In vitro evaluation of new 4-thiazolidinones on invasion and growth of Toxoplasma gondii

Article abstract of DOI:10.1016/j.ijpddr.2021.05.004

Treatments for toxoplasmosis such as pyrimethamine have shown numerous side effects. It has been reported that the likelihood of relapse associated with pyrimethamine-based therapy in patients with HIV and toxoplasmic encephalitis (TE) can have significant implications, even for patients who often develop new lesions in areas of the brain previously free of infection. This led us to research for new agents against Toxoplasma gondii. Recent findings have shown the potent biological activity of 4-thiazolidinones. We proposed to design and synthesize a new series of 2-hydrazono-4-thiazolidinones derivatives to evaluate the in vitro growth inhibition effect on T. gondii. The growth rates of T. gondii tachyzoites in Human Foreskin Fibroblast (HFF) cell culture were identified by two in vitro methodologies. The first one was by fluorescence in which green fluorescent RH parasites and cherry-red fluorescent ME49 parasites were used. The second one was a colorimetric methodology using β-Gal parasites of the RH strain constitutively expressing the enzyme beta-galactosidase. The 4-thiazolidinone derivatives 1B, 2B and 3B showed growth inhibition at the same level of Pyrimethamine. These compounds showed IC₅₀ values of 1B (0.468–0.952 μM), 2B (0.204–0.349 μM) and 3B (0.661–1.015 μM) against T. gondii. As a measure of cytotoxicity the compounds showed a TD₅₀ values of: 1B (60 μM), 2B (206 μM) and 3B (125 μM). The in vitro assays and molecular modeling results suggest that these compounds could act as possible inhibitors of the Calcium-Dependent Protein Kinase 1 of T. gondii. Further, our results support the fact that of combining appropriate detection technologies, combinatorial chemistry and computational biology is a good strategy for efficient drug discovery. These compounds merit in vivo analysis for anti-parasitic drug detection.

Full text of DOI:10.1016/j.ijpddr.2021.05.004

International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
Contents lists available at ScienceDirect
International Journal for Parasitology:
Drugs and Drug Resistance
journal homepage: www.elsevier.com/locate/ijpddr
In vitro evaluation of new 4-thiazolidinones on invasion and growth of
Toxoplasma gondii
,
*
b
a
b,**
c,d
Diego A. Molinaa , Gerardo A. Ramos , Alejandro Zamora-Velez , Gina M. Gallego-Lopez
,
´
´
e
a
´
Cristian Rocha-Roa , Jorge Enrique Gomez-Marin , Edwar Cortes
a
´
Grupo GEPAMOL, Centro de Investigaciones Biomedicas, Universidad del Quindío, Armenia, 630004, Colombia
b Grupo GICOC, Universidad del Quindío, Armenia
c Morgridge Institute for Research, Madison, WI, 53715, USA
d Department of Medical Microbiology and Immunology, University of Wisconsin–Madison, Madison, WI, 53706, USA
e Biophysics of Tropical Diseases, Max Planck Tandem Group, University of Antioquia, Medellin, 050010, Colombia
A R T I C L E I N F O
A B S T R A C T
Keywords:
Treatments for toxoplasmosis such as pyrimethamine have shown numerous side effects. It has been reported
that the likelihood of relapse associated with pyrimethamine-based therapy in patients with HIV and toxoplasmic
encephalitis (TE) can have significant implications, even for patients who often develop new lesions in areas of
the brain previously free of infection. This led us to research for new agents against Toxoplasma gondii. Recent
findings have shown the potent biological activity of 4-thiazolidinones. We proposed to design and synthesize a
new series of 2-hydrazono-4-thiazolidinones derivatives to evaluate the in vitro growth inhibition effect on
T. gondii. The growth rates of T. gondii tachyzoites in Human Foreskin Fibroblast (HFF) cell culture were iden-
tified by two in vitro methodologies. The first one was by fluorescence in which green fluorescent RH parasites
and cherry-red fluorescent ME49 parasites were used. The second one was a colorimetric methodology using
β-Gal parasites of the RH strain constitutively expressing the enzyme beta-galactosidase. The 4-thiazolidinone
derivatives 1B, 2B and 3B showed growth inhibition at the same level of Pyrimethamine. These compounds
4-Thiazolidinones
Toxoplasma gondii
Cytotoxicity
Invasion
Growth
showed IC50 values of 1B (0.468–0.952
μ
M), 2B (0.204–0.349
μ
M) and 3B (0.661–1.015
μ
M) against T. gondii. As
M) and 3B (125 M).
a measure of cytotoxicity the compounds showed a TD50 values of: 1B (60
μ
M), 2B (206
μ
μ
The in vitro assays and molecular modeling results suggest that these compounds could act as possible inhibitors
of the Calcium-Dependent Protein Kinase 1 of T. gondii. Further, our results support the fact that of combining
appropriate detection technologies, combinatorial chemistry and computational biology is a good strategy for
efficient drug discovery. These compounds merit in vivo analysis for anti-parasitic drug detection.
1. Introduction
treatment is supplemented with folinic acid kwon as Leucovorin (Mon-
toya and Liesenfeld, 2004). The idea of this treatment is to reduce the
Toxoplasmosis is a disease produced by the Toxoplasma gondii pro-
tozoan parasite, which is a cosmopolitan food and waterborne infection
with an estimated 1 to 2 billion (approximately 30%) of the world’s
population infected (Bahia-Oliveira et al., 2017). The most efficacious
treatment for this infection is based on Pyrimethamine with Sulfadia-
zine. Pyrimethamine belongs to the group of amino-pyrimidines, and
Sulfadiazine is related to sulfonamides. They inhibit two different re-
actions on the same metabolic pathway, and thus exhibit synergistic
activity associated with nucleic acid synthesis. For this reason, this
growth of the parasite considering that the parasite divides faster than
the host cell and that T. gondii cannot synthesize nucleic acids from
exogenous folinic acid, while humans can do it. However, this treatment
produces severe secondary effects like hypersensitivity, hematological
toxicity, teratogenicity, and allergic reactions. The development of
adverse effects leads to interruption of the therapy and change for less
efficacious antiparasitic drugs (de la Torre et al., 2011). Although the
mix from these compounds can actively control the tachyzoite growth,
they have little effect against the bradyzoite stage that forms the cyst
* Corresponding author.
** Corresponding author.
E-mail addresses: damolinal@uqvirtual.edu.co (D.A. Molina), ecortes@uniquindio.edu.co (E. Cortes).
https://doi.org/10.1016/j.ijpddr.2021.05.004
Received 28 April 2021; Received in revised form 17 May 2021; Accepted 20 May 2021
Available online 2 June 2021
2211-3207/© 2021 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

D.A. Molina et al.
International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
Fig. 1. 4-Thiazolidinones derivatives with anti-T. gondii activity.
tissue and therefore they does not cure the infection. The adverse event
(AE) related profiles associated with Pyrimethamine-based treatments
differ by Toxoplasma manifestation. In ocular toxoplasmosis and TE, the
most frequent AEs were dermatologic, while in congenital toxoplas-
mosis, the most frequent AEs were associated with bone marrow sup-
pression. Steven–Johnson syndrome was uncommon and reported in
only three patients (0.1%). Hematologic AEs occurred in all manifesta-
tions and highlight the importance of monitoring the blood of patients
administered Pyrimethamine-based regimens (Ben-Harari et al., 2011;
Shammaa et al., 2021). Thus, there is a need to find new anti-Toxoplasma
agents with better inhibitory activity and low or no cellular toxicity. The
4-thiazolidinone scaffold has been shown as a promising drug-like
compound against T. gondii due to its demonstrated biological effects
and the experimental information reported by several authors showing
high therapeutic index (TI) values in vitro (Fig. 1) (Tenorio et al., 2005;
de Aquino et al., 2008; Carvalho et al., 2010; Liesen et al., 2010;
D’Ascenzio et al., 2014; Carradori et al., 2017).
electron donor region (D’Ascenzio et al., 2014; Carradori et al., 2017).
Because of it, the objectives of this study were to design and synthesize a
new series of 2-hydrazono-4-thiazolidinones derivatives to evaluate if
the molecules have in vitro growth effect of T. gondii as an alternative of
new molecular entities with possible anti-Toxoplasma activity.
2. Methods
2.1. Chemistry
The solvents and chemicals were procured from commercial chem-
ical suppliers (Merck.KGaA, 64271 Darmstadt, EDM Millipore Corpo-
ration (Germany), Alfa Aesar, 30 Bond Street, Ward Hill) and used
without further purification. Melting points were recorded using an
Electrothermal IA-9100 digital fuser. Thermo Scientific Nicolet 380
spectrophotometer was used for FTIR spectra. 1H (400 MHz) and 13C
NMR (100 MHz) spectra were recorded on Bruker Avance 400 instru-
ment in DMSO‑d6. HRMS spectra were recorded using the Shimadzu
GCMS-QP2010 Ultra instrument. All reactions were monitored by TLC
performed on silica gel 60 F254 plates. The detail of synthesis experi-
ments are given in the Supplementary data.
Modifications within the nucleus of 4-thiazolidinone greatly affect
the physical-chemical properties of the molecule (e.g. solubility), for this
reason, structural diversity is very important (Shelat and Guy, 2007).
Modifications on positions 2 and 5 of the 4-thiazolidinone ring have
been considered as a reference point to enhance the biological activity of
compounds that contain this core. Additionally, the moiety hydrazone
conserved in this position of the heterocyclic ring has demonstrated to
enhance the effect of the 4-thiazolidinones against T. gondii, since the
molecules that have this group showed high effectiveness on the host
cell invasion and replication of the parasite with low cytotoxicity, to
concentrations in the micro molar scale (Hamama et al., 2009;
Rocha-Roa et al., 2018). On the other hand, the 2,5-disubstituted 1,
3-thiazole nucleus could be an important pharmacological requirement,
so the position N-3 in the 4-thiazolidinones would be important as an
2.2. Cell line, parasites and culture conditions
2.2.1. HFF cells
Human foreskin fibroblasts (HFF; Fibroblast, Skin; foreskin, normal,
human. ATCC No: SCRC-1041) obtained from the American Type Cul-
ture Collection (ATCC), Rockville, Maryland. HFF were maintained in
DMEM media supplemented with 10% FBS (Fetal Bovine Serum-Gibco,
USA), 1X of GlutaMAX 100X, 1 mM of sodium pyruvate 100 mM and 50
μL of penicillin (10,000 Units/mL) and streptomycin (10,000 μg/mL)
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D.A. Molina et al.
International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
per mL at 37 ◦C with 5% CO2.
medium. T. gondii tachyzoites (5 × 105) were treated for 24h directly
with molecules and the Pyrimethamine at 10 μM as a control. The par-
2.2.2. Parasites
asites that were glowing green were considered viable. The percentage
of viable cells was calculated using the following formula: (Fluorescence
of the sample with treatment URF * 100)/fluorescence of the control
without treatment, where URF = unit relatives of fluorescence calcu-
lated with the formula: (sample fluorescence/fluorescence in blank
well) *100. Parasites in 96-well plates were directly studied by fluo-
rescence microscopy by using a standard fluorescence EVOS Light Cube
GFP. Excitation 470/22 Emission: 526/60 INVITROGEN LIFE TECH-
NOLOGIES with a 10× objective on EVOS FL Color Imaging System.
Images were recorded with and adjusted for contrast with ImageJ. The
fluorescence of the wells with parasites treated as compared with those
of the control without treatment. For all the methodologies the com-
pounds were dissolved in DMSO (Fischer Scientific). The concentration
for DMSO did not exceed 1% in all assays to prevent cytotoxicity.
Toxoplasma gondii strains modified genetically that express enzyme
substrate or fluorescent labels were used for the in vitro experiments:
T. gondii RHβ1 strain (β-galactosidase tagged, kindly donated by Dr. John
Boothroyd from Stanford University), T. gondii RH GFP strain (green
fluorescent protein-tagged, kindly donated by Dr. David Sibley, Uni-
versity of Washington) and the T. gondii ME49 cherry strain (cherry
fluorescent-tagged, kindly donated by Dr. Laura Knoll, University of
Wisconsin) were maintained in DMEM media supplemented with 10%
heat-inactivated bovine serum (GE Healthcare Life Sciences).
2.3. Infection of human cell lines and measurement of the effect of
compounds on T. gondii growth
T. gondii RH-GFP and RHβ1 were passaged in confluent human
foreskin fibroblast (HFF) monolayers. RH-GFP (Green Fluorescent Pro-
tein) does not require external factors and is stable throughout a wide
spectrum of conditions (Striepen et al., 1998). RHβ1 is the RH strain
carrying the Escherichia coli lacZ (β-galactosidase) gene under the con-
trol of the SAG1 promoter (Seeber et al., 1996). To identify the growth
rates of T. gondii tachyzoites in HFF cell culture, three methodologies
were used. To establish a direct effect over the parasite and its growth
T. gondii GFP parasites of the strain RH (virulent) were used with the
ability to emit fluorescence upon receiving excitation at 395–475 nm, so
a green fluorescence can be generated and observed in a fluorescence
microscope. The other two are quantitative methodologies, the first one
using T. gondii β-Gal parasites of the RH (virulent) strain that is genet-
ically modified that constitutively express beta-galactosidase, then the
substrate X-Gal is added, producing a blue coloration that can be
quantified by spectrophotometric reading at 615 nm wavelength. The
second one is by using ME49 cherry fluorescence quantified by the
IncuCyte Performing analysis, without removing cells from the incu-
bator or disturbing cultures that automatically acquire and analyze
fluorescence and phase-contrast images around the time.
2.6. T. gondii RH GFP tachyzoites in 48h growth assay
HFF cells were plated in 96 well plates at approximately 20,000 cells
per well and allowed to grow until confluent. Parasite cultures that had
completely lysed an HFF monolayer were recollected into a centrifuge
tube and were washed with 10 mL of PBS 1X centrifuging to 500 g × 5
min at room temperature to remove host cell debris and parasite ag-
gregates. The supernatants were passed to another centrifuge tube and
the pellets were collected after centrifuging to 1,800 g × 10 minutes at
room temperature. The resulting parasites were resuspended, counted,
and diluted in media to a concentration of 2 × 106 per mL, and 5 μL of
this suspension was added to the appropriate wells of the microtiter
plates, which already contained the compounds to be tested, to give an
inoculum of approximately 10,000 parasites per well then we added the
molecules. For the assays with the RH GFP strain, the fluorescence was
quantified by fluorescence microscopy using a standard fluorescence
EVOS Light Cube GFP. Excitation 470/22 Emission: 526/60 INVI-
TROGEN LIFE TECHNOLOGIES with a 10× objective on EVOS FL Color
Imaging System. Images were recorded with and adjusted for contrast
with ImageJ. The plates were then incubated at 37 ◦C with 5% CO2 for
48h. After this time each well was gently washed and media replaced.
The fluorescence of the well with parasites treated was compared with
the control without treatment. GFP fluorescence was quantified by
fluorescence microscopy using a standard fluoresce EVOS Light Cube
GFP. Excitation 470/22 Emission 526/60 INVITROGEN LIFE TECH-
NOLOGIES with a 10× objective on EVOS FL Color Imaging System.
Images were recorded with and adjusted for contrast with ImageJ. The
plates were then incubated at 37 ◦C with 5% CO2 for 48h.
2.4. HFF cell viability by Alamar blue assay
HFF cells were cultured as monolayers and maintained in Dulbecco’s
modified Eagle medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin in a
humidified atmosphere with 5% CO2 at 37 ◦C in T-25 flasks and were
subcultured twice a week. Cell lines were plated with approximately
20,000 cells per well and let grown until confluence. Once confluent, the
cells were treated with a compound concentration of 10 μM for 24 h.
This was followed by an Alamar Blue assay with 0.5 mM resazurin at
37 ◦C with 5% CO2 for 4 h. All concentrations were performed in trip-
licated. A fluorescent reading was then taken with a BioTek Synergy HT
plate reader at 530/25 excitation and 590/25 emission. Host cell
viability was determined by comparing the treatments with no com-
pound treatment as 100% viability. A color change from blue to pink
indicated the metabolically active cells. A color change in the growth
control well to pink indicated proper cell growth and no color change in
the sterile control well-indicated absence of contaminants.
2.7. T. gondii expressing β-galactosidase for colorimetric assessment
We used microtiter assay for drug evaluation with a strain of T. gondii
that expresses bacterial β-galactosidase. Parasites were kindly donated
by John Boothroyd. This assay provides a high-throughput and nonra-
dioactive alternative for the identification of anti-T. gondii compounds.
An inoculum of approximately 10,000 parasites per well was used in
wells that already contained the compounds, compared with the control
without treatment. The plates were then incubated at 37 ◦C with 5% CO2
for 96 h. After this time 2 μl of X-Gal 30 mg per mL (Sigma-USA), was
2.5. T. gondii RH GFP tachyzoites 24h viability assay
added to give a final concentration of 100 μM of X-gal. The plates were
incubated at 37 ◦C with 5% CO2 for an additional 24 h and were then
read at 420 and 615 nm on a Bio-Tek microtiter plate reader. Note that:
RH-strain parasites were grown in HFF cells. Parasites to be used in
the assay were prepared as follows. Cultures that had completely lysed
an HFF monolayer were recollected into a centrifuge tube and were
washed with 10 mL of PBS 1X centrifuging to 500 g × 5 min at room
temperature to remove host cell debris and parasite aggregates. The
supernatants were passed to another centrifuge tube and the pellets were
collected after centrifuging to 1800 g × 10 min at room temperature.
The resulting parasites were resuspended, counted, and diluted in the
● Previously a suitable number of parasites was standardized to
perform the experiments since this number allowed to find differ-
ences in the readings and coloration produced by the X-Gal substrate.
Thus, for each experiment 10,000 parasites per well were used in
plates of 96 well to perform the HFF cell growth experiments after
adding 4-thiazolidinone molecules.
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D.A. Molina et al.
International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
Scheme 1. General synthesis of the derivatives 1–16B. Reagents and conditions: (a) EtOH, microwave at 40W of power; (b) MeOH, stirring at 500 rpm and room
temperature.
● For the colorimetric technique like X-Gal we have to take into ac-
count that the molecules can produce color when they were diluted
in DMSO and mixed with the DMEM media. Then the absorbance
values produced were rested with the control color of each molecule
(media culture and molecules).
IC50 after the transformation and normalization of data (Supplementary
data).
2.11. Molecular docking
Molecular docking calculations were carried out to obtain the
possible binding mode of the best compounds with two molecular tar-
gets from T. gondii, the Calcium-Dependent Protein Kinase 1 (TgCDPK1)
with PDB code 4JBV and the ROP18 kinase (TgROP18) with PDB code
4JRN. Previously, we suggest that these proteins may act as a target for
compounds derived from the 4-thiazolidinone nucleus (Molina et al.,
2018; Rocha-Roa et al., 2018). The three-dimensional structure of the
compounds was drawn and optimized using Avogadro software (Han-
well et al., 2012). Both receptors (proteins) and ligands (compounds 1B,
2B, and 3B) were prepared using Autodock Tools v1.5.6 software
(Morris et al., 2009), for this, polar hydrogens and charges were added;
additionally, for the ligands, the torsions were also added. Molecular
docking calculations were performed in triplicates and carried out using
the Autodock Vina (Trott et al., 2010) and Smina (Koes et al., 2013)
software. All the search boxes used had a dimension of 25 Å × 25 Å x 25
Å and were centered on the co-crystallized ligands. A value of 15 for the
exhaustiveness parameter was used. To set the boxes, we did a
re-docking of the co-crystallized ligands in each receptor.
2.8. Effect on T. gondii ME49 strain 5 days growth assay
T. gondii growth inhibition in vitro screens: HFF cells were plated in
24 well plates and allowed to grow until confluent monolayer. Once
confluency was reached, 40.000 ME49-cherry-cherry tachyzoites were
added to each well and the plates were incubated at 37 ◦C for 24h to
allow for infection. Media was then replaced, and compounds dissolved
in DMSO (Fischer Scientific) were added at a concentration of 10 and 5
μ
M according to the HFF cytotoxicity results. The concentration of
DMSO did not exceed 1% in all assays to prevent cytotoxicity. All con-
centrations were performed in duplicate and Pyrimethamine 10 μM was
used as a positive control. A fluorescent reading was then taken with an
IncuCyte S3 (Sartorius-Germany) plate reader at 530/25 excitation and
590/25 emission for 5 days.
2.9. Host cell and extracellular parasite pre-treatment assay
HFF cells were grown in 24 well plates to confluency. 10 μM of the
respective compound was added to the wells (performed in triplicate).
After 24 h of compound exposure, cells were then washed twice with
media. Cells were then infected with 4 × 104 ME49 cherry tachyzoites/
well and parasite growth was quantified by fluorescence for 5 days post-
infection in the IncuCyte S3. To evaluate the effect of compound expo-
3. Results and discussion
3.1. Chemistry
Previously, different research groups have reported 4-thiazolidinone
molecules with increased anti-T. gondii activity (Liesen et al., 2010;
D’Ascenzio et al., 2014; Molina et al., 2018; Abdizadeh et al., 2020).
Thus, we designed compounds with the 4-thiazolidinone ring and
different substituents in the 2-hydrazonyl fragment and the 5 positions
of the main nucleus, to increase their activity anti-parasitic.
sure to free tachyzoites, 10 μM of the respective compound was added to
ME49-cherry tachyzoites isolated from culture and resuspended in
media at 4 × 106 tachyzoites/mL. Exposure to the compound lasted 4 h
at 37 ◦C. After treatment, tachyzoites were centrifuged and washed
twice. Confluent HFF cells in 24 well plates were then infected with 4 ×
104 treated tachyzoites per well. Tachyzoite growth was quantified by
fluorescence at 5 days post-infection in the IncuCyte S3.
In the first step (Scheme 1) for the synthesis of modified compounds,
the aryl thiosemicarbazones (3) were synthesized with 80–90% yield
through condensation of aldehyde and acetophenones (1) with thio-
semicarbazide (2) in a mixture of dry ethyl alcohol containing a catalytic
amount of glacial acetic acid (Hussein et al., 2019; Xun-Zhong et al.,
2020). The procedure for the synthesis of 2-(hydrazono)-4-thiazolidi-
n-5-ylidene acetate derivatives (5) consisted on an intramolecular
cyclocondensation of thiosemicarbazones (2) in methanol solution with
dimethyl acetylenedicarboxylate (DMAD) (Yavari et al., 2007; Aly et al.,
2014). Initially, the stirring of an equimolar mixture of both compounds
in methanol at room temperature, after 1 h of stirring afforded a
precipitated. The orange solid (9B) was isolated and purified after
several washes with ice water. To explore the generality and scope of the
approach previously described, intramolecular cyclocondensation of
DMAD with various thiosemicarbazones were tested under the optimal
conditions found.
2.10. IC50 calculation and statistical analysis
The IC50 calculation and statistical analysis were performed on
Graph pad prism 6.0. The Kruskall Wallis test and the Dunn test were
applied to make multiple comparisons between the treatments and the
negative control. Also, the Mann Whitney test to compare given con-
centrations versus the control. The Cytotoxicity assay increases con-
centrations to obtain the Median toxicity dose (TD50), as the
concentration increases, the viability of HFF cells decreases. The cyto-
toxicity test allows us to approximate the mean TD50 toxicity, a measure
of cytotoxicity, and helps to find the therapeutic index calculated by
TD50/IC50. We use the data obtained with the strain ME49 to have a
concentration gradient of the effect of molecules so that calculate the
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International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
Table 1
Main characteristics of the 2-hydrazono-4-thiazolidinones compounds.
Compound
Reaction time
Melting points (◦C)
M. weight (g/mol)
Yield (%)
Color
1B
2B
2h-40′
1h-13′
258–260
254–256
284129
304.32
91.4
91.3
Beige
Yellow
3B
4B
2h-30′
2h-35′
296–298
295–297
305.31
405.11
91.5
90.7
Yellow
Orange
5B
6B
7B
10h-30′
2h-45′
2h
278–280
289–291
288–289
368.75
305.31
349.36
78.9
88.8
95.2
Beige
Orange
Yellow
8B
1h-10′
265–267
319,34
94.3
Yellow
9B
1h
229–231
238–240
349.36
317.00
88.1
92.8
Orange
Yellow
10B
1h-35′
11B
12B
13B
14B
1h-20′
1h-45′
2h-30′
1h-25′
284–286
230–232
344–346
299–301
348.33
319.34
390.8
86.6
90.5
96
Yellow
Yellow
Yellow
Gray
333.32
92.5
15B
16B
2h-15′
1h-30′
295–297
290–291
335.34
330.32
76.4
87
Yellow
Orange
In all cases reactions proceeded with the same behavior, affording
the expected 2-(hydrazono)-4-thiazolidin-5-ylidene acetate derivates
(1–16B) in excellent yields, and characterized based on their physical,
analytical, and spectral data (Table 1). The structures of the synthesized
compounds were confirmed by FTIR, 1H, and 13C NMR spectroscopy and
HRMS. The IR spectrum of 9B exhibited a characteristic absorption at
3467 cmꢀ 1 y 3136 cmꢀ 1 due to the stretching of the OH and NH,
exhibited characteristic resonances in the appropriate regions of the
spectra. Finally, the mass spectrum of 9B exhibited an intense molecular
ion peak at m/z = 349.36 consistent with the molecular formula of
C15H15N3O5S.
3.2. Evaluation of biological activities
respectively at about 1713 and 1608 cmꢀ 1 due to carbonyl groups. The
–
In vitro assay systems to screen drugs for anti-T. gondii is focused on
growth assays of the parasite or infection of host cells. It is very
important to be able to identify anti-T. gondii candidates using different
in vitro assay systems. In this evaluation, we used different systems to
filter molecules, taking into account that each technique has its effec-
tiveness and limitations (Carey et al., 2004; Jin et al., 2012).
presence of C N group was confirmed by the characteristic for the
–
stretching band at 1515 cmꢀ 1. The 1H NMR spectrum of 9B showed
downfield two singlets at 12.83 ppm for NH and 9.58 ppm for the OH
group; one doublet at 7.49 ppm, one doublet of doublets at 7.37 ppm,
and on doublet at 6.89 ppm for aromatic protons. The vinyl proton was
observed at 6.67 ppm and three CH3 groups at 3.86–2.42 ppm. The
13CNMR spectrum of 9B showed 15 signals, which is in agreement with
the proposed structure. The 1H and 13C NMR spectra of 1–16B were
similar to those of 9B, except for the substituted phenyl moiety that
3.2.1. HFF cell viability by Alamar blue assay
The treatment of HFF cells with molecules 4B, 10B, 11B and 15B at a
single compound concentration of 10 μM for 24 h were toxic because
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D.A. Molina et al.
International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
Fig. 2. Evaluation of cell viability by Alamar Blue
assay. Cells were treated with a single compound
concentration of 10 μM for 24 h. Molecules 4B, 10B,
11B, 15B reduced the percentage of viability of the
HFF cells. The results in the bar graph are the
average ± standard deviation of the percentage of
viability in triplicate of a representative experiment.
Once confluent, cells were treated with a compound
concentration of 10 μM for 24 h. This was followed
by an Alamar Blue assay with 0.5 mM resazurin at
37 ◦C with 5% CO2 for 4 h. The results in the bar
graph are the average ± standard deviation of the
percentage of triplicate viability of a representative
experiment.
Fig. 3. Viability of RH-GFP tachyzoites in
host-cell free medium, parasites were treated
with compounds at 10 μM and 5 μM for 24 h
at 37 ◦C with 5% CO2. Pyrimethamine (Pyr)
and DMSO at 1% were used as controls.
Molecules 1B, 2B, 3B, 4B, 6B, 9B and 12B
reduce the viability of parasites. Pyrimeth-
amine didn’t reduce the viability of the par-
asites. The results in the bar graph are the
average ± standard deviation of the per-
centage of the percentage of viability in
triplicated of a representative experiment.
Fig. 4. 48h growth assay of RH-GFP tachyzoites.
Microtiter plates with the compounds were infected
with an inoculum of approximately 10,000 para-
sites per well. Molecules 1B, 2B, 3B, 4B, 6B and
12B reduce the percentage of growth similarly to
pyrimethamine. Fluorescence of GFP RH parasites
was quantified by fluorescent microscopy after 48h
of treatment by using a standard fluorescence EVOS
Light Cube GFP. Excitation 470/22 Emission: 526/
60 with a 10× objective on EVOS FL Color Imaging
System. Images were recorded with and adjusted for
contrast with ImageJ.
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International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
Fig. 5. Colorimetric assessment using T. gondii expressing β-galactosidase. (A). Growth inhibition screen with T. gondii β-galactosidase parasites (1B, 2B, 3B) from
the group 2-hydrazono-4-thiazolidinone reduce more that 50% of the growth of the parasite. 4B, 6B, 9B, 12B didn’t have an effect on the strain β-galactosidase. (B)
We identified promising derivatives (1B, 2B, 3B) and assessed for further profiling in ME49 strain.
reduced more than 50% percent of the cell viability when compared to
the negative control (Fig. 2). However, treatment with these same
3.2.4. Effect of compounds on T. gondii expressing beta-galactosidase
Toxoplasma strains stably expressing bacterial β-galactosidase makes
possible to analyze after 96h of incubation using a colorimetric growth
assays to evaluate a given drug’s activity (McFadden et al., 1997). We
analyzed the same molecules with both techniques T. gondii GFP growth
(Figs. 3–4) and β-galactosidase RH strain (Fig. 5A). The 4B, 6B and 12B
molecules reduced more than 50% of the viability of the parasites and
the percentage of growth, at the same level that the control Pyrimeth-
amine by GFP assay, but they were not selected for further experiments
with ME49 because they did not reduce the growth in more than 50% in
β-galactosidase assay. The molecules 1B, 2B and 3B were selected for
further profiling using T. gondii ME49 because they show interesting
results; low toxicity in HFF measure by resazurin, direct effect and
proliferation effect, assessed by fluorimetric and colorimetric technique
(Fig. 5B).
molecules at a concentration of 5 μM were not toxic.
3.2.2. Viability assay of T. gondii RH GFP tachyzoite at 24h
T. gondii RH GFP tachyzoites were treated with compounds at the
concentration of 5 μM and 10 μM for 24 h according to with the previous
HFF cell viability test (Fig. 2) and Pyrimethamine at the concentration of
10 μM as seen in Fig. 3. The parasites that were glowing green were
considered viable and the percentage of tachyzoites viability was
compared with parasites without treatment. Molecules labeled as 1B,
2B, 3B, 4B, 6B, 9B and 12B reduced in more than 50% the viability of
the parasites.
3.2.3. Effect of compounds on T. gondii RH GFP tachyzoites 48h growth
assay
Molecules with group 2-hydrazono-4-thiazolidinone (1B, 2B, 3B, 4B,
6B and 12B) reduced the percentage of growth at the same level of the
control Pyrimethamine measuring GFP fluorescence in T. gondii RH-GFP
tachyzoites (Fig. 4).
3.2.5. Effect on T. gondii RH cherry and ME49 strains at 5 days assay
Then, the RH cherry parasites were used to test the reproducibility of
1B, 2B, and 3B molecules (Fig. 6A). These molecules reduce the pro-
liferation of a 24h after established infection. The molecules 1B, 2B, 3B
were selected for further profiling using T. gondii ME49 (Type II lineage)
which is considered less virulent and more representative of natural
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International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
were treated with 2B, there was no significant change in growth.
However, when tachyzoites were treated with molecule 2B before
infection, the growth percentage was reduced to 40.3% (Fig. 7). From
these data, it can be reasoned that 2B molecule can be taken up by the
host cell to affect the growth of T. gondii while that molecules, 1B and 3B
can have a direct effect on the extracellular parasite before the invasion
and on the host cell.
3.2.7. Inhibitory concentration calculation and statistical analysis
Sixteen N-functionalized derivatives-4-thiazolidinones 5-acetates
were synthesized and evaluated in vitro to determine their antipara-
sitic effect against T. gondii in different strains such as TgRH GFP and
TgME49 cherry-red. The results of HFF cell viability show that com-
pounds 1B, 2B and 3B at a concentration of 10 μM do not alter cellular
metabolism on HFF cells measure by Alamar Blue (Fig. 2). Likewise, the
TgRH GFP parasite viability assay after 24 h of incubation shows that
molecules 1B, 2B, 3B and 4B significantly reduce tachyzoites viability
between 60 and 85% (Fig. 3).
The parasite intracellular growth assay shows that compounds 1-4B,
6B and 12B significantly reduce the multiplication of the parasite at a
concentration of 10 μM and are similar to Pyrimethamine by GFP (Fig. 4)
The graph of the growth percentage of T. gondii in strain ME49 high-
lights that molecules 1B, 2B and 3B at a concentration of 10 M pre-
sented a similar effect as the Pyrimethamine control (10 M), with
μ
μ
which, the correlation and reproducibility of the activity results of the 4-
thiazolidinone derivatives in TgRH and TgME49 (Fig. 6A and B).
Due to the good correlation that the previous tests presented, the
intracellular growth effect test on TgME49 was carried out in 5 days of
exposure, this experiment determined that compounds 1B, 2B and 3B
presented similar effects than Pyrimethamine control at a concentration
of 10 μM.
To know the mode of action of the molecules synthesized molecules
in both HFF cells and tachyzoites, pre-treatment tests were carried out
Fig. 6. (A) Effect on T. gondii RH cherry strain 5 days assay. The RH cherry
parasites were used to test reproducibility of 1B, 2B, and 3B molecules. All
with the best compounds at a concentration of 10
μ
M. The mean toxic
M), 2B (206
μM) (Fig. 8). Similarly, the IC50 was calculated for 1B
concentrations were performed in duplicate at 10 μM this concentration also
dose (TD50) was calculated for the three molecules 1B (60
M) and 3B (125
(0.46 M), 2B (0.20 μM) and 3B (0.66 μM). The lower a therapeutic
μ
was used for Pyrimethamine as a positive control. The results on the bar chart
are the percentage of growth of a representative experiment. (B) Effect on
T. gondii ME49 strain 5 days growth assay. The molecules 1B, 2B and 3B were
selected for further profiling using T. gondii ME49 (Type II lineage) which is
considered less virulent and more representative of natural infections. (Sup-
plementary data Figure 6 concentration gradient to find IC50).
μ
μ
index (TI), the greater the toxicity of the molecule on the host cell. On
the contrary, a high TI indicates that the drug is less toxic to HFF cells.
The therapeutic index as a measure of efficacy is calculated by TD50
/
IC50. The TI for the molecules were: 1B (128), 2B (1,000) and 3B (188).
The maximum mean inhibitory concentration is a measure of the po-
tency of a substance to inhibit a specific biological or biochemical
function. The fluorescence units for the cherry-ME49 strain were
plotted. The transformation and normalization of the data were made to
find the IC50 (Table 2). The results indicate that the 2B molecule exhibits
greater selectivity for tachyzoites relative to host cells.
Table 2
Anti-Toxoplasma activity, Median toxicity dose, Therapeutic index and molec-
ular docking results for the best 4-thiazolidinone compounds.
Molecule
IC50
M)
TD50
M)
TI
Affinity TgCDPK1
Affinity TgROP18
(
μ
(
μ
(kcal/mol)
(kcal/mol)
A. Vina
Smina
A. Vina
Smina
3.3. Molecular docking
1B
2B
3B
0.46
0.20
0.66
60
128
ꢀ 7.16
± 0.03
ꢀ 7.90
± 0.00
ꢀ 7.63
± 0.12
ꢀ 7.26
± 0.03
ꢀ 7.90
± 0.00
ꢀ 7.93
± 0.23
ꢀ 6.30
± 0.00
ꢀ 6.56
± 0.03
ꢀ 6.96
± 0.03
ꢀ 6.40
± 0.10
ꢀ 6.60
± 0.00
ꢀ 7.10
± 0.00
We have carried out molecular docking to describe a possible binding
mode of our best compounds in the parasite T. gondii. We use proteins for
which some reports suggest that they may act as a target for compounds
derived from the 4-thiazolidinone nucleus (Molina et al., 2018;
Rocha-Roa et al., 2018). The results for molecular dockings (Table 2)
suggest that compounds 1B, 2B and 3B would have a greater affinity
(more negative value) for TgCDPK1 protein than for TgROP18. TgCDPK1
protein is one of the most studied molecular targets in drug design
against T. gondii, this can be reflected in the most of 40 crystallizations
deposited in the Protein Data Bank. The chemical structures of the
compounds that have been most studied as inhibitors of TgCDPK1 are
derivatives of the pyrazolopyrimidine nucleus (Deng et al., 2019).
However, there were are also some crystallizations with inhibitors that
contain thiazole rings in their structures (PDB codes 5T6K, 5T6I and
5T6A), which allows us to think that possibly our 4-thiazolidinone de-
rivatives could also interact with TgCDPK1. Since T. gondii is an obligate
206
125
1000
188
infections (Howe and Sibley, 1995). Fig. 6B shows the reduction of
proliferation of a 24h established infection and we also use this strain to
calculate the IC50 (Table 2).
3.2.6. Host cell pre-treatment and extracellular tachyzoite exposure results
To describe a possible mode of action, both HFF cells and tachyzoites
were pre-treated with 10 μM of the molecules. When HFF cells were pre-
treated with molecules before infection, there was no change in parasite
growth; when the parasite was pre-treated with molecules before
infection, there was decrease growth of the parasite. When HFF cells
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International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
Fig. 7. Parasite Growth inhibition with Pre-treatment of Host cell and extracellular tachyzoite. (A) HFF cells were pre-treated with compounds at 10 μM and
incubated at 37 ◦C for 24 h. Cells were then washed and infected with 40000 ME49 cherry tachyzoites. (B) ME49 cherry tachyzoites in host-cell free medium were
pre-treated with compounds at 10
infection for both experiments.
μM for 4 h at room temperature, then 40,000 parasites were used to infect HFF. Fluorescent readings were taken 5 days post-
Fig. 8. HFF Cytotoxicity assay by increasing concentrations in order to obtain the TD50 Median toxicity dose, a measure of cytotoxicity and helps to find the
therapeutic index.
intracellular parasite, steps such as invasion and egress from the host cell
are crucial for its survival. Inhibition of proteins such as TgCDPK1 has
been shown to alter these and other processes of the parasite (Lourido
et al., 2010; Sugi et al., 2013; Shrestha et al., 2019). TgCDPK1 has
particular characteristics that make it a promising molecular target
against T. gondii, for example, it is not expressed in animals and has
changes in its binding site to ATP that make it different from human
kinase proteins. Its gatekeeper residue has low bulky (glycine),
compared to the bulky gatekeeper of human kinases (generally methi-
onine, leucine, or phenylalanine) (Billker et al., 2009; Hui et al., 2015),
this allows that a possible drug presents specific interactions with the
parasite. Thus, it is possible to associate the in vitro effects of our com-
pounds on the parasite with a possible inhibitory effect of the TgCDPK1
protein.
important fragment in 2B. Given that this region is mainly hydrophobic,
it is feasible to think that modifications of the same type on the pyridine
ring could contribute to increasing its affinity for this site. Upon entering
the hydrophobic region of the ATP-binding pocket, 2B would be ex-
pected to act as a competitive inhibitor.
In general, this research shows that the presence of fragments such as
2-pyridinyl, 2-hydroxyphenyl, and monoxime groups on the 2-hydra-
zonyl position substantially enhanced anti-T. gondii activity compared
to the compounds developed by D’Ascenzio et al. (2014) and Carradori
et al. (2017). Furthermore, the presence of a fragment of α,β-unsaturated
ester on the 5-position of the 4-thiazolidinone ring was shown to
improve the anti-Toxoplasma activity since a compound labeled 27A by
D’Ascenzio et al. (2014), whose only structural difference was the
absence of this ester group that showed an IC50 value of 2.9
μM
Based on the above hypothesis, we analyzed the possible molecular
interactions between compounds with the best in vitro results and
TgCDPK1 (Fig. 9). All three compounds were docked right in the elon-
gated area of the ATP-binding pocket available thanks to the presence of
the small gatekeeper (Gly128), occupying it mainly with the fragments
attached to the hydrazone moiety. Specifically, compound 2B (a com-
pound with the best in vitro results) would be using its pyridine fragment
to form mainly hydrophobic interactions with amino acids such as
Leu96, Leu103, Leu114, Leu126, and Phe202, which would be favoring
the anchoring of the compound, turning the pyridine ring into an
compared to 0.2 μM obtained for our compound 2B, which suggests that
fragments of this type on position 5 of the 4-thiazolidinone ring would
be an interesting moiety to conserve or modify in future studies. Simi-
larly, Carvalho et l., (2010) reported a similar series of 4-thiazolidinone
derivatives with a potential effect on T. gondii, however, the effects there
reached concentration scales of mM, showing that our structural mod-
ifications effectively improve the anti-Toxoplasma effect of 4-thiazolidi-
none core derivatives.
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International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
Fig. 9. Predicted binding modes of the best in vitro compounds in TgCDPK1. (A) General view of the binding mode of compounds 1B (cyan), 2B (orange) and 3B
(green) in the TgCDPK1 protein. 2D interactions between 1B (B), 2B (C) and 3B (D) compounds with the TgCDPK1 protein. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)
4. Conclusion
Declaration of competing interest
The authors declare no competing interests.
Acknowledgements
In this study, different functional groups were used to modify the
structure of 4-thiazolidinone analogs and were then identified by 1H
NMR, 13C NMR, IR, and HR-MS analyses. The anti-Toxoplasma activity of
the synthesized compounds was measured by TgGFP and TgME49 assays
in vitro. The 1B, 2B and 3B target compounds had not only activity
against T. gondii but also lower toxicity towards the host cells. For these
molecules, the fluorescence methods showed correlation with the
β-galactosidase assay. The 2B molecule had an IC50 value for parasite
This project was funded by the Colombian Agency for Science,
Technology and Innovation, COLCIENCIAS, grant number 57104, and
contract 777–2017. The authors acknowledge to Dr. Laura Knoll for the
internship, reagents and lab space. C.R.R. has been supported by Min-
Ciencias, University of Antioquia and Ruta N, Colombia, the Max Planck
Society, Germany.
growth of 0.204 μM, and had no effect on host cell viability at 10 μM and
this compound could be considered in vitro leads for anti-Toxoplasma
therapy. Also, based on our in vitro and silico results we suggest that our
best compounds could act as inhibitors of the TgCDPK1 protein. The use
of different methods to analyze the inhibition of T. gondii growth on
human cells by small molecules increases the power of selectivity tests.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ijpddr.2021.05.004.
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International Journal for Parasitology: Drugs and Drug Resistance 16 (2021) 129–139
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Author contributions statement
D.A.M. and L.K., J.E.G. and E.C. conceived and designed the study. D.
A.M., G.A.R and E.C. performed chemical experiment part. D.A.M., A.O.
Z. and G.M.G.L performed biological experiment part. D.A.M. and C.R.R.
performed the molecular docking part. D.A.M., C.R.R. and E.C. wrote the
manuscript. All authors have read and approved the final version of the
manuscript. University of Costa, Research Group in Natural and Exact
Sciences, GICNEX.
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antimicrobial activities of thiosemicarbazides, 4-thiazolidinones and 1,3,4-
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