A fluorinated phenylbenzothiazole arrests the trypanosoma cruzi cell cycle and diminishes the infection of mammalian host cells

Article abstract of DOI:10.1128/AAC.01742-19

Chagas disease (CD) is a human infection caused by Trypanosoma cruzi. CD was traditionally endemic to the Americas; however, due to migration it has spread to countries where it is not endemic. The current chemotherapy to treat CD induces several side eff

Full text of DOI:10.1128/AAC.01742-19

AAC Accepted Manuscript Posted Online 11 November 2019
Antimicrob. Agents Chemother. doi:10.1128/AAC.01742-19
Copyright © 2019 American Society for Microbiology. All Rights Reserved.
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A fluorinated phenylbenzothiazole arrest the Trypanosoma cruzi cell cycle and
diminishes the mammalian host-cell infection
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Roberto I. Cuevas-Hernándeza,b, Richard M.B.M. Girardb, Sarai Martínez-Ceróna,
Marcelo Santos da Silvac,d, Maria Carolina Eliasc,d, Marcell Crispimb, José G. Trujillo-
Ferraraa# and Ariel Mariano Silberb#.
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aLaboratory of Biochemistry Research, Escuela Superior de Medicina, Instituto
Politécnico Nacional, México. Plan de San Luis Y Díaz Mirón S/N, Casco de Santo
Tomas, Miguel Hidalgo, ZIP: 11340 Mexico City, Mexico.
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bLaboratory of Biochemistry of Tryps - LaBTryps, Department of Parasitology, Institute
of Biomedical Sciences, University of São Paulo. Av. Lineu Prestes 1374, Cidade
Universitária CEP: 05508-000, Butantã, São Paulo - SP - Brazil.
cSpecial Laboratory of Cell Cycle, Butantan Institute, Av. Vital Brasil 1500 (05503-
900), São Paulo - SP – BRAZIL.
dCentre of Toxins, Immune Response and Cell Signalling – CeTICS, Butantan Institute,
Av. Vital Brasil 1500 (05503-900), São Paulo - SP - BRAZIL.
# Both are corresponding authors together:
Ariel Mariano Silber (AMS) Tel.: +55-11-3091-7335; e-mail: asilber@usp.br
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José G. Trujillo-Ferrara (JGTF) Tel.: +52 1 55-5729-6000 ext. 62747; e-mail:
jtrujillo@ipn.mx
Running title: BT10 triggers cell cycle arrest in T. cruzi
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Keywords: Phenylbenzothiazole, Chemotherapy, Trypanosoma cruzi, kDNA,
Antiparasitic agents
Abstract
Chagas disease (CD) is a human infection caused by Trypanosoma cruzi. CD was
traditionally endemic to the Americas, however, due to migration it has spread to non-
endemic countries. The current chemotherapy to treat CD induce several side effects
and its effectiveness in the chronic phase of the disease is controversial. In this
contribution, substituted phenylbenzothiazole derivatives were synthesized and
biologically evaluated as trypanocidal agents against Trypanosoma cruzi. The
trypanocidal activities of the most promising compounds were determined through
systematic in vitro screening and their mode of action as well. The physicochemical-
structural characteristics responsible for the trypanocidal effects were identified, and
their possible therapeutic application in Chagas disease is discussed. Our results show
that the fluorinated compound, 2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl]
phenol (BT10) has the ability to inhibit the proliferation of epimastigotes (IC50(Epi)
=
23.1 ± 1.75 μM) and intracellular forms trypomastigote (IC50(Tryp) = 8.5 ± 2.9 μM) and
diminishes the infection index by more than 80%. In addition, BT10 has the ability to
selectively fragment 68% of the kinetoplastid DNA compared with 5% of nucleus
DNA. The mode of action for BT10 on T. cruzi suggests that the development of
fluorinated phenylbenzothiazole with electron-withdrawing substituent could be a
promising strategy for the design of trypanocidal drugs.
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Introduction
American trypanosomiasis or Chagas disease (CD) is a zoonosis caused by the
flagellated intracellular protist Trypanosoma cruzi. According to the World Health
Organization (WHO), CD is endemic in the Americas; however, due to migration, it has
spread to non-endemic countries such as Canada and several European and Western
Pacific countries (1, 2). This disease, with approximately 7 million affected people,
causes approximately 14 thousand deaths annually, and there are 70 million at risk of
becoming infected (3). T. cruzi, which is naturally transmitted by hematophagous
insects of the Reduviidae family, can also be transmitted by other routes, such as
congenitally (from an infected pregnant woman to the foetus), orally (through
contaminated foods and liquids), and through organ transplantations and blood
transfusions (4).
T. cruzi has a complex life cycle, which occurs within invertebrate and vertebrate hosts
(5–7). Replicative, non-infective epimastigotes present in the insect vector give rise to
non-replicative infective metacyclic trypomastigotes. These forms invade the host cell,
establish the infection and differentiate into replicative amastigotes. Amastigote forms
give rise to a transient stage named intracellular epimastigotes, which subsequently
differentiate into trypomastigotes that can disseminate in the mammalian host through
the bloodstream after causing lysis of host cells and are capable of infecting other cells.
The insect vector can acquire these forms during its bloodmeal, becoming infected and
able to infect a new mammalian host (8, 9).
Human infection is characterized by two sequential clinical phases: acute and chronic.
The acute phase is often asymptomatic and usually remains undiagnosed. Most infected
individuals proceed to the chronic phase, which lasts for the rest of the patient´s life.
The chronic phase presents several clinical forms: 70% of patients are asymptomatic,
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while the remaining 30% present clinical manifestations, with the most frequent being
chagasic chronic cardiomyopathy and, more rarely, megavisceras (mainly megacolon
and megaoesophagus). Over years, the cardiac form of the infection can cause sudden
death or heart failure due to the progressive destruction of the cardiac muscle (9).
A century after the discovery of the disease by Carlos Chagas in 1909, only two
nitroheterocyclic drugs, nifurtimox, and benznidazole, are used to treat T. cruzi
infection. These drugs are effective during acute infection. However, their effectiveness
during the chronic phase of CD is controversial due to their toxicity (10). Therefore,
more secure, effective and accessible alternatives are currently being sought to treat CD.
T. cruzi, along with other trypanosomatids, possesses unique morphological and
metabolic features, offering opportunities for looking for selective inhibitors. Among
them, it is worth mentioning the kinetoplast, a complex structure bearing the
mitochondrial genome referred to as kinetoplast DNA or kDNA. The kDNA consists of
a large number of relaxed circular DNA molecules interlocked with each other to form a
catenated DNA network, and this feature is among those that have frequently been
considered promising therapeutic targets because they are exclusive to trypanosomatids
(11).
In the last two decades, large libraries of compounds of diverse chemical nature have
been screened for trypanocidal agents (12). Among the selected chemical structures,
benzothiazoles (BZTs) have been studied in detail and have been suggested as
trypanocides (13–15). BZTs are a class of bicyclic compounds with a broad spectrum of
biological applications, such as neuroprotectors (16), anticonvulsants (17), antioxidants
(18), kinase inhibitors (19), anticancer agents (20, 21), antimicrobials (22) and
leishmanicidal (23). In particular, it has been suggested that the trypanocidal effects of
benzothiazoles are related to the inhibition of triosephosphate isomerase (TIM) (24, 25).
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In previous work, we reported the development of 4-[5-(trifluoromethyl)-1,3-
benzothiazol-2-yl] benzoic acid, named BT3, which showed excellent trypanocidal
activity on bloodstream trypomastigotes of T. cruzi (14); thus, it was proposed as a new
nucleus for the development of trypanocidal agents. The aim of this contribution was to
determine the trypanocidal activity of a collection of 14 benzothiazoles structurally
related to BT3. We evaluated their anti-T. cruzi activity and we analyse their
physicochemical-structural characteristics responsible for its biological activity. BT10,
the most promising molecule due to its higher selectivity index than the other
derivatives, was selected for further analysing its effects on different aspects of T. cruzi
biology. We propose BT10 as a promising pharmacological hit compound for
developing a treatment against CD.
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RESULTS
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BT10 affects the proliferation of T. cruzi epimastigotes
A collection of 14 structurally related molecules, named BT1 to BT14 was synthesized
(Figure 1A). The identification of the synthesized molecules and their chemical
characterization were performed by melting point (mp), nuclear magnetic resonance
(NMR) and electrospray ionization – mass spectrometry spectra determination (ESI–
MS). These compounds were initially evaluated for their ability to inhibit epimastigote
proliferation in the presence of 25 M BT1-14. We attributed a positive inhibition
activity to those compounds that, at the concentration used, were able to diminish the
cell density at the mid-exponential growth phase (which was measured on the 5th day
from the beginning of incubation) by 50% or more. Epimastigotes cultured in the
absence of drugs in the presence or absence of DMSO (which did not show significant
differences) were used as negative controls for inhibition, and their cell density was
considered as 100% proliferation. For a positive control for the inhibition of cell
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proliferation, the parasites were incubated in the presence of a combination of 60 μM
rotenone + 0.5 μM antimycin (RA). The compound BT10 was identified as having
trypanocidal or trypanostatic activity. BT10 produced a diminution of cell density of
50% when compared to that of control (untreated) cultures. Despite not reaching the
criterion for selecting them as trypanocidal/trypanostatic, it is worth mentioning that
compounds BT3, BT9, and BT11 showed a modest but statistically significant decrease
in the cell density concentration when compared to that of controls (Figure 1B and
Figure S1). Because BT3 was used as lead (14) for choosing and synthesizing BT1-14,
it was selected for further experiments to use this information to unveil the
structure/activity relationship of these compounds.
To further evaluate the potency of BT3 and BT10 as anti-T. cruzi agents, we initially
determined their IC50 on epimastigote proliferation through dose-response experiments.
Cells were cultured in liver infusion tryptose (LIT) in the presence of different
concentrations of BT3 and BT10. As previously described, epimastigotes cultured in the
absence of drugs were used as negative controls for inhibition, and their cell density was
considered as 100% proliferation; parasites treated with RA were used as positive
controls for the inhibition of cell proliferation. As expected, BT3 and BT10 showed a
dose-dependent inhibition of epimastigote proliferation with IC50 values of 48.8 ± 5.77
μM and 23.1 ± 1.75 μM, respectively (Figure 2A, 2B).
BT10 does not induce programmed cell death (PCD)
To characterize the mechanism of action of BT10, we initially investigated its ability to
trigger programmed cell death (PCD) in T. cruzi epimastigotes. For this analysis, we
investigated the typical PCD morphological, cellular and biochemical hallmarks in
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trypanosomatids, such as ROS production, as well as Ca2+ and ΔΨm imbalance (26, 27).
We initially looked for exposure of phosphatidylserine in the external leaflet of the
plasma membrane. Parasites were treated with 25 M and 50 M BT10 (approximately
1x and 2x the IC50). After washing, they were incubated with Annexin V-FITC to assess
external exposure of phosphatidylserine and propidium iodide to assess plasma
membrane permeabilization. The cells were then subjected to analysis by flow
cytometry. The results showed that BT10 did not induce alterations in the plasma
membrane; no exposure of phosphatidylserine or signs of membrane permeabilization
were evidenced at both concentrations used in the assay when compared with that of the
controls (Figure 3A, 3B, 3C). We also investigated whether BT10 triggered other PCD
hallmarks, such as the dissipation of the mitochondrial inner membrane potential (ΔΨm),
production of reactive oxygen species (ROS), and changes in the cytosolic Ca2+ levels
(28). To analyse membrane depolarization (ΔΨm), parasites treated with 25 or 50 M
BT10 (or not, control) during five days were stained with Rh123 for 20 min at 28 ºC
and further analysed by flow cytometry. Treated parasites showed a shift in the obtained
fluorescence values, showing an alteration of ΔΨm (Figure 3D). To determine the
possible variations of the intracellular Ca2+ concentrations, epimastigotes were
incubated with 25 M BT10 (or not, control) for five days. After treatment, the parasites
were incubated with Fluo-4 and analysed by flow cytometry. The results showed that
treated parasites exhibited increased intracellular Ca2+ concentrations compared with
those of untreated parasites (control) (Figure 3E). Finally, to evaluate possible changes
in the production of H2O2 due to treatment with BT10, epimastigotes treated with 25
M BT10 for 24 hours (or not, control) were labelled with carboxy-DCFDA. The
results showed that the treated parasites did not produce a greater amount of H2O2 than
untreated parasites (Figure 3F). Taken together, these results indicate that BT10 does
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not trigger any of the types of classic cell death as a primary effect, even at high
concentrations. However, mitochondrial inner membrane depolarization and alterations
in Ca2+ concentrations could be critical factors affecting the proliferation or long-term
survival of parasites.
As the previous results suggest that BT10 did not trigger cell death, we hypothesized
that the drug interferes with the T. cruzi cell cycle. To confirm this possibility, we
evaluated the reversibility of the effect of BT10 on epimastigote proliferation.
Epimastigotes were treated or not (control) with 25 μM and 100 μM BT10. The treated
parasites showed diminished proliferation during the treatment with respect to that of
the controls. This alteration was reversed by washing out BT10, indicating that BT10
reversibly inhibited epimastigote proliferation (Figure S2). On this basis, we further
analysed possible alterations in the cell cycle. Parasites treated with 25 μM and 100 μM
BT10 or left untreated (control) for five days were labelled with propidium iodide and
submitted to cell cycle analysis by flow cytometry (Figure 4A). The data collected
showed a significant decrease in cells in the G0/G1 phases and an accumulation in the
G2/M phase with significant alterations in the S phase at 100 μM BT10 compared to
those in the control cells (Figure 4B).
Importantly, an accumulation of cells in the G2/M phases is usually due to an arrest of
the cell cycle at G2 checkpoint, in which DNA integrity is sensed and checked (29).
Because cell cycle arrest can be induced at G2 checkpoint by DNA damage (30, 31), we
investigated whether BT10 causes DNA damage to treated parasites. We analysed the
effect of BT10 on both genomic DNA (gDNA) and kDNA integrity using a TUNEL
assay. Epimastigotes treated for 5 days with 25 M BT10 (or left untreated as a control)
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were submitted to the TUNEL assay. The parasites were initially analysed by flow
cytometry, showing differences in fluorescence intensity in the treated population with
respect to that of the control (Figure 5B). When the parasites were analysed by
microscopy, we found that the kDNA was intensively labelled when compared to the
gDNA in the parasites treated with BT10. Untreated parasites and treated parasites
exhibited 35.2  6.2 % and 67.6  8.6 % of cells with labelled kDNA, respectively. On
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the other hand, the percentage of treated parasited with labelled gDNA was 5.4  1.2%
while the untreated parasites remain unlabelled. These data indicate that BT10
selectively triggers kDNA damage (Figure 5A). Taken together, these data indicate that
BT10 induces DNA double-strand breaks preferentially on kDNA.
Cytotoxicity of BT3 and BT10 to mammalian cells
To further evaluate the effect of BT10 on the stages corresponding to mammalian host
cell infection, it is necessary first to determine the range in which the compounds are
non-toxic for the host cells. Therefore, CHO-K1 cells were incubated in the presence of
different concentrations of BT3 (ranging from 16 to 800 μM) or BT10 (ranging from 8
to 288 μM), and cytotoxicity was evaluated by MTT assay after 48 h of treatment. The
concentrations corresponding to a measurement of 50% cytotoxicity (CC50) were
obtained from the typical sigmoidal concentration-response curves, resulting in 95.3 ±
3.3 μM for BT10 and 127.6 ± 0.9 μM for BT3 (Figures 6A, 6B).
BT10 selectively inhibits the intracellular cycle of T. cruzi
According to the results obtained from the cytotoxicity experiments, a range of BT10
concentrations from 0.1 to 32 μM was selected to evaluate the IC50 for trypomastigote
release after an entire infection cycle on CHO-K1 cells. To measure the effect of BT10
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on trypomastigote production by infected host cells, CHO-K1 cells were incubated with
trypomastigotes for 4 h. The cells were washed to eliminate the non-internalized
parasites and then incubated with culture medium at different concentrations of BT10 or
not (control). On the 5th day post-infection, the trypomastigotes released into the culture
media were counted. We observed a dose-dependent decrease in trypomastigote release,
which allowed us to measure the IC50 for trypomastigote bursting (8.5  2.9 M)
(Figure 7A, 7B). Based on this value and the CC50 obtained for the cytotoxicity of
BT10 on CHO-K1 cells, we obtained a selectivity index (SI; CC50/IC50(Tryp)) of 11.21. It
is expected that the diminished number of burst trypomastigotes in the BT10-treated
cells is a reflection of the diminished proliferation of intracellular forms. To verify this
hypothesis, CHO-K1 cells were incubated with trypomastigotes for 4 h. The cells were
washed to eliminate the non-internalized parasites, and then they were incubated with
culture medium supplemented or not (control) with 8.5 μM BT10 (the concentration
corresponding to the IC50 obtained for trypomastigote bursting). On the second day
post-infection, the cultures were fixed and stained, and the nuclei corresponding to the
total number of cells, the number of infected cells, and the number of amastigotes per
infected cell were counted. The percentage of infected cells and the number of
intracellular amastigotes per cell were 8.6  0.88 and 0.16  0.05 for infected cells
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treated with 8.5 μM of BT10 and 13.1  3.7 and 0.6  0.13 for untreated conditions,
respectively (Figure 7C, 7D). BT10 treatment diminished the number of infected cells
by 33.8% and the average number of intracellular amastigotes per cell by 70.3%. The
resulting infection index was 1.4  0.48 for the treated infected cells and 8.9  1.34 for
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the controls, indicating that the treatment reduced the infection index by 81.3 %
(Figure 7E, 7F). Taken together, these results indicate that treatment with 8.5 M BT10
interferes with proliferation and/or differentiation of intracellular stages (Figure 7).
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DISCUSSION
In the present work, we obtained and evaluated a collection of 14 benzothiazoles (BT1-
14) related to previously studied 4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl] benzoic
acid (BT3), which showed relevant anti-T. cruzi trypomastigote activity (14). The anti-
proliferation activity for all these compounds was initially evaluated in a screening with
25 M each compound. BT10 was the only one that diminish the epimastigote growth
by 50%, as previously mentioned. Although BT3 did not pass these criteria, it was the
second best in terms of potency. Thus, BT10 and BT3 were used for some experiments
that allowed us to infer some structure-activity relationships. Both compounds contain a
trifluoromethyl group (-CF3) in the R1 position of the benzothiazole moiety.
Additionally, BT3 has a carboxyl group (-COOH) on R3 of the phenyl moiety, while
BT10 contains a methoxy group (-OCH3) on R2 and a hydroxyl group (-OH) on R3.
These data suggest that the inclusion of a fluorinated group on 2-phenylbenzothiazole
derivatives contributes significantly to the anti-proliferation activity. All these
inferences are supported by the fact that benzothiazole derivatives such as (S)-2-(3,4-
difluorophenyl)-5-(3-fluoro-N-pyrrolidylamido)
benzothiazole
possess
anti-
Trypanosoma brucei activities (15). BT2 and BT6 compounds are closely related to
BT3 and BT10, respectively, with the only difference being the absence of the -CF3
substituent. The relevance of the fluorinated group in BT2 and BT6 could explain their
lack of anti-T. cruzi activity (Figure 1A, 1B, 1C). In fact, this finding is not surprising
since it is well known that fluorine atoms confer a set of physicochemical properties to
organic compounds. Among them, it is worth mentioning improvement of lipophilicity,
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increased velocity of diffusion through biological membranes (32), an improved affinity
for receptors (by favouring electrostatic interactions) and an increased binding affinity
to active sites (33). In addition, the presence of fluorinated groups can also modify the
acidity or basicity of the molecules, affecting the processes of absorption, metabolism
and bioavailability (34). To analyse the correlation of the activity of BT1-14 to in silico
drug-likeness analysis (rule of five – RO5) (35), we determined whether the compounds
in the collection follow these rules. Regarding the quantitative parameters of the RO5,
we calculated the lipophilicity (expressed as the partition coefficient - Log P) and
topological polar surface area (TPSA - defined as the integration of the surfaces of polar
atoms (in this case oxygen, nitrogen, and attached hydrogen) (36). The computed
parameters showed that BT3 and BT10 have Log P values of 3.83 and 3.92 and TPSA
values of 78.43 and 70.59, respectively. The presence of the -CF3 group affected the
predicted lipophilicity (comparing BT2 with BT3 and BT6 with BT10), resulting in
intermediate Log P values for the most active compounds. However, BT3 and BT10
were among those presenting the highest TPSA values in the collection. The increased
TPSA values are probably due to the -COOH, -OH and -OCH3 groups on the phenyl
moiety, which trigger electron withdrawal in combination with the –CF3 group in the
case of BT10 and BT3. This fact could have a positive effect on anti-proliferation
activity. As the drug-likeness depends on both values, we propose herein a parameter
consisting of the product between Log P and TPSA (Table S1), which showed for our
compounds a good prediction capacity in relation to their in vitro activity.
Among the drugs analysed in our collection, BT10 was the only one that passed through
our criteria for being selected as an anti-T. cruzi drug for further studies, resulting in 2-
fold more potent activity than that of BT3. Despite its anti-proliferation effect, we could
not detect signals of cell death in BT10-treated parasites, such as exposure of
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phosphatidylserine or loss of cytoplasmic membrane integrity. Thus, we hypothesized
that BT10 would act as a trypanostatic compound rather than a trypanocide. Despite
having a lower activity, we observed the same profile for BT3-treated parasites
(Figure S3), indicating that the mode of action of these structurally related
compounds in T. cruzi is other than cytotoxicity, as previously reported for other
benzothiazoles when evaluated on different cancer cell lines (37, 38). To obtain more
clues about the inhibitory activity of BT10, we explored other parameters related to the
maintenance of cell viability: the status of the mitochondrial inner membrane potential
(ΔΨm), intracellular Ca2+ levels and endogenous production of ROS (26, 28). Our
results show that a fraction of the BT10-treated cells had a diminished ΔΨm with
respect to that of the controls. This result indicates that their mitochondria are at least
partially depolarized, which is consistent with the observed increase in intracellular Ca2+
concentration.
Altogether, our results agree in part with the effects reported for benzothiazoles with
antimicrobial activity in both Gram-positive and Gram-negative bacteria, although the
effects are observed with 4-fold the minimum inhibitory concentration (between 3.91 -
15.6 μg / mL) (39). Notably, these effects by themselves point to a trypanostatic rather
than trypanocidal activity. This possibility was confirmed by the fact that the effect of
BT10 was reversible and produced alterations in the epimastigote cell cycle. Indeed,
BT10 triggered a decrease of parasites in G0/G1 phases and an accumulation in the G/M
phase at a concentration of 100 μM.
An alteration of the cell cycle consisting of the accumulation of parasites in the G/M
phase could be a hallmark for DNA damage (31, 40). Our results revealed that BT10-
and BT3-treated cells (Figure S4) had selectively damaged kDNA, raising three
possibilities: i. kDNA has a higher sensitivity to damage than gDNA; ii. the
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mitochondrial DNA repair machinery (in the presence of the drug) is less efficient than
that of the nuclear DNA; or iii. the drug accumulates at higher concentrations in the
mitochondria, submitting kDNA to higher concentrations of the drug than those for
gDNA. Of course, a combination of the three possibilities cannot be ruled out. In any
case, the mechanism by which damage occurs to kDNA remains elusive; it is possible
that BT10 and BT3 will inhibit some DNA-dependent enzyme or inhibitor directly by
inhibition of transcription and replication enzymes through direct interaction with DNA,
as suggested by the helically arranged relationship of BT6 in crystal formation (41).
This work support the fact that the kDNA seems to be the main target to explain the
anti-T. cruzi effect of BT3 and BT10. Indeed, the concentrations required to inhibit the
triosephosphate isomerase are higher than those required to kill trypanosomatids (14,
15, 23). These data break the paradigm that the trypanocidal effect of benzothiazoles is
due to the inhibition of triosephosphate isomerase as traditionally proposed (13, 25, 42).
Regardless of the mechanism of action, the anti-T. cruzi activity on the parasite stages
that are relevant for mammalian infection is a sine qua non condition for any compound
to be proposed as a possible drug to develop a treatment against Chagas disease.
Remarkably, the treatment of infected cells with 8.5 µM BT10 caused a significant
reduction in the number of intracellular amastigotes, in the trypomastigote burst and in
the infection index. These results, together with the drug selectivity (SI of 11.21) (Table
1), are particularly promising for the development of chemotherapy against the chronic
phase of the disease.
To conclude, BT10 is a remarkable fluorinated hit compound for the development of
new and better anti-T. cruzi compounds, which would be in accordance with the
initiative proposed by the Drugs for Neglected Diseases initiative (DNDi) (43). Because
the kinetoplast is the preferred target for BT10, other compounds based on its structure
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could have potential therapeutic applications for other diseases caused by kinetoplastid
parasites, such as Leishmania spp. and T. brucei.
Materials and methods
Reagents. All chemicals, reagents, and solvents for synthesis and MTT
(methylthiazolyldiphenyl-tetrazolium bromide) were purchased from Sigma-Aldrich
(St. Louis, MO, USA); Fluo-4 AM and Annexin V-FITC were purchased from
Invitrogen (Eugene, Oregon, USA); and culture media and foetal calf serum (FCS) were
purchased from Cultilab (Campinas, SP, Brazil).
Synthesis of drugs and chemical characterization. The 2-phenylbenzothiazole
derivatives were synthesized by the following reaction conditions previously described
(14). Briefly, the appropriate 2-aminothiophenol (1.98 mmol) and substituted
benzaldehyde (2.1 mmol) were reacted with an equimolar amount of Na2S2O5 (2.1
mmol). The mixture, was stirred refluxing in DMSO at ~120 °C for 40 - 60 min. The
product was precipitate by adding cool water and collected by vacuum filtration. The
filtrate was then washed with an excess of water and left to dry. The remaining traces of
sodium metabisulfite were extracted with brine and CH2Cl2, and the solvent was
removed under vacuum. Finally, the resulting product was purified and recrystallized in
ethanol/water (1:3). The progress of the reaction was monitored by TLC analysis using
a mix of ethyl acetate/hexane (1:1) as the eluent. All synthesized products were
1
chemically characterized by H, 13C NMR spectra on a Jeol GSX-300 spectrometer (1H
300 MHz, 13C 75 MHz) or Bruker-400 (1H 400 MHz, 13C 101 MHz) or Bruker-750
biospin Rheinstetten, Germany (1H 750 MHz, 13C 189 MHz) using DMSO-d6 and
CDCl3 as the solvent and TMS as the internal reference. Chemical shift values (ax) are
15

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presented in parts per million (ppm), and coupling constants (J values) are presented in
Hertz (Hz). ESI – MS spectra were recorded on a Bruker micrOTOF-Q II. The
uncorrected melting points were obtained in open-ended capillary tubes in
Electrothermal 9300 digital equipment.
1
BT1 (2-phenyl-1,3-benzothiazole): white needles, 77 % yield. mp 95-96 ºC. H NMR
3
4
(400 MHz, CDCl3) δ 8.08 (m, 3H), 7.89 (dd, 1H, J= 8 Hz, J= 0.8 Hz), 7.5 (t, 1H,
3
J=1.2 Hz), 7.48 (m, 3H), 7.37 (td, 1H, J= 1.2 Hz); 13C NMR (101 MHz, CDCl3) δ
168.0, 154.1, 135.1, 133.6, 130.9, 129.0 (2C), 127.5 (2C), 126.3, 125.1, 123.2, 121.6;
MS(ESI) C13H9NS, [M + H]+, m/z calcd 212.0528, found 212.0520.
BT2 (4-(1,3-benzothiazol-2-yl)benzoic acid): white powder, 63 % yield. mp 332 ºC. 1H
NMR (400 MHz, DMSO-d6) δ 8.20 (d, 2H, 3J = 8.4 Hz), 8.17 (s, 1H), 8.12 (d, 3H, 3J =
3
3
8.0 Hz), 7.58 (t, 1H, J = 7.6 Hz), 7.50 (t, 1H, J = 7.6 Hz); 13C NMR (101 MHz,
DMSO-d6) δ 167.1, 166.6, 154.0, 136.8, 135.2, 133.7, 130.7 (2C), 127.7 (2C), 127.3,
126.4, 123.6, 122.9; MS(ESI) C14H9NO2S, [M + H]+, m/z calcd 256.0426, found
256.0394.
BT3 (4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl]benzoic acid): white needles, 82 %
1
3
yield. mp 257 ºC H NMR (300 MHz, DMSO-d6) δ 13.2 (s, 1H), 8.36 (d, 1H, J= 6.6
3
Hz), 8.15 (d, 2H, 3J= 8.4 Hz), 8.06 (d, 2H, J= 8.7 Hz), 7.73 (dd, 1H, 3J= 8.7, 4J= 1.5
Hz); 13C NMR (75 MHz, DMSO-d6) δ 169.2, 166.9, 153.4, 139.2, 136.1, 133.8, 130.7
(2C), 128.3, 127.9 (2), 124.4, 122.2, 120.3; MS(ESI) C15H8F3NO2S, [M + H]-, m/z calcd
322.0144, found 322.0226.
16

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BT4 (2-phenyl-5-(trifluoromethyl)-1,3-benzothiazole): yellow scaly crystals, 53 %
1
yield. mp 127-129 ºC. NMR (400 MHz, CDCl3) δ 8.33 (t, 1H, J= 0.8 Hz), 8.09 (m,
2H), 8.0 (dt, 1H, 3J= 8.6 Hz, 4J= 0.4 Hz), 7.52 (m, 3H); 13C NMR (101 MHz, CDCl3) δ
170.1, 153.7, 138.4, 133.0, 131.5, 129.1 (2C), 127.7 (2C), 125.5, 122.2, 121.5, 120.4;
MS(ESI) C14H8F3NS, [M + H]+, m/z calcd 280.0402, found 280.0379.
BT5 (methyl 4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl]-benzoate): white needles,
1
41 % yield. mp 242 ºC. H NMR (300 MHz, DMSO-d6) δ 8.43 (s, 1H), 8.41 (s, 1H),
8.21 (d, 2H, 3J= 11.2 Hz), 8.09 (d, 2H, 3J= 11.2 Hz), 7.78 (d, 1H, 3J= 11.2 Hz), 3.87 (s,
3H); 13C NMR (75 MHz, DMSO-d6) δ 169.1, 165.9, 153.6, 139.3, 136.7, 132.8, 130.5
(2C), 128.6, 128.1 (2C), 124.3, 122.3, 120.4, 52.7; MS(ESI) C16H10F3NO2S, [M + H]+,
m/z calcd 338.0457, found 338.0452.
BT6 (2-(4-hydroxy-3-methoxyphenyl)-benzothiazole): grey powder, 87 % yield. mp
1
3
162-163 ºC. H NMR (300 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.03 (d, 1H, J=8.25 Hz),
3
4
3
4
7.97 (d, 1H, J= 8.4 ppm), 7.61 (d, 1H, J= 2.1 Hz), 7.47 (dd, 1H, J= 8.1 Hz, J= 2.1
Hz), 7.47 (m, 1H), 7.37 (td, 1H, J= 7.6 Hz), 6.91 (d, 1H, J= 8.1 Hz), 3.87 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) δ 167.9, 154.0, 150.4, 148.5, 134.6, 126.8, 125.3, 124.7,
122.7, 122.5, 121.7, 116.3, 110.4, 56.1; MS(ESI) C14H11NO2S, [M + H]+, m/z calcd
258.0583, found 258.0583.
BT7 (3-(1,3-benzothiazol-2-yl)-benzoic acid): grey crystals, 77 % yield. mp 257 ºC. 1H
3
NMR (750 MHz, DMSO-d6) δ 13.40 (s, 1H), 8.63 (s, 1H), 8.31 (d, 1H, J= 4.4 Hz),
3
3
3
8.18 (d, 1H, J= 4 Hz), 8.12 (d, 1H, J= 4.4 Hz), 8.1 (d, 1H, J= 4.4 Hz), 7.72 (t, 1H,
3J= 4), 7.57 (t, 1H, 3J= 4), 7.49 (t, 1H, 3J= 3.8 Hz); 13C NMR (189 MHz, DMSO-d6) δ
17

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167.0, 166.7, 153.9, 134.9, 133.6, 132.3, 131.7, 130.4, 128, 127.3, 126.3, 123.5, 122.9;
MS(ESI) C14H9NO2S, [M + H]+, m/z calcd 256.0426, found 256.0407.
BT8 (methyl 4-(1,3-benzothiazol-2-yl)-benzoate): solid brown, 67 % yield. mp 158 ºC
1H NMR (400 MHz, DMSO-d6) δ 8.25 (d, 2H, 3J= 8.8 Hz), 8.21 (dd, 1H, J=1), 8.14 (d,
2H, 3J= 8.8 Hz), 7.6 (td, 1H, 3J= 7.6 Hz, 4J= 1.2 Hz), 7.52 (td, 1H, 3J= 7.7 Hz, 4J= 1.2
Hz), 3.91 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 166.4, 166.0, 153.9, 137.2, 135.2,
132.1, 130.6 (2C), 127.9 (2C), 127.4, 126.5, 123.7, 123.0, 52.8; MS(ESI) C15H11NO2S,
[M + H]+, m/z calcd 270.0583, found 270.0563.
BT9 (methyl 3-(1,3-benzothiazol-2-yl)-benzoate): white powder, 87 % yield. mp 248
1
3
ºC. H NMR (750 MHz, DMSO-d6) δ 8.62 (s, 1H), 8.32 (d,1H J= 8.25 Hz,), 8.17
(d,1H, 3J= 7.5 Hz,), 8.11 (t, 2H, 3J= 8.25 Hz), 7.73 (t, 1H, 3J= 7.5 Hz,), 7.57 (t, 1H, 3J=
3
7.87 Hz), 7.49 (t, 1H, J= 7.87 Hz), 3.92 (s, 3H); 13C NMR (189 MHz, DMSO-d6) δ
166.5, 166, 153.9, 134.9, 133.7, 132.2, 132.0, 131.1, 130.6, 127.6, 127.3, 126.3, 123.5,
122.9, 52.9; MS(ESI) C15H11NO2S, [M + H]+, m/z calcd 270.0583, found 270.0575.
BT10 (2-methoxy-4-[5-(trifluoromethyl)-1,3-benzothiazol-2-yl]phenol): Brownish
1
crystalline powder, 22 % yield. mp 137 ºC. H NMR (300 MHz, DMSO-d6) 8.3 (d, 1H,
3
4
3
3J= 6 Hz), 8.28 (s, 1H), 7.68 (dd, 1H, J= 9 Hz, J= 3 Hz), 7.6 (d, 1H, J= 3 Hz), 7.51
3
4
3
(dd, 1H, J= 8.4 Hz, J= 2.1 Hz), 6.94 (d, 1H, J= 8.1 Hz), 3.87 (s, 3H); 13C NMR (75
MHz, DMSO- d6) δ 170.6, 153.7, 151.0, 148.5, 138.8, 128, 124.1, 123.9, 122.1, 121.3,
119.3, 116.4, 110.6, 56.1; MS(ESI) C15H10F3NO2S, [M + H]+, m/z calcd 326.0457,
found 326.0429.
18

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BT11 (4-(1,3-benzothiazol-2-yl)-2,6-dimethoxyphenol): grey crystals, 85 % yield. mp
1
3
3
135 ºC H NMR (750 MHz, CDCl3) δ 8.03 (d, 1H, J= 8.25 Hz), 7.87 (d, 1H, J= 7.5
Hz), 7.47 (m, 1H), 7.35 (td, 1H, 3J= 7.5 Hz), 7.34 (s, 2H), 3.99 (s, 6H); 13C NMR (189
MHz, CDCl3) δ 168.1, 154.0, 147.3 (2C), 137.7, 134.8, 126.3, 125.1, 124.9, 122.8,
121.5, 104.5 (2C), 56.5 (2C); MS(ESI) C15H13NO3S, [M + H]+, m/z calcd 288.0689,
found 288.0695.
BT12 (4-(1,3-benzothiazol-2-yl)benzonitrile): white crystalline powder, 88 % yield. mp
158 ºC. 1H NMR (300 MHz, DMSO-d6) δ 8.21 (d, 2H, J = 8.4 Hz), 8.16 (d, 1H, J = 8.1
Hz), 8.08 (d, 1H, J = 8.07 Hz), 7.98 (d, 2H, J = 8.4 Hz), 7.56 (m, 1H), 7.49 (t, 1H, J=
7.5 Hz); 13C NMR (75 MHz, DMSO-d6) δ 165.7, 153.8, 137.0, 135.3, 133.7 (2C), 128.2
(2C), 127.4, 126.6, 123.8, 123.0, 118.7, 113.7; MS(ESI) C14H8N2S, [M + H]+, m/z calcd
237.0481, found 237.0482.
BT13 (2-(4-chlorophenyl)-1,3-benzothiazole): grey crystals, 72 % yield. mp 105 ºC. 1H
3
3
4
NMR (750 MHz, CDCl3) δ 8.06 (d, 1H, J= 8.2 Hz), 8.02 (d, 2H, J= 9 Hz, J= 2.25
Hz), 7.9 (d, 1H, 3J= 8.2 Hz), 7.5 (t, 1H, 3J= 7.5 Hz), 7.46 (d, 2H, 3J= 9 Hz, 4J= 2.25 Hz),
7.39 (td, 1H, J= 7.12 Hz); 13C NMR (189 MHz, CDCl3) δ 166.6, 154.0, 137.0, 135.0,
132.1, 129.2 (2C), 128.7 (2C), 126.4, 125.4, 123.2, 121.6; MS(ESI) C13H8NSCl, [M +
H]+, m/z calcd 246.0139, found 246.0134.
BT14 (2-(4-bromophenyl)-1,3-benzothiazole): yellow crystals, 38 % yield. mp 124 ºC.
1H NMR (750 MHz, CDCl3) δ 8.06 (d, 1H, 3J = 8.2 Hz), 7.95 (d, 2H, 3J= 8 Hz), 7.89 (d,
1H, 3J= 8.2 Hz), 7.62 (d, 2H, 3J= 8.2 Hz), 7.49 (t, 1H, 3J= 7.5 Hz), 7.39 (td, 1H, 3J= 7.5
Hz); 13C NMR (189 MHz, CDCl3) δ 166.6, 154.0, 135.0, 132.5, 132.2 (2C), 128.9 (2C),
19

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126.5, 125.4, 123.3, 121.6; MS(ESI) C13H8NSBr, [M + H]+, m/z calcd 289.9634, found
289.9613.
Cells and parasite cultures. T. cruzi epimastigotes (CL strain clone 14) were
maintained in the exponential growth phase by subculturing every 48 h in LIT medium
at 28 °C (44). The Chinese Hamster Ovary cell line (CHO-K1) was cultivated in RPMI-
1640 medium supplemented with 10% heat-inactivated FCS, 0.15% (w/v) NaHCO3,
100 units/mL penicillin and 100 mg/mL streptomycin at 37 °C in a humidified
atmosphere containing 5% CO2. Trypomastigotes were obtained by infection in CHO-
K1 cells with trypomastigotes at 37 °C in the presence of 10% FCS. After 24 h, the cells
were maintained at 33 °C and 2% FCS (45). Trypomastigotes were collected from the
extracellular medium five days after infection.
In vitro inhibition of proliferation assays. The cell density of exponentially
proliferating epimastigotes (approximately 50×106 parasites/mL) was adjusted to
2.5×106 cells/mL and transferred (200 µL/well) into 96-well plates (46). Epimastigote
proliferation was measured by reading the optical density (OD) at 620 nm every 24 h
through the exponential and stationary phases (9 days). The OD values were converted
to cell density values (cells per millilitre by using a calibration curve obtained by
measuring the OD values at 620 nm of parasite suspensions at different known
densities. The concentrations of the compound that inhibited 50% of epimastigote
proliferation (IC50) were determined in the exponential growth phase (5th day) by fitting
the cell density data to a sigmoidal concentration-response curve using GraphPad Prism
v.6. A combination of 60 µM rotenone and 0.5 µM antimycin A (RA) was used as a
20

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positive control for proliferation inhibition. Untreated parasites supplemented with
DMSO (the vehicle for the drugs) and unsupplemented parasites were used as negative
controls. The compounds were evaluated in quadruplicate in each experiment, and the
results correspond to three independent experiments.
The effect of BT3 and BT10 compounds on mammalian cell viability. CHO-K1 cells
(1 x 105 cells/well) in 100 μL of RPMI medium supplemented with FCS (10%) were
seeded in 96-well plates with or without (control) different concentrations of the most
active compounds, BT3 (16 to 800 μM) and BT10 (8 to 288 μM). Cell viability was
determined by the MTT assay as previously described (47). The produced formazan was
solubilized in 50 μL of DMSO, and the optical density (OD) was measured at 540 nm
using 690 nm as a reference. The IC50 was determined by fitting the data to a sigmoidal
dose-response curve using GraphPad Prism v.6. Each assay was developed in
quadruplicate, and the results correspond to the mean of three independent experiments.
Analysis of phosphatidylserine exposure, mitochondrial inner membrane (ΔΨm)
depolarization, intracellular Ca2+ levels, and hydrogen peroxide production.
Epimastigotes (2.5 x 106 cell/mL) were incubated for five days, unless otherwise stated,
in the presence or absence (control) of 25 M and 50 M BT10 (approximately 1x and
2x the IC50, respectively). To determine phosphatidylserine exposure, the cells were
labelled with propidium iodide (PI) and Annexin-V FITC (Molecular Probes) according
to the manufacturer’s instructions. As positive controls for cytoplasmic membrane
permeabilisation and extracellular exposure of phosphatidylserine, the parasites were
treated with 150 μM digitonin or 1M staurosporine for 30 min, respectively (48). For
determining variations in ΔΨm, cells were aliquoted in fractions at densities of 1.0 x 106
21

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cells/mL. One half of the aliquot was incubated for 15 min with 1 μM FCCP in PBS,
while the other half was left untreated. Then, all samples were centrifuged for 10 min at
2,700 x g and resuspended in HEPES-glucose buffer (50 mM HEPES (pH 7.4), 116 mM
NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM glucose, and 2 mM CaCl2). The cells
were labelled by the addition of 256 nM Rhodamine 123 (Rh123) for 20 min at 28 °C
(49). To analyse variations in the intracellular Ca2+ levels, the parasites were incubated
with 5 mM Fluo-4 AM (Invitrogen) for one hour at 28 °C. After this period, the cells
were washed twice with HEPES-glucose and resuspended in the same buffer (50). To
evaluate the production of hydrogen peroxide, the parasites treated with 1xIC50 or
2xIC50 of BT10 during 24 h were incubated for 30 min at 28 °C in HEPES-glucose
buffer in the presence of 10 μM of carboxy-DCFDA. In all cases, the cells were
analysed by flow cytometry on a Guava cytometer (General Electric), with 10,000
events collected, and analysed using FlowJo software (v10.1r7).
DNA content and cell cycle analysis. Parasites (2.5 x 106 cells/mL) treated with 25 and
100 μM BT10 (or not, negative control) for five days were collected by centrifugation
(2,700 x g for 5 min), washed in PBS, fixed in 70% ethanol for 12 h, and incubated with
10 μg/mL RNase A (Thermo Scientific) for 30 min at 37 °C. To measure the DNA
content, parasite cells were stained with 40 μg/mL propidium iodide (Molecular
Probes/Invitrogen) and analysed by flow cytometry on an acoustic focusing cytometer
(Attune; Applied Biosystems), with 50,000 events collected (31). Histograms (number
of counts by BL2 area) scatter plots (side scatter [SSC] area by forward scatter [FSC]
area) and gates for each cell cycle phase were analysed using FlowJo software
(v10.1r7). Cell cycle data were fitted using Dean-Jett Fox (DJF) model included in the
FlowJo software.
22

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Analysis of DNA damage by TUNEL assay. Epimastigotes treated with 25 M BT10
(or not, control) in the exponential growth phase were collected by centrifugation,
washed with PBS and fixed by incubation with 4% paraformaldehyde for 10 min at
room temperature. After washing and resuspension in PBS, the cells were permeabilized
by treatment with 0.1% Triton X-100 for 10 min at 26 ºC and were treated with 0.1 M
glycine for 5 min to neutralize the remaining aldehyde groups. The TUNEL assay was
performed by using the DeadEnd™ Fluorometric TUNEL System Promega kit (G3250)
according to the manufacturer’s protocol. VECTASHIELD® Mounting Medium with
DAPI (Vector Labs) was added to be used as an anti-fade mounting solution and to stain
nuclear and kinetoplast DNA. For each group analysed, we analysed at least 100 cells in
at least three different image fields. This assay was carried out in triplicate. To confirm
these results, we analysed an aliquot of the cells in each condition by cytometry.
Histograms (counts x BL1 area), scatter plots (SSC-area x FSC-area) and gates to
exclude cell debris/doublets were performed using Attune Cytometric Software (v.1.2.5)
and FlowJo software (v10.1r7). In total, 10,000 events were analysed for each sample.
Effect of BT10 on amastigote replication and trypomastigote release. CHO-K1 cells
(5.0 x 104 per well) were maintained in 24-well plates in RPMI medium supplemented
with 10% FCS at 37 °C. To perform the infections, the cells were incubated with
trypomastigote forms (2.5 x 106 per well) for 4 h. After this period, parasites in the
supernatant were removed by washing the plates twice with PBS, and the cells were
incubated overnight in RPMI medium supplemented with 10% FCS at 37 °C in the
presence of different concentrations of BT10 or left untreated (control). The plates were
then incubated at 33 °C to allow the parasite to complete the infection cycle, as
23

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previously reported (46). To measure the effect on amastigote replication, after 48 h, the
CHO-K1 cells and parasites were fixed with 4% paraformaldehyde, and nuclei were
stained with Hoechst 33342 (Invitrogen) for counting. We acquired fluorescence
microscopy images corresponding to 30 fields per biological sample at a magnification
of 200x (EVOS FL Cell Imaging System, Thermo Fisher Scientific). Cells, parasites,
and infected cells were counted using ImageJ software. The infection index was
calculated as the arithmetical product of the percentage of infected cells and the number
of parasites per cell. The effect of BT10 on trypomastigote production was determined
by collecting samples of the extracellular medium on the fifth day post-infection and
counting the number of trypomastigotes in a Neubauer chamber (40). The results
correspond to the mean of three independent experiments, each performed in triplicate.
Data treatment and statistical analysis. Curve adjustments, regressions, and statistical
analyses were performed with the GraphPad Prism 7 analysis tools. All assays were
performed at least in biological triplicates. The specific details of the statistical analysis
for each experiment are described in the corresponding figure legend. In general, The
Student’s t-test was used to analyse differences between the two groups. P values of less
than 0.05 were considered statistically significant.
Author contributions
Synthesised and characterized compounds: RICH, JGTF, SMC. Conceived and
designed the biological experiments: RG, RICH, AMS. Performed the experiments:
RICH, RG, MC, AMS, MSS. Analysed the data: RICH, RG, AMS, MCE. Contributed
reagents/materials/analysis tools: JGTF, AMS. Wrote the paper: RICH, AMS, RG. All
authors have given approval to the final version of the paper.
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Acknowledgements
This work was supported by Fundação de Amparo à Pesquisa do Estado de São
Paulo grant 2016/06034-2 (awarded to AMS), (www.fapesp.br); Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq) grants 301971/2017-0 and
404769/2018-7 (awarded to AMS) (www.cnpq.br), and Research Council United
Kingdom Global Challenges Research Fund under grant agreement “A Global Network
for Neglected Tropical Diseases” (grant MR/P027989/1) (awarded to AMS)
(https://www.ukri.org). Consejo Nacional de Ciencia y Tecnología (CONACyT) grant:
2011-1 No.168116 (awarded to JGTF); Secretaria de Investigación y Posgrado del
Instituto Politécnico Nacional (SIP-IPN) grants: 20120151, 20130488, 20140109,
20150309 and 20160126 (awarded to JGTF). Red Macrouniversidades and CONACyT
(student grants CRA/027-28/2016 and 246663). We acknowledge Alexandre Moura for
technical assistance during this work.
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