (MDA) as
a biomarker, after 24 h of exposure to PFUR 4a and 4b, both at
6.25 µM. Control (untreated trophozoites), DMSO (vehicle for
solubilization), MTZ (metronidazole at 100 µM). Data are presented
as the mean standard deviation of at least three independent
experiments
Discussion
Trichomoniasis has a signifcant impact on public health
due to its high incidence and prevalence worldwide [1].
Te infection is associated with HIV acquisition and
transmission, a higher risk of preterm delivery, and a

Alves et al. Parasites Vectors
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Page 7 of 13
Fig. 5 Representation of 2D projection and predicted binding mode of PFUR 4a with the T. vaginalis enzymes TvPNP (a, b) and TvLDH (c, d). The
distance (Å) of the hydrogen bonds between specifc residues and PFUR 4a is shown in green
higher prevalence of HSV-1 and HSV-2 [37–40], aggra- a kinetic growth analysis showed that PFUR 4a and
vating its efect on people’s health. In addition, with an 4b, both at 6.25 µM (MIC), were able to fully reduce T.
increase in treatment failures resulting in recurrent vaginalis viability to a level that was not signifcantly
infections, there is an obvious need to develop new alter- diferent from MTZ after a 24 h treatment (Fig. 3c, d).
natives for treating trichomoniasis. Our data showed Similar kinetic growth inhibition profles have been dem-
two furanyl N-acylhydrazone derivatives, PFUR4a and onstrated in previous studies [28, 36]. Te furanyl N-acyl-
4b, presented in vitro antiprotozoal activity against T. hydrazone derivatives tested in this study were previously
vaginalis (Fig. 2) in promising concentrations and with investigated for their antitubercular potential and PFUR
a considerably good selectivity towards the parasite. Our 4b exhibited moderate activity against Mycobacterium
in silico and in vitro analyses indicated that these two tuberculosis with a MIC of 100.3 µM [26]. When com-
compounds are promising treatment alternatives, which pared, these results suggest that these compounds might
may act slightly diferently from the reference drug, and be more active/selective against protozoan species.
should be further explored.
Other N-acylhydrazone derivatives have been reported
In this context, we highlight that both PFUR 4a and 4b, as antiparasitic agents against Trypanosoma cruzi
which demonstrated a high trichomonacidal activity, pre- in vitro and in vivo in promising concentrations and also
sented a MIC value of 6.25 µM (Fig. 3a, b) and IC50 of showed low cytotoxicity against mammalian cells in both
1.69 µM and 1.98 µM, respectively (Table 1). Moreover, cases [41], consequently demonstrating a great level of

Alves et al. Parasites Vectors
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Table 2 Binding energies and molecular interactions predicted in silico between PFUR 4a, PFUR 4b, and MTZ, and enzymes important
to T. vaginalis survival
Compound Target enzyme Binding
No. of
Hydrogen bond residues
Hydrogen bond length (Å)
Non‑covalent interactions
energy (kcal/ hydrogen
mol)
bonds
4
PFUR 4a
TvPNP
−6.4
GLY92, PHE159, ANS161,
ALA167
3.22, 3.54, 3.17, 3.23
THR90, CYS91, THR156,
PHE159, GLN163, THR164,
LEU166, MET170, VAL178,
GLU179, MET180, SER203,
ASP204
PFUR 4a
PFUR 4b
TvLDH
TvPNP
−5.8
−6.8
8
4
ARG97, ARG161, HIS186,
THR239
2.09, 3.06, 1.95, 2.71, 2.49,
2.93, 2.17, 2.92
ILE16, SER88, MET89, PRO90,
ASN130, LEU157, ALA229
GLY20, ARG87, GLY89, THR90 3.02, 2.80, 3.57, 3.02
CYS19, ASP21, HIS62, VAL88,
GLY89, CYS91, GLY92,
THR156, PHE159, GLU179,
ILE206
PFUR 4b
TvLDH
−5.9
7
ARG97, ARG161, HIS186,
SER240
2.12, 3.02, 2.24, 2.83, 2.54,
3.09, 2.09
GLY14, GLN15, ILE16, SER88,
LEU91, ASN130, LEU157,
ALA229, TRP230
PFUR 4b
PFUR 4b
TvTrxR
TvMGL
−6.0
−5.7
6
3
GLN82, LYS167, SER172,
ALA173
3.35, 2.98, 3.19, 3.38, 2.87,
3.36
TYR85, THR86, LYS154, ALA155,
TYR157, ASN168, ASP178
TYR111, ANS158, LYS209
3.04, 2.80, 3.27
SER85, GLY86, MET87, ILE90,
ASP184, THR186, SER206,
THR208, VAL218
MTZ
TvPNP
−5.0
3
ASN161, GLN163, ALA167
2.89, 3.07, 2.98
PHE154, THR156, LEU158,
PHE159, THR164, LEU166,
MET170, VAL178, ILE206
MTZ
MTZ
TvLDH
TvMGL
−5.2
−4.2
5
4
LEU157, ARG161, HIS186,
SER240
2.08, 2.49, 2.30, 2.30, 2.34
2.99, 3.06, 2.98, 3.27
ILE16, ILE128, ASN130, LEU154,
ALA229, THR239, PRO244
SER206, LYS209
SER85, GLY86, MET87, TYR111,
THR208, VAL218, VAL337,
SER338
Abbreviations: MTZ, metronidazole; TvPNP, T. vaginalis purine nucleoside phosphorylase; TvLDH, T. vaginalis lactate dehydrogenase; TvTrxR, T. vaginalis thioredoxin
reductase; TvMGL, T. vaginalis methionine gamma-lyase
selectivity towards protozoan species. However, to the reported here. Similarly, a previous study on N-acyl-
best of authors’ knowledge there are no prior reports hydrazones containing 4-hydroxy-3-methoxyphenyl
of compounds containing the N-acylhydrazone moiety showed antiparasitic activity against Plasmodium falcipa-
being active against T. vaginalis, or a species of the genus rum demonstrating that the addition of a nitro group sig-
Trichomonas. As for the furan moiety, previous studies nifcantly increased the compound’s activity, indicating
have shown that compounds containing this pharmaco- that nitro groups could either induce or enhance antipar-
phoric group are active against T. vaginalis and Giardia asitic activity against protozoan species [19]. In addition,
lamblia [42, 43]. Tese studies substantiate the antipro- the structural resemblance of compounds PFUR 4a and
tozoan potential of furanyl N-acylhydrazone derivatives 4b could help elucidate the similar results observed in the
demonstrated in our results.
in vitro antiparasitic and biochemical assays performed
Te compounds PFUR 4a and 4b are quite similar in here.
structure, the former having a 5-nitrofuran-2-yl radi-
Moreover, the TBARS assay demonstrated that T. vagi-
cal and the latter a 5-nitrothiopen-2-yl radical, and both nalis trophozoites exposed to PFUR 4a and 4b for 24 h
compounds are distinguished from the other 10 com- did not present a level of lipid peroxidation signifcantly
pounds by having this nitro (NO2) substituent in their higher than the one observed for the control group
structure. Similarly, MTZ also has a nitro group in its (Fig. 4). Considering that lipid peroxidation is a chain of
structure and this group plays an essential role in the reactions mediated by free radicals that results in an oxi-
production of toxic intermediates after its reduction and dative deterioration of polyunsaturated lipids, which are
MTZ activation [44]. Considering these similarities, it abundant in components of biological membranes [45],
has been speculated that nitro groups of PFUR 4a and these results indicate that membrane damage is not a sig-
4b might play a role in altering the redox equilibrium in nifcant factor to be considered as a mechanism by which
hydrogenosomes leading to the trichomonacidal activity these compounds induce T. vaginalis death.

Alves et al. Parasites Vectors
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Fig. 6 Representation of 2D projection and predicted binding mode of PFUR 4b with the T. vaginalis enzymes TvPNP (a, b), TvTrxR (c, d), TvLDH (e, f)
and TvMGL (g, h). The distance (Å) of the hydrogen bonds between specifc residues and PFUR 4b is shown in green

Alves et al. Parasites Vectors
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Fig. 7 Representation of 2D projection and predicted binding mode of metronidazole (MTZ) with the T. vaginalis enzymes TvPNP (a, b), TvLDH (c, d)
and TvMGL (e, f). The distance (Å) of the hydrogen bonds between specifc residues and MTZ is shown in green

Alves et al. Parasites Vectors
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Fig. 8 Cytotoxicity efect of PFUR 4a (a) and PFUR 4b (b) at 1.5625, 3.125, 6.25 and 12.5 µM on CHO‑K1 cells through MTT assay after 24 h of
exposure; 0.6% DMSO (vehicle for solubilization) and MTZ (metronidazole at 100 µM). Data are presented as the mean standard deviation of at
least three independent experiments. Diferent letters show a signifcant diference between treatments at P <0.05
Docking studies were performed to gain insight into is a potential drug target for anti-T. vaginalis chemo-
the binding mode of compounds, PFUR 4a and PFUR 4b, therapy since it is not found in mammals, and the struc-
with enzymes required for T. vaginalis survival. PFUR 4a ture of TvTrxR is very diferent from human TrxR [18],
presented a satisfactory binding mode with TvPNP and highlighting the biological application of PFUR 4b. None-
TvLDH, while PFUR 4b interacted in silico with TvPNP, theless, more studies must be conducted to confrm this
TvTrxR, TvLDH and TvMGL (Figs. 5, 6 and Table 2). Te hypothesis and improve our understanding of the mecha-
results indicate that these enzymes could be somehow nism of action for PFUR 4a and PFUR 4b.
involved in these compounds’ action against T. vaginalis.
In order to establish a comparison between the puta-
Trichomonas vaginalis PNP is a well-known potential tive efects of PFUR 4a and 4b and the known activity
target for chemotherapy [46, 47] and both compounds of MTZ, we also analyzed the possible binding mode of
interact in silico with key residues in the active site of the MTZ with the enzymes investigated here. Interestingly,
enzyme, which are relevant for the inhibition of its activ- the in silico interactions of TvPNP, TvLDH, and TvMGL
ity, indicating they may act as TvPNP inhibitors. Based were stronger with PFUR 4a and 4b than with MTZ
on the metabolic defciency of T. vaginalis towards the (Fig. 7 and Table 2). In T. vaginalis, among the actions
lack of de novo synthesis of purine nucleosides and the described [11, 44, 48], MTZ is said to disrupt the DNA
requirement of purine salvage to maintain their purine helical structure inhibiting the synthesis of proteins
nucleotide pools in order to survive [16], modulation of that result in cell death [44, 48]; therefore, inhibiting
this pathway may be involved in the mechanism of action the enzymes investigated here may not be required for
of PFUR 4a and PFUR 4b against T. vaginalis, highlight- the action of MTZ. Tis indicates that the mechanism
ing the signifcance of this predicted interaction. Further- of action of PFUR 4a and 4b might be somewhat difer-
more, based on the signifcance of TvLDH for parasite ent from the mechanism of action of MTZ, highlighting
survival and in silico interactions described, impairment that PFUR 4a and 4b may act via modulation of energy
of energy metabolism could also explain the reduction in metabolism, antioxidant defenses, and the content of
viability of the fagellated parasitic protozoan T. vaginalis sulfur-containing amino acids. Nonetheless, more studies
by PFUR 4a and PFUR 4b.
are needed to support this hypothesis.
Likewise, in silico interaction of PFUR 4b with key
Te cytotoxicity assay performed in CHO-K1 cells
residues in the active site of TvTrxR and TvMGL could showed that at their MIC (6.25 µM), both compounds
indicate a possible modulation of both enzymes, prob- exhibited no cytotoxic efect on non-tumor/normal cells
ably through their inhibition, by this compound. Con- (Fig. 8a, b) according to the standards set by the Inter-
sidering the exclusivity of in silico interaction of PFUR national Organization for Standardization (ISO) at ISO
4b with TvTrxR and TvMGL when compared to PFUR 10993-5:2009, which states that a decrease of cell viabil-
4a, we may hypothesize that modulation of antioxidant ity by more than 30% is considered a cytotoxic efect
defense (through TvTrxR) and the content of sulfur- [49, 50]. Terefore, since PFUR 4a and PFUR 4b reduced
containing amino acids (via TvMGL) could account for viability by 18.6% and 8.8% at their MIC, respectively,
the faster onset of action of PFUR 4b when compared to this inhibition of growth does not represent a cytotoxic
PFUR 4b (Fig. 3c, d). It is worth mentioning that TvMGL efect. In addition, levels of cell growth inhibition are

Alves et al. Parasites Vectors
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Page 12 of 13
Funding
signifcantly the same as the one found for MTZ, dem-
onstrating that these compounds do not afect cells more
than the commercial drugs used for trichomoniasis.
Lastly, both compounds showed a promising SI (Table 1),
which can be much better than currently presented con-
sidering that the higher concentration tested (12.5 µM)
inhibited less than 20% of the mammalian cells. Alto-
gether, these results emphasize the potential application
of these molecules.
This study was partially fnanced by the Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior‑Brazil (CAPES)‑Finance Code 001‑Masters Scholar‑
ship (MSDA, Grant Number 1693097).
Availability of data and materials
Data supporting the conclusions of this article are provided within the article.
The datasets used and/or analyzed during the present study are available from
the corresponding author upon reasonable request.
Ethics approval and consent to participate
Not applicable.
Finally, the need for new and efective molecules for
treating trichomoniasis is emerging exponentially and all
data gathered here show that furanyl N-acylhydrazone
derivatives, PFUR 4a and 4b, have a highly signifcant
antiparasitic activity against T. vaginalis with a great level
of selectivity for the parasite over non-tumor/normal
cells and are, therefore, promising molecules for studies
on new trichomonacidal agents.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Laboratório de Biotecnologia Infecto‑parasitária, Centro de Desenvolvimento
Tecnológico, Biotecnologia, UFPel, Pelotas, RS 96010‑900, Brazil. 2 Laboratório
de Neurobiotecnologia, Centro de Desenvolvimento Tecnológico, Biotec‑
nologia, UFPel, Pelotas, RS 96010‑900, Brazil. 3 Laboratório de Biotecnologia
do Câncer, Centro de Desenvolvimento Tecnológico, Biotecnologia, UFPel,
Pelotas, RS 96010‑900, Brazil. 4 Instituto de Tecnologia em Fármacos‑Far‑Man‑
guinhos, Fiocruz‑Fundação Oswaldo Cruz, Rio de Janeiro, RJ 21041‑250, Brazil.
5 Programa de Pós‑Graduação em Química, Instituto de Química, Universidade
Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ 21945‑970,
Brazil.
Conclusions
Our study demonstrates that furanyl N-acylhydrazone
derivatives PFUR 4a and PFUR 4b have a potent anti-T.
vaginalis activity in vitro, which is statistically similar to
the reference drug. At low concentrations, these com-
pounds show good and promising selectivity towards T.
vaginalis. Moreover, molecular docking analyses point
to the modulation of enzymes TvPNP, TvLDH, TvTrxR,
and TvMGL by these compounds. In conclusion, our data
characterized PFUR 4a and PFUR 4b as promising trich-
omonacidal agents and support further studies not only
with these compounds but also other N-acylhydrazone
derivatives, as alternatives for treating trichomoniasis.
Received: 19 August 2019 Accepted: 1 February 2020
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