Antitrichomonal activity and docking analysis of thiazole derivatives as TvMP50 protease inhibitors

Article abstract of DOI:10.1007/s00436-020-06931-w

Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is the most prevalent non-viral sexually transmitted infection that affects over 170 million people worldwide. The only type of drug recommended for the therapeutic control of trichomoniasis i

Full text of DOI:10.1007/s00436-020-06931-w

Parasitology Research
https://doi.org/10.1007/s00436-020-06931-w
PROTOZOOLOGY - ORIGINAL PAPER
Antitrichomonal activity and docking analysis of thiazole derivatives
as TvMP50 protease inhibitors
Gonzalo Mena-Rejón1 & Yussel Pérez-Navarro2 & Julio César Torres-Romero1 & Laura Vázquez-Carrillo2
&
Rubén M. Carballo1 & Rodrigo Arreola3 & Ángel Herrera-España1 & Victor Arana-Argáez1 & Ramiro Quijano-Quiñones1
Jose Manuel Fernández-Sánchez4 & María Elizbeth Alvarez-Sánchez2
&
Received: 22 June 2020 /Accepted: 11 October 2020
#
Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is the most prevalent non-viral sexually transmitted infection
that affects over 170 million people worldwide. The only type of drug recommended for the therapeutic control of trichomoniasis
is the 5-nitroimidazoles, although there have been reports of some undesirable side effects and clinical resistance. Hence, the need
for the search for new tricomonicidal agents is necessary. In a previous work, we demonstrated that two 2-amino-4-aryl thiazole
derivatives (ATZ-1 and ATZ-2) possess a portent antigiardial effect. In the current paper, we investigated the in vitro
antitrichomonal activity of these thiazole compounds. Both ATZ-1 and ATZ-2 reduced the viability and growth of parasites in
a dose-dependent manner, with an IC50 value of 0.15 μg/mL and 0.18 μg/mL, respectively. Furthermore, both thiazole com-
pounds were able to decrease the proteolytic activity in T. vaginalis trophozoites compared with untreated parasites. Interestingly,
a full proteolytic inhibition profile was observed in the 50-kDa region which was associated with the decreased expression of the
gene that codes for the trichomonad protease TvMP50. The docking simulations predicted strong interactions of the thiazole
compounds in the TvMP50 protease’s active site, suggesting a possible role as protease inhibitors. Our results demonstrate the
potential of 2-amino-4-aryl thiazole derivatives as trichomonicidal compounds and could be, mechanistically, involved in the
inhibition of key trichomonad proteases.
.
.
.
.
Keywords Trichomoniasis T. vaginalis Thiazole Anti-proteolytic TvMP50
Introduction
Sexually transmitted infections (STIs) are a leading cause of
Section Editor: Kevin S.W. Tan
acute illness, sterility, long-term incapacity, and even death,
with several and serious consequences among millions of in-
dividuals around the world. These infections include those
sexually transmitted bacterial, mycological, viral, and parasit-
ic pathogens (Gewirtzman et al. 2011). Trichomonas
vaginalis is the only known parasite to cause the most frequent
non-viral STI worldwide, denominated trichomoniasis
(Kissinger 2015). The World Health Organization (WHO)
estimates an incidence of 170–190 million new cases of
trichomoniasis worldwide every year, affecting both men
and women. The only class of antimicrobial drug known to
be viable against T. vaginalis infections is the nitroimidazoles.
Even with adequate available therapy, the trichomoniasis has
remained a public health concern on both industrialized and
developing countries, partially because of the dependence on a
single drug class for its treatment, which could become
* María Elizbeth Alvarez-Sánchez
elizbeth2@gmail.com; maria.alvarez@uacm.edu.mx
1
Facultad de Química, Universidad Autónoma de Yucatán (UADY),
Calle 43 S/N entre calle 96 y calle 40, Colonia Inalámbrica, C.P.
97069 Mérida, Yucatán, México
2
Posgrado en Ciencias Genómicas, Universidad Autónoma de la
Ciudad de México (UACM), San Lorenzo # 290, Col. Del Valle, CP
03100 México City, México
3
Psychiatric Genetics Department, Clinical Research Branch,
National Institute of Psychiatry, Ramón de la Fuente, Calzada
México-Xochimilco 101, Colonia San Lorenzo Huipulco, Tlalpan,
14370 México City, DF, México
4
División de Ingeniería en Gestión Empresarial, Tecnológico de
Estudios Superiores de Ecatepec, Avenida Tecnológico S/N, Colonia
Valle de Anahuac, Ecatepec de Morelos, Estado de México, Mexico

Parasitol Res
problematic if nitroimidazole resistance between T. vaginalis
strains increased (Cudmore and Garber 2009; Kirkcaldy et al.
2012). With this situation in mind, research goals for address-
ing trichomoniasis must include designing and testing new
antitrichomonal agents.
Materials and methods
Chemistry
Synthesis of the 2-Amino-4-arylthiazole derivatives used
herein was described previously (Mocelo-Castell et al.
2015). Production of these derivatives was carried out in the
Pharmaceutical Chemistry Laboratory of the Chemistry
Faculty of the Autonomous University of Yucatan, in three
steps (Fig. 1). During the first step, the thiazole core was
obtained by reaction between 1-(4-bromophenyl) ethan-1-
one or 1-(p-tolyl)ethan-1-one, with thiourea and iodine under
microwave irradiation. In the second step, the compounds
obtained, 4-(p-tolyl)thiazol-2-amine and 4-(4-
chlorophenyl)thiazol-2-amine, were treated with acetic anhy-
dride to obtain N-(4-(4-chlorophenyl)thiazol-2-yl)acetamide
and N-(4-(p-tolyl)thiazol-2-yl)acetamide, respectively.
Finally, these latter compounds were treated with molecular
bromine to obtain the desired compounds.
T. vaginalis infection is a multifactorial process where
some proteinases, such as metalloproteinases, cysteine pro-
teinases, and a rhomboid proteinase (TvROM1), have been
implicated as major components on host cell adhesion and
cytotoxicity (Mercer and Johnson 2018. The TvMP50 metal-
loproteinase is an immunogenic protein that is recognized by
sera from male patients with trichomoniasis (Quintas-
Granados et al. 2013). In previous reports, TvMP50 was iden-
tified as a proteolytic band of ~ 50 kDa in gelatin zymogram,
either as a native or recombinant protein, indicating that it acts
as an active monomer, presumably derived from the ancestral
aminopeptidase P-like enzymes (Puente-Rivera et al. 2017;
Arreola et al. 2018). Interestingly, the proteolytic activity of
TvMP50 is upregulated by zinc and is associated with the
cytotoxicity of T. vaginalis to the host cell, which was reduced
when specific metalloproteinase inhibitors were used (Puente-
Rivera et al. 2017). Therefore, TvMP50 is an attractive target
for the development of new antitrichomonal drugs.
In vitro susceptibility assays against T. vaginalis
trophozoites
Thiazole derivatives are an important class of compounds
with a wide range of medical applications (Chhabria et al.
2016). Regarding protease inhibition, it is important to men-
tion two drugs: ritonavir and simeprevir, which contain thia-
zole rings and are potent, are FDA-approved, and are protease
inhibitors for the treatment of HIV and HCV infections, re-
spectively (Kempf et al. 1998; Li and De Clercq 2017). In a
similar way, thiazole derivatives have been shown to be potent
antiparasitic agents through anti-proteolytic activity. For ex-
ample, trisubstituted thiazole analogs were shown capable of
inhibiting the cysteine proteinases falcipain-2 and falcipain-3
from Plasmodium falciparum (Goud et al. 2005). In addition,
previous reports have shown that proteases from some
trypanosomatid species are thiazole derivative targets, which
block their action and cause the death of parasites (de Oliveira
Cardoso et al. 2014; de Moraes Gomes et al. 2016; Gomes de
Oliveira et al. 2020).
Our group conducts research on the identification and de-
velopment of potent antiparasitic compounds. An example of
this was the one performed by Mocelo-Castell et al. in 2015,
where a set of 50 thiazole derivatives (2-amino-4-
arylthiazoles) were obtained, and their activity against
Giardia intestinalis was tested. Among them, two bromo-
analogues bearing an N-acetyl group exhibited the highest
in vitro activity against G. intestinalis trophozoites.
Considering these findings, we decided to evaluate the extend-
ed antiparasitic capability of these two compounds and further
deepen in their mechanism of action. Here, we show that these
thiazole derivatives exhibit potent activity against T. vaginalis
as well as the inhibition of the TvMP50 metalloproteinase.
To determine the effect of 2-amino-4-aryl thiazole derivatives
on T. vaginalis growth, in vitro susceptibility assays were
performed as previously described (Cáceres-Castillo et al.
2019). Briefly, all compounds were dissolved in DMSO at a
stock concentration of 1 mg/mL. For the assay, 12.5 × 106
T. vaginalis trophozoites were incubated in the presence of
serially diluted thiazole derivatives (0.1 to 1.0 μg/mL) in
TYM medium for 24 h at 37 °C. After different time intervals,
cells were counted with an automated cell counter (TC10,
Bio-Rad). Following incubation, parasite viability was
checked by Trypan Blue exclusion assay. Trophozoites treat-
ed with either 0.1% DMSO or 6.0 μM metronidazole (MTZ)
for 24 h were used as vehicle control and positive control,
respectively. All experiments were performed by triplicate.
IC50 (concentration causing 50% inhibition) values were cal-
culated by probit analysis. The analyses were made by using
one-way ANOVA, followed by a post hoc Dunnett’s test.
Statistical significance was accepted at p < 0.05.
Gelatin substrate gel electrophoresis
To evaluate the potential anti-proteolytic activity of the 2-
amino-4-aryl thiazole derivatives, a gelatin substrate gel elec-
trophoresis was performed as previously described (Vazquez
Carrillo et al. 2011). Briefly, T. vaginalis trophozoites were
grown in the presence of thiazole derivatives (0.1–0.2 μg/
mL), vehicle control (0.1% DMSO), or positive control
(6.0 μM MTZ) in TYM medium for 24 h at 37 °C.
Following incubation, 2 × 107 cells were harvested by centri-
fugation at 500×g for 10 min at 4 °C and washed twice with

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Fig. 1 Reaction scheme for synthesis of the 2-amino-4-arylthiazole derivatives used in this study. In the compounds, R was methyl for N-(5-bromo-4-p-
tolyl-thiazol-2-yl)-acetamide or chloro for N-[5-bromo-4-(4-chloro-phenyl)-thiazol-2-yl]-acetamide
cold PBS pH 7.0. The pellet was resuspended in 30 μl of
Laemmli buffer without boiling, and the protein concentration
was determined by Bradford method, and then 50 μg of the
protein was charged for each well onto a 0.2% gelatin
copolymerized 10% SDS PAGE and run at 35 mA/gel until
the tracking dye reached the bottom of the gel. The gel was
soaked in 2.5% Triton-X 100 for 1 h at room temperature to
eliminate SDS. The renatured gel was further incubated for 3 h
at 37 °C in 0.2 M Tris–HCl buffer pH 8.2 with 0.1% β-
mercaptoethanol to activate the proteinases. Finally, the gel
was stained with Coomassie blue, and the clear bands were
considered as positive proteolytic activity. The degree of pro-
teolytic activity was semi-quantified by densitometric analysis
of clear bands using version 4.6.3 of the Quantity One soft-
ware (BioRad, Hercules, CA). The analyses were made by
using one-way ANOVA, followed by post hoc Dunnett’s test.
Statistical significance was accepted at p < 0.05.
in 1 mL of TRIzol® reagent, and total RNA was extracted
according to the manufacturer’s instructions.
Following extraction, 1 μg of total RNA was treated with
DNAse I to eliminate genomic DNA remnants. Next, 200 ng of
DNAse I treated-RNA was retro-transcribed using the
SuperScript™ II Reverse Transcriptase Kit. To evaluate the
mRNA level of mp50 gene (TVAG_403460), a reverse tran-
scription polymerase chain reaction was performed as previous-
ly described (Puente-Rivera et al. 2017), using 0.2 μL cDNA
preparation as a template. The constitutively expressed β-tubulin
gene in T. vaginalis was used as an internal control to normalize
the results. PCR amplicons were visualized on 1% agarose gels
stained with ethidium bromide, and the semi-quantitative mea-
surement was achieved by digital analysis using version 4.6.3 of
the Quantity One software (BioRad, Hercules, CA).
Molecular docking and cavities
Docking calculation was carried out in the Swissdock, an
online docking web service accessible via the ExPASy web
server from the Swiss Institute of Bioinformatics based on the
EADock DSS docking software (Grosdidier et al. 2011a; b).
Parameters used in Swissdock were docking type: accurate;
passive_flexibility_distance 5.0; wanted_confs 5000;
nb_facts_eval 5000; nb_seeds 250; sd_steps 100; abnrsteps
250; clusterin_gradius 2.0 and max_cluster_size 8. The coor-
dinate files (protein and ligands) were prepared with the pro-
gram Openbabel v. 2.4.0 (O'Boyle et al. 2011), and the visu-
alization of the data result provided by SwissDock was
Gene expression
A semi-quantitative reverse transcription polymerase chain
reaction was performed to evaluate the mRNA levels of
mp50 gene on thiazole derivative-treated T. vaginalis tropho-
zoites incubated for 24 h. Trophozoites incubated with vehicle
control (0.1% DMSO) or positive control (6.0 μM MTZ) were
used for comparison. Briefly, 1 × 106 parasites were harvested
by centrifugation at 500×g for 10 min at 4 °C and washed
twice with cold PBS pH 7.0, and the pellet was resuspended

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Fig. 2 Chemical structure of thiazole derivatives (ATZ-1 and ATZ-2) evaluated against T. vaginalis
analyzed with v. 1.13.1 of the Chimera software (Pettersen
et al. 2004) using the Viewdock tool. Protein coordinates of
the final published model from TvMP50 were obtained
(structure not deposited in the Protein Data Bank. See
publication Arreola et al. 2018), and the compounds (thiazole
derivatives, MTZ, and fluoxetine) were built in ACD/
ChemSketch freeware 5.12 software (www.acdlabs.com)
and affined in Chimera with the Molecular Dynamics
Simulation Tool. Each compound of interest was tested with
three docking conditions: (1) TvMP50 with empty active site
(free of metals and inactive), (2) TvMP50 with a Ca2+ in the
active site (inactive enzyme), and (3) TvMP50 with two Zn2+
in the active site (active enzyme). Fluoxetine was used as
control molecule not related with the enzyme and metronida-
zole as active compound against T. vaginalis. The crystal
structure showed a Ca2+ and a Ni2+ on the active site; the
enzyme was active with Zn2+. We modeled the hypothetical
correct functional metals with two Zn2+ and a nonfunctional
d
Fig. 3 Trichomonicidal activity of thiazole derivatives. The viability of
T. vaginalis trophozoites was measured in the presence of increasing
concentrations of ATZ-1 compound (a) or ATZ-2 compound (b) for 24
h. Mean standard deviation from three biological replicate experiments
are shown. ap < 0.01; versus vehicle control (DMSO at 0.1%); bp < 0.05;
treated group (MTZ at 6 μM); p < 0.05; versus metronidazole-treated
e
group (MTZ at 6 μM); p < 0.05; versus preceding concentration of
thiazol compound. c Growth kinetics of T. vaginalis trophozoites during
24 h of incubation in the presence of both thiazole compounds at 0.2
μg7mL
c
versus vehicle con(DMSO at 0.1%); p < 0.01; versus metronidazole-

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enzyme with one Ca2+. The selection criteria for the cluster
candidates were using the 40 best predictions considering the
Delta G (DG) values and the Fullfitness (FFN) values (20 best
predictions for each one). The initial images were built with
Chimera and edited with Gimp v 2.10.18.
the trophozoites viability by > 95% at 1.0 μg/mL after 24-h
exposure (Fig. 3a, b), revealing a concentration-dependent
inhibition, with an IC50 value of 0.15 μg/mL for ATZ-1 and
0.18 μg/mL for ATZ-2. Incubation with vehicle (DMSO,
0.1%) did not exhibit significant differences in parasite viabil-
ity compared with untreated trophozoites (control).
Considering that the IC50 values for both compounds were
close to the tested concentration of 0.2 μg/mL, these were used
to evaluate a kinetic growth curve for a period of 24 h by mea-
suring the number of parasites. Both compounds decreased the
T. vaginalis growth in a similar way, exhibiting a reduction of
approximately 35% in comparison with the corresponding ve-
hicle control group after 6-h exposure, and the maximal growth
inhibition effect was observed after 24-h exposure, decreasing
T. vaginalis growth by approximately 63% (Fig. 3c).
Since there is a correlation between the proteolytic activity
with the T. vaginalis virulence, as well as with the anti-
proteolytic capability of the thiazole derivatives, we searched
for possible protease inhibitory activity by ATZ-1 and ATZ-2.
Figure 4 shows representative photographs of gelatin
zymography, which reveal several gelatinolytic proteins in
the T. vaginalis lysate with different molecular weights
(panels A and B, DMSO vehicle control lane 1). ATZ-1 at
the highest concentration tested (0.2 μg/mL) was able to
Results
Previous work in our lab established a high-throughput
screening of thiazole derivatives to identify anti-Giardia com-
pounds. Through this screening, we identified two potent 2-
amino-4-arylthiazoles derivatives, N-(5-Bromo-4-p-tolyl-
thiazol-2-yl)-acetamide and N-[5-bromo-4-(4-chloro-phe-
nyl)-thiazol-2-yl]-acetamide, which were named here ATZ-1
and ATZ-2, respectively (Fig. 2).
To confirm the antiparasitic activity of the 2-amino-4-
arylthiazoles derivatives mentioned above, we tested their
ability to inhibit T. vaginalis growth using the in vitro suscep-
tibility assay. Results showed that treatment with 0.1 μg/mL
of ATZ-1 or ATZ-2 decreased T. vaginalis viability by 31%
and 20%, respectively, whereas treatment with 0.2 μg/mL of
each compound decreased T. vaginalis viability by approxi-
mately 65% (Fig. 3a, b). Moreover, both compounds reduced
Fig. 4 Effect of ATZ-1 and ATZ-2 compounds on proteolytic activity of
total cellular extract of T. vaginalis. Gelatin substrate gel electrophoresis
showing a decrease of the proteolytic activity of the parasites incubated in
the presence of ATZ-1 compound (a) or ATZ-2 compound (b) for 24 h, in
comparison with the corresponding vehicle control group (DMSO at
0.1%). Metronidazole (MTZ) was used as a control to show protease
activity inhibition. The complete inhibition of the 50-kDa region is indi-
cated by an arrowhead. c, d show the densitometric analysis (mean
standard deviation of at least three independent measurements) to the
corresponding inhibition by ATZ-1 and AT-2, respectively.
Significances are shown in comparison with the vehicle control. *p <
0.05, **p < 0.01

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inhibit the trichomonal protease activity (Fig. 4a, lane 4) com-
parable with the classical trichomonicidal drug metronidazole
(Fig. 4a, MTZ 6 μM, lane 2). Moreover, the observed protease
inhibition activity by ATZ-2 at 0.2 μg/mL concentration was
approximately 3-fold lower than the one exhibited with the
vehicle (Fig. 4b, lane 4). Also, at a lower concentration of 0.1
μg/mL of the 2-amino-4-arylthiazoles derivatives, a signifi-
cant reduction in proteolytic activity was observed (Fig.
4a, b, lanes 3). Interestingly, the proteolytic activity in the
50-kDa region was inhibited by both ATZ-1 and ATZ-2
(Fig 4a and b, black arrow head).
These latter results suggest that the mechanism of action of
these thiazole derivatives could be related to enzymatic dis-
ruption via inhibition of key proteases in T. vaginalis, as the
metalloproteinase TvMP50, involved in their cytotoxic effect
to host cells. To test this hypothesis, we performed gene ex-
pression assays to evaluate the transcription levels of the mp50
gene. The results revealed that the expression levels of the
mp50 transcript were reduced after the treatment with both
ATZ-1 and ATZ-2 for 24 h (Fig. 5). Interestingly, the thiazole
derivatives exhibited stronger reduction in the transcription of
mp50 rather than the corresponding vehicle control group
(Fig. 5a,b). Notably, the expression of mp50 was almost
completely inhibited by ATZ-1, at the highest concentration
tested (0.2 μg/mL), which was able to reduce the transcript
levels approximately 2-fold lower than the one exhibited by
MTZ (Fig. 5a, lane 4).
To determine the possible mechanism for TvMP50 inhibi-
tion through their possible interaction with the ATZ-1 and
ATZ-2 derivatives, a molecular docking was carried out.
The selected prediction results suggest that ATZ-1 and
ATZ-2 compounds can bind to the active site of the
TvMP50 metalloproteinase (Fig. 6). The presence of ATZ-1
and ATZ-2 in the active site of TvMP50 can be observed only
in the complex with Zn2+ and, accordingly, the DG and FFN
values increase (Table 1). The TvMP50 empty and the com-
plex with Ca2+ do not show a binding mode, and the DG and
FFN values are lower than in the TvMP50-Zn2+ complex. The
control molecule fluoxetine, as expected for a non-related
compound, is not observed in the active site in presences of
metals. However, we observed that it could bind to the active
site when it is empty, probably due to the presence of the
acidic cluster residues with negative charges. Regarding
MTZ, both TvMP50 metal complexes showed the presence
of the molecule with elevated DG and FFN values unlike with
the empty enzyme, although TvMP50-Zn2+ complex exhibit-
ed the higher DG values. Docking suggests that MTZ is ca-
pable of binding different metal clusters on proteins, probably
a secondary drug activity, not described before.
Discussion
Two indisputable trichomoniasis facts are that it is the most
common non-viral STI and that it is the only known STI
caused by a protozoan parasite. Nowadays, chemotherapy
against trichomoniasis involves mainly the 5-
nitroimidazole members, either metronidazole or
tinidazole. However, all of these have been reported having
undesirable side effects, and antibiotic resistance has al-
ready increased considerably because of treatment failure
(Alessio and Nyirjesy 2019). Therefore, the identification
of new molecules with trichomonicidal activity is still nec-
essary to develop new chemotherapeutic alternatives
against this parasite.
Thiazole and its derivatives have established as sources of
lid compounds for new promises of medicinal agents. In a
previous work, we showed a significant antigiardial effect of
two 2-amino-4-arylthiazoles derivatives, N-(5-bromo-4-p-tol-
yl-thiazol-2-yl)-acetamide (ATZ-1) and N-[5-Bromo-4-(4-
chloro-phenyl)-thiazol-2-yl]-acetamide (ATZ-2) (Mocelo-
Castell et al. 2015). Considering the close relationship be-
tween the metabolisms and treatment for Giardia and
Fig. 5 The expression of TvMP50 is affected by ATZ-1 and ATZ-2
compounds. a RT-PCR reaction showing the differential expression of
mp50 after the growth of T. vaginalis in presence of thiazole compounds
for 24 h. ATZ-1 and ATZ-2 treated-parasites at 0.2 μg/mL showed a
significant decrease of mp50 expression, similar to the one observed in
MTZ-treated parasites. b Relative expression was analyzed by measuring
the RT-PCR band intensity using the Quantity One software version 4.6.3
and represented as relative fold change in expression

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Table 1 Docking predictions. Delta G (DG) and Fullfitness (FFN) values of the 40 best predictions
Active site empty
TvMP50-Ca2+
TvMP50-Zn2+
ATZ-1
256 predictions
− 7.5165 to − 7.1492
− 2738.79 to − 2734.98
Neither
256 predictions
255 predictions
Delta G (kcal/mol)
Fullfitness
− 7.5134 to − 7.1686
− 3141.96 to − 3138.20
Neither
− 8.1060 to − 7.8420
− 3121.26 to − 3115.34
Yes
DG 20
FFN 15
ATZ-1 presence on the active site,
in the 40 best predictions
ATZ-2
254 predictions
− 7.2714 to − 7.1418
− 2737.57 to − 2735.26
Neither
256 predictions
256 predictions
Delta G (Kcal/mol)
Fullfitness
− 7.3707 to − 7.2086
− 3140.17 to − 3137.73
Neither
− 8.8475 to − 8.0580
− 3127.58 to − 3115.31
Yes
DG 20
FFN 20
ATZ-2 presence on the active site,
in the 40 best predictions
Metronidazole
Delta G (Kcal/mol)
Fullfitness
256 predictions
− 7.1060 to − 6.2916
− 2674.40 to − 2672.43
neither
255 predictions
256 predictions
− 11.7528 to − 6.7329
− 3086.21 to − 3076.66
Yes
DG:2
FFN:11
− 15.2291 to − 14.0447
− 3152.62 to − 3135.38
Yes
DG:20
FFN:20
Metronidazole presence on the active site,
in the 40 best predictions
Fluoxetine
256 predictions
256 predictions
250 predictions
− 9.2339 to − 8.8467
− 3105.93 to − 3099.9
Neither
Delta G (Kcal/mol)
Fullfitness
− 11.453 to − 9.9405
− 2726.42 to − 2721.21
Yes
DG 20
FFN 5
− 9.2410 to − 8.6421
− 3129.83 to − 3124.12
Neither
Fluoxetine presence on the active site,
in the 40 best predictions
T. vaginalis, the present study was performed to test the
trichomonicidal effect of these thiazole derivatives.
previously reported that protozoan parasite proteases are
considered good drug targets to develop a therapeutic strat-
egy against diverse parasitic diseases (McKerrow et al.
2008; Das et al. 2013; McKerrow 2018).
Our results show that ATZ-1 and ATZ-2 were able to ex-
hibit an anti-T. vaginalis activity in a dose-dependent manner
(Fig. 3, panels A and B), presenting an IC50 value of 0.15 μg/
mL and 0.18 μg/mL, respectively. In addition, both com-
pounds were able to reduce the T. vaginalis trophozoites
growth in a time course of 24 h, which was not significantly
different from that of MTZ (Fig. 3, panels C). These results
highlight the antiparasitic activity of the arylthiazoles deriva-
tives and agree with previously mentioned reports that have
shown that these compounds are effective against diverse par-
asites (Brito et al. 2019; Pereira et al. 2019; Georgiadis et al.
2020).
Considering that the thiazole derivatives have been re-
ported as anti-proteolytic agents, we speculated that the
ATZ-1 and ATZ-2 compounds might play a role in altering
the proteolytic activity leading to the anti-T. vaginalis activ-
ity. In the present study, the anti-proteolytic activity of both
the ATZ-1 and ATZ-2 compounds was demonstrated, by
using gelatin substrate gel electrophoresis. ATZ-1 was able
to reduce the T. vaginalis proteolytic activity to a level that
was not significantly different to the one exhibited by MTZ
(Fig. 4, panel A). A similar proteolytic inhibition profile was
observed with ATZ-2 (Fig. 4, panel B). It has been
Interestingly, both thiazole derivatives were able to fully
reduce the proteolytic activity in a range of 46 to 50 kDa,
considering the possibility that these compounds could, in
part, affect the activity of a key T. vaginalis protease locat-
ed at that region, TvMP50. The transcription levels of the
mp50 gene exhibit downregulation associated with ATZ-1
and ATZ-2 treatment, mainly at the 0.2 μg/mL concentra-
tion (Fig. 5a). Based on the biological assays, docking stud-
ies were conducted to determine the plausible binding mode
of ATZ-1 and ATZ-2 with the TvMP50 protease. Notably,
the docking simulations for both thiazole derivatives
showed their interaction principally with the dinuclear
Zn2+ cluster and coordinated residues in the active site of
TvMP50, mainly in the presence of zinc, in a stronger way
(Fig. 6, panel E). Respect the MTZ-docked binding mode
results in comparison with ATZ1 and ATZ2 an interesting
chemical property cloud be of interest, the atoms in contact
with the Zn2+ cluster share a trigonal planar geometry (acet-
amide oxygen of thiazole derivatives and imidazole nitro-
gen of MTZ) with a predicted hybridized sp2 orbital and
unhybridized p2 orbital. Although, the electronic

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Fig. 6 Predictions of the docking TVMP50-Zn2+ conditions for ATZ-1
ƒ
and ATZ-2 compounds. a ATZ-1 docking. The 40 predictions selected by
DG and FFN better values depicted on the TvMP50 structure. Most of the
predictions appear on the active site cavity in similar binding modes and
few predictions on the other two cavities generated by three loops in the
N-terminal domain. The dashed circle depicts the active site cavity. The
residues of the metal cluster and two Zn2+ are represented but are hidden
by the predictions. The asterisk (*) represents the sites where CASTp
program detects the most relevant cavities on the N-terminal. b ATZ-2
docking. The 40 predictions selected by Delta G and FFN better values
depicted on the TvMP50 structure. All predictions appear on the active
site cavity in a similar binding mode. The variation on the binding modes
is related to the position of the two rings of ATZ-2 and ATZ-1. c ATZ-1
docking. All ATZ-1 docking predictions depicted on the TvMP50 struc-
ture. Many predictions are in the active site cavity and the close cavity
between both domains, although a considerable number of predictions
appear on the two cavities of the N-terminal domain. An additional small
cavity on the back of the structure appears with few predictions. d ATZ-2
docking. All ATZ-2 docking predictions depicted on the TvMP50 struc-
ture. Many predictions are in the active site cavity and few predictions on
the close cavity between both domains; considerable predictions are lo-
cated on the two cavities of the N-terminal domain. The double bar line
indicates the N-terminal domain, and the C-terminal domain is represent-
ed by the solid bar. Colors key: in cyan DG predictions, in pink FFN
predictions, all predictions on green. e The binding mode of ATZ-2 top
prediction (higher DG, sFFN, and energy values), as a representative, to
show the hypothetical acetamide interactions with Zn2+ with 2.2–2.3 Å.
The other atoms interactions of ATZ-2 are maintained between 3.2 and 4
Å
probably contributing to the reductive activation of metro-
nidazole (cytotoxic form produces in the hydrogenosome),
which suggest a secondary mode activation mediated by an
enzyme as TvMP50, reduction of MTZ, or an enzyme inhi-
bition. However, it is less well known the mechanism that
leads to the treatment of ATZ-1 and ATZ-2 to downregu-
lation of the mp50 gene transcription. The identification of
the mechanism could be an interesting additional research
way to find other targets of thiazole derivatives with anti-
trichomonal activity.
However, further studies are necessary to evaluate structur-
al modifications that could improve trichomonicidal activity,
as well as studies to elucidate the mechanisms associated and
the possible anti-proteolytic effects. In conclusion, our results
demonstrate that the 2-amino-4-arylthiazoles derivatives pos-
sess potent antiparasitic activity against T. vaginalis tropho-
zoites as plausible trichomonad TvMP50 protease inhibitors,
revealing the potential use of these thiazoles as novel
antitrichomonal agents.
distribution of the sp2 orbitals is different and hence the
interaction with Zn2+ cluster. However, future studies
should include these observations to re-design ATZ1 and
ATZ2 compounds to search for better properties to find
alternative new drugs based on these compounds. The
TvMP50 protease plays a major role during the
T. vaginalis pathogenesis process; therefore, it is worth
mentioning that the results suggest that the mechanism of
action of thiazole derivatives tested here may be associated,
in part, with the inhibition of this key protease and lead to
damage to parasites. Although, it was reported that the ad-
dition of iron to in vitro culture of T. vaginalis decreases
MTZ minimal lethal concentration (Elwakil et al. 2017),
Acknowledgments This work was supported by grant from Autonomous
University of Mexico City (UACM) awarded to MEAS. We acknowl-
edge Brenda Herrera Villalobos and Alfredo Padilla Barberi from the
UACM for their technical assistance.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.

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