In vitro activity and mode of action of distamycin analogues against African trypanosomes

Article abstract of DOI:10.1016/j.ejmech.2016.12.002

Distamycin, a natural polyamide containing three heterocycle rings with a polar end, has inspired several groups to prepare synthetic analogues, which proved to have anti-trypanosomal and anti-tumoral activity. We describe the synthesis of bi and tri thiazoles amides that harbor different substitutions at their ends and the evaluation of their anti-Trypanosoma brucei activity. The most active compound 10b showed better biological activity (EC₅₀310 nM and selectivity index 16) than the control drug nifurtimox (EC₅₀15 μM and selectivity index 10). Studies on the mode of action show that the parasiticidal activity of 10b originates from disruption of lysosomal homeostasis, which is followed by release of redox active iron, an increase in oxidizing species and collapse of cell membrane integrity. In this respect, our study suggests that non-charged lipophylic distamycins destabilize cell membranes.

Full text of DOI:10.1016/j.ejmech.2016.12.002

Accepted Manuscript
In vitro activity and mode of action of distamycin analogues against African
trypanosomes
Jaime Franco, Andrea Medeiros, Diego Benitez, Karen Perelmuter, Gloria Serra,
Marcelo A. Comini, Laura Scarone
PII:
S0223-5234(16)31008-X
10.1016/j.ejmech.2016.12.002
EJMECH 9093
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 September 2016
Revised Date: 29 October 2016
Accepted Date: 1 December 2016
Please cite this article as: J. Franco, A. Medeiros, D. Benitez, K. Perelmuter, G. Serra, M.A. Comini, L.
Scarone, In vitro activity and mode of action of distamycin analogues against African trypanosomes,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2016.12.002.
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In vitro activity and mode of action of Distamycin
analogues against African trypanosomes
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Jaime Francoa,b, Andrea Medeirosb,c, Diego Benitezb, Karen Perelmuterd, Gloria Serraa,
Marcelo A. Comini*b, Laura Scarone*a
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Cátedra de Química Farmacéutica, Departamento de Química Orgánica, Facultad de
Química, Universidad de la República, Montevideo, Uruguay.
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Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo,
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Uruguay.
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Departamento de Bioquímica, Facultad de Medicina, Universidad de la República,
Montevideo, Uruguay
d Cell Biology Unit, Institut Pasteur de Montevideo, Montevideo, Uruguay.
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* Corresponding authors:
(1) Laura Scarone
Cátedra de Química Farmacéutica, Departamento de Química Orgánica
Facultad de Química, Universidad de la República
Gral. Flores 2124
Montevideo, CP 11800
Uruguay
E-mail: laurito@fq.edu.uy; Fax: +598 29241906; Phone: +598 29290290
(2) Marcelo Comini
Group Redox Biology of Trypanosomes
Institut Pasteur de Montevideo
Mataojo 2020
Montevideo, CP 11400
Uruguay
E-mail: mcomini@pasteur.edu.uy; Fax: +598 2522 4185; Phone: +598 2522 0910 ext. 164
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Graphical abstract
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Highlights
• tri-thiazoles resembling distamycin present anti-Trypanosoma brucei activity
• the most lipophilic derivative shows higher potency and selectivity than nifurtimox
• cytocidal action involves lysosomal damage, release of free-iron and oxidation
• subtle substitutions confer selectivity for the molecular target of tri thiazoles
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Abstract
Distamycin, a natural polyamide containing three heterocycle rings with a polar end, has
inspired several groups to prepare synthetic analogues, which proved to have anti-
trypanosomal and anti-tumoral activity. We describe the synthesis of bi and tri thiazoles
amides that harbor different substitutions at their ends and the evaluation of their anti-
Trypanosoma brucei activity. The most active compound 10b showed better biological
activity (EC50 310 nM and selectivity index 16) than the control drug nifurtimox (EC50 15 µM
and selectivity index 10). Studies on the mode of action show that the parasiticidal activity of
10b originates from disruption of lysosomal homeostasis, which is followed by release of
redox active iron, an increase in oxidizing species and collapse of cell membrane integrity. In
this respect, our study suggests that non-charged lipophylic distamycins destabilize cell
membranes.
Keywords: thiazole, Trypanosoma brucei, lysosome, iron, membrane damage
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1. Introduction
Human African Trypanosomiasis (HAT or sleeping sickness) is caused by the hemoflagellated
parasites Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense whereas
Trypanosoma brucei brucei is the etiological agent of the cattle disease Nagana. The parasites
are transmitted by the bite of an infected tsetse fly (Glossina genus) that infests 37 sub-
Saharan countries. Human and cattle infections by African trypanosomes are either fatal or
highly disabling if left untreated [1]. Although control programs implemented years ago
contributed to reduce the number of HAT cases to less than 10000 per year, Nagana remains a
major obstacle for the nutritional and economical development of the rural communities from
endemic areas [2]. For instance, per year about 3 million cattle dye, 35 million doses of
trypanocidal drugs are administered and direct losses in cattle production alone account for 6
to 12 billion US$ [3].
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Present-day chemotherapy for HAT includes: suramin (1916), pentamidine (1937),
melarsoprol (1949), eflornithine (1990) and nifurtimox-eflornithine combined therapy (NECT,
2009), all of them suffering from poor efficacy, undesirable route of administration,
unacceptable toxicity and emergence of drug resistance [4-6]. Nagana treatment includes:
isometamidium chloride, diminazene aceturate, homidium bromide and chloride, both proven
mutagenic agents [7-9]. The easy accession to these drugs and lack of administration controls
are considered the main causes for the increasing drug resistance reported in 17 African
countries [10]. Thus, the significant limitations of the current chemotherapy prompt the urgent
discovery of safer and effective anti-trypanosomal drugs.
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Natural products are widely recognized for playing important roles in drug development,
particularly as anticancer, antibiotic and antiparasitic drugs [11,12]. Furthermore, natural
polyamides containing heterocycles as distamycin and netropsin analogues have been
investigated as molecules with helical topology that form strong complexes with the minor
groove of DNA [13]. Many research groups have developed methodologies to obtain
distamycin (1, Figure 1) analogues and study their DNA binding affinity and sequence
selectivity as anti-infective agents [14-17]. Recently, Suckling’s group reported a series of
oligoamides of 2-amino-5-alkylthiazole 4-carboxylic acids (2, Figure 1), synthesized as
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distamycin and netropsin analogues, with interesting anti-trypanosomal activities [18].
Although this work did not address the mode of action of the most active compounds, possible
mechanisms of action were proposed based on a structure-activity relationship analysis and
the structural/physicochemical properties of the compounds. Similarly to diamidines, these
compounds may intercalate and disrupt the mitochondrial DNA (kinetoplast) [13] and/or, as
certain anti-microbial peptides called α- and β-defensins, may permeabilize cell membranes
due to the presence of ionizable groups [19].
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As part of our search for candidate compounds to develop novel antiparasitic drugs employing
molecular simplification [20-22], here we present the results on the synthesis of thiazoles
linked by amide bonds as distamycin analogues (3, figure 1) and their biological activity
against bloodstream T. brucei brucei and murine macrophages (cell line J774). Furthermore,
we investigated the potential antitrypanosomal mode of action of the most potent and selective
analogue identified.
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Figure 1. Distamycin and oligoamide analogues
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2. Results and Discussion
2.1. Synthesis of distamycin analogues
The synthetic strategy started with Hantszch’s thiazoles synthesis, obtained from ethyl bromo
piruvate and acetylthioamide to obtain thiazole 4a or thiourea to obtain thiazole 4b (Scheme
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1). Then, ethyl ester hydrolysis of thiazole 4a allowed us to obtain thiazole acid 4e. The amine
group in position 5 of thiazole 4b was protected with di-tert-butyl dicarbonate (4c) and the
ethyl ester group was hydrolyzed to obtained thiazole acid 4d.
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Bis-thiazole ester 5a was obtained from thiazole amine 4b and thiazole acid 4e, employing
HBTU (O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) as
coupling reagent, in 90% yield (Scheme 1).
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Boger’s and Brucoli’s groups reported bis-thiazole 5b synthesis employing EDCI (N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride) as coupling reagent with 58%
and 71% yield, respectively [23, 24]. In our hands, this coupling reagent resulted in 5b with
44% yield from both thiazole amine 4b and thiazole acid 4d. However, employing HBTU as
coupling reagent, the yield was increased to 70%. The bis-thiazoles analogues 6a and 6b were
obtained quantitatively by ester hydrolysis of thiazole esters 5a and 5b, respectively. N-Boc
cleavage of bis-thiazole 5b gave bis-thiazole 7 (quantitative) and allowed us to prepare the N-
acetyl derivate 8 (90% yield). Compound 8 was converted quantitatively into the bis-thiazole
9 by classical ester hydrolysis.
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Following the same serie of reactions we prepared tri-thiazoles 10a and 10b. Despite the well
known poor reactivity of the amine thiazole, we were able to obtain a 66% yield for the
synthesis of 10a from 4b and 6a. For preparation of the tri-thiazole 10b (synthesis reported in
reference [25] with EDCI as coupling reagent in 52% yield) two strategies were explored: 1)
from thiazole acid 4d and amine bithiazole 7 with HBTU, we obtained tri-thiazole 10b in 33%
yield and 2) from amine thiazole 4b and bithiazole acid 6b with HBTU, we obtained tri-
thiazole 10b in 45% yield. Coupling reagents as EDCI and DEPBT resulted in a poor
formation of tri-thiazole 10b. Then, analogues 11 and 12 were obtained in good yields by N-
deprotection and N-acetylation reactions, respectively. Tri-thiazole 13 and 14 were prepared
by ester hydrolisis of tri-thiazole 10b and 12, respectively.
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Scheme 1. Synthesis of distamycin analogues.
2.2. Biological activity
Bi- and tri-thiazoles linked by amide bond were evaluated in vitro against the infective form
of T. b. brucei, which is also a suitable laboratory model of the human pathogen T. b.
rhodesiense. An initial screening was performed at 5 and 30 µM compound followed by the
assessment of the EC50 for compounds displaying a cytotoxicity against the parasite > 40% at
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5 µM. In addition, the selectivity of their biological activity was tested towards murine
macrophages (cell line J774). The results are summarized in Table 1.
Table 1. Biological activity of distamycin analogues.
Infective T. b. bruceib
Selectivity
Entry
Compounda
indexc
growth inhibition
EC50 (µM)
13.7 ± 1.4
---
at 5 µM (%)
1
2
BocHN-Tz-Tz-COOEt (5b)
BocHN-Tz-Tz-COOH (6b)
3.5
---
10.8 ± 7.3
ND
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H2N-Tz-Tz-COOEt (7)
---
25.6 ± 7.4
ND
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5
AcHN-Tz-Tz-COOEt (8)
AcHN-Tz-Tz-COOH (9)
7.4
ND
16.5
ND
19.6
29.1
ND
ND
ND
9.6 ± 5.6
---
---
20.0 ± 2.5
---
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BocHN-Tz-Tz-Tz-COOEt (10b)
BocHN-Tz-Tz-Tz-COOH (13)
H2N-Tz-Tz-Tz-COOEt (11)
AcHN-Tz-Tz-Tz- COOEt (12)
AcHN-Tz-Tz-Tz-COOH (14)
H3C-Tz-Tz-COOEt (5a)
0.31 ± 0.07
---
7
22.7 ± 8.4
---
8
5.5 ± 0.1
5.4 ± 1.2
---
9
---
10
11
12
no effect
no effect
8.1 ± 2.0
---
H3C-Tz-Tz-COOH (6a)
---
13
14
H3C-Tz-Tz-Tz-COOEt (10a)
10.5
10.0
≅ 5
---
Nifurtimox
---
15.0 ± 2.5
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ND, not determined.
a Tz, thiazole.
b the values are expressed as mean ± SD (n=3).
c Ratio EC50 murine macrophage/EC50 T. b. brucei
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Overall, the tri-thiazole derivatives presented higher anti-trypanosomal activity (4 out of 6
with EC50 ≤10 µM) and selectivity (SI ≥10) than the bi-thiazole analogues (1 out of 7 with
EC50 ≤10 µM and SI <10). For instance, the addition of a heterocycle to 5a (entry 11, not
active) and 7 (entry 3, 26% growth inhibition at 5 µM) produced the more potent analogues
10a (entry 13, EC50 ≈ 5 µM) and 11 (entry 8, EC50 = 5.5 µM) respectively, and conversion of
the bi-thiazole 5b (entry 1, EC50 = 13.7 µM) into the tri-thiazole 10b increased near 40 times
its anti-trypanosomal activity (entry 6, EC50 = 0.31 µM).
For the bi-thiazoles, there appear to be a need for fulfilling an steric demand on the molecular
target since the 2-amino substituted (position R2) thiazole 5b and 8 (entries 1 and 4,
respectively, average EC50 of 11 µM) were more active than the 2-methyl analogue 5a (entry
11, no cytotoxic at 5 µM) and the N-unsubstituted bis-thiazole 7 (entry 3, 26% growth
inhibition at 5 µM). In contrast, the nature of the substituent at R2 does not affect significantly
the activity of bi-thiazoles when the structure contains a carboxylic acid at R1 (compounds 6a,
6b and 9 entries 12, 2 and 5, average growth inhibition of 15% at 5 µM).
The most active compound against T. b. brucei, the N-Boc trithiazole ester 10b (entry 6),
displayed a potency almost 50-fold (EC50 = 0.31 µM) higher than that of the control drug
nifurtimox (EC50 = 15 µM) and a similar selectivity index (SI) (SI = 16.5 for 10b vs. SI = 10
for nifurtimox). Interestingly, compound 14 (entry 10), the closest analogue to the most active
tri-heterocycle derivative reported by Lang et al. (MIC = 64 nM) [18] is inactive towards
trypanosomes when tested at 30 µM. The major differences between these compounds are: a)
thiazole moiety (14) vs. N-methyl pyrrole, and b) 5-unsubstituted (14) vs. 5-isopropyl
thiazoles. This suggests that hydrophobicity may play an important role in molecular
recognition of the cellular target and, hence, in the biological activity of distamycin analogues.
In line with this, all the molecules that can be charged at physiological pH (compounds 6a, 6b,
9, 13 and 14) presented null or marginal activity against the parasite (average 10% growth
inhibition at 5 µM). Furthermore, almost all esters derivatives exhibited higher activities than
the corresponding compounds with a free carboxylate moiety: i.e. compare 5b with 6b (entries
1 and 2), 8 with 9 (entries 4 and 5), 10b with 13 (entries 6 and 7) and 12 with 14 (entries 9 and
10). In addition, compounds with blocked amino groups presented higher activity than their
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congeners with a free amine ‒ i.e. compare the tri-thiazoles 10b and 11 (entries 6 and 8) and
the bi-thiazoles 5b and 8 ‒ suggesting that increased lipophilicity may contribute to their
activity. In fact, 10b presented the highest log P values among the different analogues tested
here (Suplementary Table S1). Interestingly, a similar correlation between lipophilicity and
anti-T. brucei activity was reported for related bisimidazolines [26].
The SAR data shows that both the number of heterocycles rings and the lack of polarity
modulate the biological activity of the distamycin analogues.
2.3. Studies on the mechanism of action
Distamycin and netropsin analogues were proposed as DNA minor groove binders that disrupt
parasite cell cycle and, consequently, inhibit parasite proliferation. Because of their chemical
nature and peptide like-structure these compounds were also suggested to act as α- and β-
defensins that interact and destabilize cell membranes (e.g. pore formation). While the first
mechanism is expected to exert a cytostatic effect, the second one should trigger cell lysis
(cytotoxic effect).
As a first approach to investigate the mode of action of the most active distamycin analogue
identified in this work, compound 10b, the integrity of the parasite membrane was monitored
using the membrane impermeant dye propidium iodide (PI).
Membrane integrity assays were performed exposing the parasites for short times (0.5 – 4 h)
to 10b at ¼-, ½- and 1-fold its EC50 value (310 nM) or to the detergent Triton X-100 (0.001%
v/v; positive control), and adding PI immediately prior to sample analysis by flow cytometry.
The assay shows a time-dependent increase in the number of cells PI positive, which, in
general, correlated also with the concentration of 10b (Figure 2). For instance, added at its
EC50, the compound triggered membrane damage in about 10% of the cell population after 1 h
of treatment, a value that increased to 80-90% for the subsequent 2-4 h exposure. At this time
point, the degree of membrane permeabilization produced by 10b (310 or 155 nM) was almost
identical to that exerted by the detergent used as control. The mode of action of nifurtimox
involves its reduction to a non-saturated open-chain nitrile that acts as potent electrophile,
which modifies different biomolecules [27]. As expected, nifurtimox tested at its EC50 (15 µM)
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did not affect membrane permeability in parasites treated for 0.5-4 h. It is worth to note that
except for a single data point (77.5 nM compound for 1 h), the effect exerted by 10b on
membrane integrity was statistically significant (p < 0.05) for all concentrations and time
points tested compared to parasites treated with the vehicle alone (DMSO) or nifurtimox
(NFX).
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Figure 2. Membrane permeability of bloodstream T. brucei treated with compound 10b.
Five x105 parasites/mL were treated with different concentrations (310, 155 and 77.5 nM) of
10b for different times: A) 0.5 h, B) 1 h, C) 2 h and D) 4 h. Control treatments included Triton
X-100 at 0.001% v/v, nifurtimox (NFX) at 15 µM and DMSO at 1% v/v. Values are expressed
as mean ± SD (n=3) and the asterisks denote the probability indexes of 10b-treated parasites
vs. DMSO control (* p < 0.05; ** p < 0.01; *** p < 0.001), calculated applying the ONE
WAY ANOVA test and the Dunnets’s multiple comparision post test.
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Different molecular mechanisms may lead to the disruption of parasite membrane integrity,
from the insertion of the compound into the cell membrane, as shown for different mammalian
antimicrobial peptides that kill African trypanosomes (e.g. α- and β-defensins and
cathelicidins) [28], to the inhibition of components from the parasite anti-oxidant system. In
this respect, it is well documented that down-regulation or gene knockout of key enzymes
from the parasite redox system triggers the oxidation of unsaturated lipids in lysosomal or
mitochondrial membranes, which releases iron that enhances the peroxidation of membranes
(via Fenton reaction) resulting in a rapid cell lysis [29-32].
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A T. b. brucei transgenic cell line expressing a GFP-based redox biosensor was used to
investigate whether the mode of action of 10b implies a deregulation of the intracellular redox
milieu. The biosensor, hGrx-roGFP2, is able to rapidly (nano second) translate changes in the
pool of reduced and oxidized glutathione (GSH/GSSG) into changes in its fluorescence
intensity at 488 nm [33].
The redox reporter T. brucei cell line was exposed to different concentrations of 10b,
menadione, NFX, the membrane permeant reducing agent dithiothreitol (DTT) or the vehicle
DMSO for 1 h, and the fluorescence intensity of the biosensor was analyzed by flow
cytometry. As shown in Figure 3 A, at all concentrations tested (77.5-310 nM) 10b lowered
by ~ 10% (p < 0.001) the fluorescence intensity of the biosensor, which indicates a higher
GSSG/GSH ratio or a more oxidative millieu, compared to parasites treated or not with
DMSO. As expected, the potent redox cycler menadione added at 1 mM caused a 21%
oxidation of the biosensor whereas 15 µM NFX or 1 mM DTT did not elicit redox changes
after 1 h treatment.
In order to confirm the redox basis of the changes in biosensor fluorescence triggered by 10b,
the cells exposed to 77.5-310 nM compound for 1 h were treated with 1 mM DTT for
additional 30 min. This treatment was able to restore the redox state of the biosensor to levels
similar to the non-treated or vehicle-treated cells (Figure 3 B), supporting the idea that 10b
alters the intracellular redox balance. Similarly, the oxidation of the biosensor in parasites
exposed to menadione was reverted by treatment with DTT. For the control conditions (NFX
or DMSO), the reducing agent did not modify the fluorescence intensity of the biosensor.
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B
***
A
100
*** ***
100
50
0
***
***
50
0
10b (nM)
10b (nM)
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Figure 3. Evaluation of the intracellular redox state of infective T. b. brucei treated
with 10b. Parasites (1x106 cells/mL) expressing the redox biosensor hGrx-roGFP2 were
treated for 1 h with 10b (77.5, 155 and 310 nM) or different control compounds such as
menadione (1 mM), nifurtimox (NFX, 15 µM), dithiothreitol (DTT, 1 mM), DMSO 1% v/v
and 1 mM DTT. After the corresponding treatments the cells were analyzed by flow
cytometry. Redox analysis was performed only for PI negative cells. The results are presented
as percentage biosensor reduction relative to DMSO for: A) parasites treated for 1 h with
different compounds, and B) followed by a 30 min treatment with 1 mM DTT. Values are
expressed as mean SD (n=3) and the asterisks indicate difference (p < 0.001) with respect to
the DMSO control according to the ONE WAY ANOVA test and the Dunnets’s multiple
comparison post test.
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In principle, the intracellular oxidation of parasites treated with 10b may be consequence of
the direct inhibition of specific component(s) from the antioxidant system. In order to test this
hypothesis, we first analyzed the potential of 10b to inhibit trypanothione synthetase (TryS),
the enzyme producing the major redox cosubstrate in trypanosomatids [29, 34], namely
trypanothione. At 30 µM 10b, as well as none of the distamycin analogues synthesized in this
work, did not inhibit to a significant level trypanothione synthetase from T. brucei,
Trypanosoma cruzi and Leishmania infantum (Supplementary Table 2). Next, mammalian
cells (cell line CHO-K1) expressing the redox biosensor rxYFP, which also detects changes in
the GSSG/GSH ratio [34], were exposed to concentrations of 10b around the EC50 determined
for macrophages (5 µM) for 1 h and analyzed by flow cytometry (Fig 4 A and B). Irrespective
of the concentration tested (1-10 µM), 10b produced about 10% oxidation of the biosensor in
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CHO-K1 cells compared to cells treated with vehicle (p < 0.01). Control treatments with
menadione or H2O2, both added at 1 mM, induced a marked oxidation (i.e. 70% and 25%,
respectively) of the biosensor (p < 0.001). Addition of 1 mM DTT for 30 min to cells treated
with 10b, menadione or H2O2 (10 µM) restored the fluorescence intensity of rxYFP to basal
levels, confirming thus the redox origin of the changes in biosensor fluorescence.
B
A
100
50
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100
50
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***
**
**
***
***
10b (µM)
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Figure 4. Evaluation of intracellular redox state of a mammalian cell line treated with
10b. Cells (8x105 cells/mL) CHO-K1 expressing the redox biosensor rxYFP were treated
mM),
for 1 h with 10b (1, 5 and 10 µM) or different control compounds such as menadione (1
hydrogen peroxide (H2O2, 1 mM), dithiotreitol (DTT, 1 mM), DMSO 1% v/v and 1
mM
DTT. After the corresponding treatments the samples were analyzed by flow cytometry and
the redox changes quantified only for PI negative cells. The results are presented as
percentage rxYFP reduction relative to DMSO for: A) cells treated for 1 h with different
compounds, and B) followed by a 30 min treatment with 1 mM DTT. Values are expressed
as mean SD (n=3) and the asterisks denote difference (** = p < 0.01 and *** = p < 0.001)
with respect to the DMSO control according to the ONE WAY ANOVA test and the
Dunnets’s multiple comparison post test.
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Taken together, these results led us to conclude that the intracellular oxidation induced by 10b
cannot be ascribed to the inhibition of a specific component of the redox metabolism of the
parasite.
Alternatively, the redox changes triggered by 10b may be consequence of a secondary process
associated to a functional or structural impairment of organelles (i.e. lysosome, endoplasmic
reticulum and/or mitochondria) that host (pro)oxidant molecules [31, 32].
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In this regard, we evaluated the integrity of the mitochondrion and lysosome, both single copy
organelles in trypanosomes, using the organelle-specific fluorophores MitoTracker and
LysoTracker, respectively. Parasites pre-treated with 310 nM 10b for 30 min were further
incubated with the mitochondrial marker for additional 30 min at 37°C and then fixed with
paraformaldehyde. Mitotracker is incorporated to the single parasite´s mitochondrion via
active transport; therefore, a functional or structural impairment of this organelle will result in
a lower MitoTracker signal. The morphology and staining (fluorescence intensity) of the
mitochondrion of parasites exposed to 10b was similar to that of cells treated with DMSO 1%
v/v (Supplementary Figure 1), indicating that the compound does not exert mitochondrial
damage and loss of function.
In trypanosomes, the lysosome is located between the nuclear and mitochondrial (kinetoplast)
DNA. Most parasites incubated at 37°C for 30 min with DMSO 1% v/v and Lysotracker
showed a discrete lysosomal staining (Figure 5A). In contrast, parasites exposed for 30 min to
10b showed a non-discrete staining with the acidotropic probe that included the labeling of
several vesicular compartments (Figure 5B) and a uniform staining of the cytosol (Figure 5C),
which is reminiscent of an early and advanced damage of the lysosome. The quantitative
analysis (Figure 5D) shows that the number of parasites presenting a discrete (single)
lysosomal signal is more than 3-fold lower in the culture treated with 10b (23 ± 4%)
compared to the DMSO-treated control (77 ± 3%). Although our experiment does not allow to
discriminate whether the dispersed LysoTracker staining is due to an enlargement of the
lysosome or to the lost of its integrity, it is important to recall that similar phenotypic changes
were reported for parasites defective in the expression of a cytosolic peroxidase that protects
against lipoperoxidation of the lysosomal membrane [30, 31] or of Rab4, a protein involved in
lysosomal trafficking [35]. In summary, our finding indicates that the rapid and lethal
cytotoxic action of 10b is probably triggered by disruption of lysosomal homeostasis.
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Figure 5. Compound 10b disrupts the lysosomal homeostasis of bloodstream T. brucei. T.
brucei brucei (2.5x106 cells/mL) was treated with 10b (0.31 µM) or DMSO 1% v/v and
LysoTracker Red DND-99 25 µM. After 30 min incubation at 37°C, the cells were fixed with
paraformaldehyde and the slides observed under a fluorescence microscope. Representative
images of parasites treated with A) DMSO and showing discrete (single red dot) lysosome
staining, B) 10b and showing early (multiple red dots) or C) late lysosomal damage (dispersed
signal). D) Percentage of parasites showing discrete lysosomal staining. A total of 90 cells
were analyzed per treatment. Values are expressed as mean ± SD (n=3), with the asterisk
denoting statistical difference with a p < 0.0001 compared to DMSO (one-tailed Student´s t-
test). For each condition are shown bright field and fluorescence (Lysotracker: red color, and
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DAPI staining for nucleus: N and mitochondrial DNA (kinetoplast): K) images from the same
cell.
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In African trypanosomes, the lysosome plays a central role in iron metabolism because in this
organelle the transition metal is freed from host transferrin, stored or further mobilized to the
cytosol [36, 37]. Free iron is able to undergo Fenton reaction with the concomitant generation
of highly oxidizing species that cause irreversible damage in different macromolecules, such
as cell membranes [31]. In order to test whether the morphological alteration of the lysosome
caused by 10b involves the release of iron, the content of intracellular free-iron was analyzed
using the fluorescent probe PhenGreen SK diacetate. This reagent is a phenanthroline
derivative conjugated to FITC (λex 488 nm), whose fluorescence is quenched by iron or
increased if the metal is sequestered by a chelator. All treatments were performed for 1 h at
37°C and the results are shown in Figure 6. First, parasites exposed to 10 µM deferoxamine
(DFO), a chelator of Fe3+ that following endocytic uptake localizes almost exclusively in
lysosomes [38], showed a 32% increase in probe fluorescence compared to the DMSO control
(p < 0.01; Figure 6A). In contrast, cells treated with 10b at 3.1 µM showed a marked
reduction (50%, p < 0.001) of probe fluorescence, which is indicative of an increase in the
pool of free-iron at intracellular level (Figure 6A). Although not statistically significant, co-
incubation with 10 µM DFO reduced by 15% fluorescence quenching triggered by 10b
(Figure 6A). As expected, a 10-fold higher concentration of DFO (100 µM) yielded a higher
unquenching effect of the iron probe in 10b-treated cells (p < 0.01; Figure 6B). Compared to
DFO, bipyridine (BiPy), a membrane-permeant Fe2+-chelator, added at 10 or 100 µM proved
more effective (p < 0.01 or p < 0.001, respectively) in preventing iron probe quenching in
cells exposed to 10b (Figure 6A and 6B). Altogether, these results confirm that 10b promotes
the release of free-iron in the cells and that the parasite´s lysosome is a major source of
intracellular iron.
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Figure 6. Evaluation of free-iron in bloodstream T. brucei treated with 10b. The
infective form of T. b. brucei (2x106 cells/mL) was treated with 3.1 µM 10b,
deferoxamine (DFO) and bipyridine (BiPy) at 10 µM (A) or 100 µM (B). Treatment
with DMSO 1% v/v was included as control. After incubation for 1 h at 37°C the
cells were analyzed by flow cytometry. Values are expressed as mean ± SD (n=3) relative
fluorescence units (urf). The probability indexes for the different treatments are
indicated with asterisks when compared to DMSO control (* p = 0.1, ** = p < 0.01
and *** = p < 0.001) or with hashtags when compared to 10b alone-treated parasites (##
= p < 0.01 and ### = p < 0.001), respectively. The ONE WAY ANOVA test and the
Dunnets’s multiple comparison post test were applied for statistical calculations.
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If the release of lysosomal iron triggered by 10b is responsible for the collapse of the cell
membrane, the sequestration of free-iron should, at least in part, counteract this effect. In
order to confirm this hypothesis, parasites were treated for 2 h at 37°C with 0.31 µM 10b or
different concentrations of the iron chelators BiPy and DFO (Figure 7). As shown in Fig. 7A,
10b added alone increased to 87% the percentage of parasites with membrane damage (PI (+)),
whereas in the presence of BiPy 100 µM the amount of PI(+) cells was significantly lower
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(47%, p < 0.001). Although to a minor extent, also the co-incubation with DFO 100 µM
reduced by 7% the number of PI (+) cells (p < 0.001). As expected, control treatments with
BiPy or DFO (100 µM) alone did not induce membrane permeabilization. As stated above,
BiPy is a lipophilic molecule that rapidly permeates eukaryotic cell membranes through
passive diffusion, whereas DFO is endocyted slowly by the parasite, which can explain why
the last was unable to reduce parasite death caused by 10b under this assay condition. Indeed,
pre-treating cultures with different concentrations of DFO (125-500 µM) 3 h prior to a 3 h-
exposure to 10b, reduced significantly (p < 0.001) and in a dose-response manner the number
of non-viable parasites (40-67% PI(+) cells) compared to cultures treated with 10b alone
(80% PI(+) cells; Figure 7B). Interestingly, under the assay conditions tested here (Figure 6
and 7) and given the preferences (Fe2+ vs. Fe3+) and capacity (bidentate vs. hexadentate, for
BiPy vs. DFO) in iron binding, BiPy appeared to be more effective than DFO in counteracting
the effects triggered by 10b, which suggest that Fe2+ is the major species at intracellular level.
Our findings strongly suggest that cell death triggered by 10b starts with the lost of lysosomal
homeostasis, accompanied by release of iron from this organelle, an increase in the
intracellular oxidative milieu and damage of cell membranes. In this respect, it is important to
note that the lysosome is a highly oxidizing and acidic organelle that host proteolytic activities.
Therefore, the collapse of the lysosome will lead to the release of these toxic components into
the cytosol, which will further exacerbate cellular damage and death.
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Figure 7. Iron chelators protect bloodstream T. brucei against membrane damage by 10b.
T. b. brucei (1x106 cells/mL) was treated with: A) 0.31 µM 10b and 100 µM deferoxamine
(DFO) or bipyridine (BiPy) for 2 h at 37°C, or B) different concentrations of DFO (125-500
µM) for 3 h and then for additional 3 h with 0.31 µM 10b. After the corresponding treatments,
membrane integrity was analyzed using PI staining and flow cytometry. Values are expressed
as mean ± SD (n=3) and the asterisks denote the probability indexes of 10b-treated parasites
vs. DMSO control (***p < 0.001). Hashtags denote the probability indexes for DFO +/- 10b
or BiPy +/- 10b treated parasites vs. 10b alone (### = p < 0.001). The ONE WAY ANOVA
and the Dunnets’s multiple comparison post tests were used for statistical calculations.
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3. Conclusion
Novel distamycin analogues were synthesized in good yields, with several trithiazoles
displaying low to sub-µM and >10-fold cytotoxic selectivity towards the bloodstream form of
African trypanosomes. SAR analysis highlights the relevance of the number of heterocycles
linked by amide bonds and the hydrophobic character of the compound ends, as major
determinants of biological activity. In line with this, investigation of the mechanism of action
for the most active and lipophilic derivative (10b) suggests that the compound kills the
parasite by affecting the integrity of the lysosomal membrane, which is accompanied by
release of labile iron, and likely proteases, that upon redox cycling shift the intracellular redox
milieu to a more oxidative state both cuasing massive membrane damage. Our study provides
the first evidence that the most potent and non-polar trithiazole identified here behaves as a
defensin- or short hydrophobic peptide-like molecule that destabilize membrane structures
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(citocidal), rather than as DNA-intercalating agent (citostatic), as recently reported for a
different series of distamycin analogues [39]. It also highlights the versatility and suitability of
distamycin-like structures to be modified in order to gain selectivity for a particular molecular
target.
4. Experimental
4.1. Chemical methods
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IR spectra were recorded on a Shimadzu FTIR 8101A spectrophotometer. H NMR and C
NMR spectra were recorded on Bruker Avance DPX- 400. Chemical shifts are related to TMS
as an internal standard. High resolution mass spectra (HRMS) were obtained on a micro Q-
TOF (ESI) (Bruker Daltonics).
Melting points were measured using a Fisher-Johns Melting Point Apparatus. Flash column
chromatography was carried out with Silica gel 60 (J.T. Baker, 40 µm average particle
diameter). All reactions and chromatographic separations were monitored by TLC, conducted
on 0.25 mm Silica gel plastic sheets (Macherey/Nagel, Polygram_ SIL G/UV 254). TLC
plates were analyzed under 254 nm UV light, iodine vapor, p-hydroxybenzaldehyde spray or
ninhydrine spray. Yields are reported for chromatographically and spectroscopically (1H and
13C NMR) pure compounds.
All solvents were purified according to literature procedures [40]. All reactions were carried
out in dry, freshly distilled solvents under anhydrous conditions unless otherwise stated.
4.1.1. General procedure for amide bond formation: HBTU (1.2 eq.), DIPEA (2.2 eq.) and 4-
DMAP (0.2 eq.) were added to a stirred solution of the respective amine (1.0 eq.) and acid
(1.0 eq.) under N2 atmosphere in dry CH2Cl2 at 0° C. The resulting mixture was stirred at
room temperature for 24-72 h. Then it was filtered through celite, washed with CHCl3 and
evaporated in vacuo. The crude was redissolved in EtOAc, washed with 5% v/v HCl and then
with a saturated solution of NaHCO3, dried with MgSO4, filtered and evaporated in vacuo.
The crude was purified by flash chromatography using the corresponding eluent to give the
amide.
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4.1.1.1 H3C-Tz-Tz-COOEt (5a, Supp. Fig. S3): Compound 5a was prepared following the
general procedure for amide bond formation from 4e and 4b. Yield 90%. White solid. M.p.
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166-169°C. Rf = 0.33 (EtOAc:hexane, 1:1), H NMR (CDCl3, 400 MHz): δ 1.43 (t, 3H, J=
7.1Hz), 2.75 (s, 3H), 4.44 (q, 2H, J= 7.1Hz), 7.91 (s, 1H), 8.16 (s, 1H), 10,73 (s, 1H). 13C
NMR (100 MHz, CDCl3): δ 14.4, 19.2, 61.5, 122.5, 125.8, 142.2, 147.0, 157.3, 158.6, 161.5,
167.0. HRMS m/z calcd for C11H11N3NaO3S2 ([M+Na]+) 320.0134, found 320.0183. IR film
ν(cm-1) 3109, 2924, 1716, 1666, 1624, 1535, 1234, 1211, 1173, 1096.
4.1.1.2 BocHN-Tz-Tz-COOEt (5b): Compound 5b was prepared following the general
procedure for amide bond formation from 4b and 4d. Yield 70%. White solid. M.p. 249-
250°C. 1H NMR ((CD3)2CO, 400 MHz): δ 1.35 (t, 3H, J = 7.3 Hz), 1.55 (s, 9H), 4.32 (q, 2H,
J = 7.3 Hz), 8.04 (s, 1H), 8.10 (s, 1H), 10.68 (s, 2H). 13C NMR ((CD3)2CO, 100 MHz): δ 14.6,
28.3, 61.4, 83.0, 121.0, 123.4, 143.1, 143.3, 153.8, 158.1, 159.7, 161.3, 161.8.
4.1.1.3 H3C-Tz-Tz-Tz-COOEt (10a, Supp. Fig. S5): Compound 10a was prepared following
the general procedure for amide bond formation from 6a and 4b. Yield 66%. Solid. M.p. 225-
229°C. Rf = 0.34 (EtOAc:hexane, 2:1), 1H NMR (400 MHz, CDCl3) δ 1.44 (t, 3H, J= 7.1Hz),
2.86 (s, 3H), 4.45 (q, 2H, J = 7.1Hz), 7.92 (s, 1H), 8.04 (s, 1H), 8.22 (s, 1H), 10.41 (s, 1H),
10.54 (s. 1H). 13C NMR (100 MHz, CDCl3) δ 14.4, 19.2, 61.4, 121.2, 122.5, 126.2, 142.1,
142.3 146.7, 157.3, 157.4, 158.5, 158.7, 161.4, 167.4. HRMS m/z calcd for C15H13N5NaO4S3
([M+Na]+) 446.0022, found 446.0025. IR film ν(cm-1) 3421, 2928, 1624, 1539, 1230, 1092.
4.1.1.4 BocHN-Tz-Tz-Tz-COOEt (10b, Supp. Fig. S2): Compound 10b was prepared
following the general procedure for amide bond formation from 6b and 4b. Yield 45%. White
solid. 1H NMR (CDCl3, 400 MHz): δ1.42 (t, 3H, J = 7.07 Hz), 1.59 (s, 9H), 4.42 (q, 2H, J =
7.07 Hz), 7.91 (s, 1H), 7.96 (s, 1H), 8.02 (s, 1H), 8.46 (s, 1H), 10.14 (s, 1H), 10.51 (s, 1H).
13C NMR (CDCl3, 100 MHz): δ 14.4, 28.1 , 61.5, 83.7, 120.8, 121.4, 122.6, 141.9, 142.1,
142.2, 157.5 , 158.7 , 159.9 , 161.5.
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4.1.2. General procedure for deprotection of the NH-Boc group: Trifluoro acetic acid (TFA)
was added on an ice bath, to a solution of NH-Boc compound in dry CH2Cl2 under nitrogen
atmosphere. The mixture was stirred at room temperature 24 h. Saturated solution of NaHCO3
was added until pH 8, extracted exhaustively in AcOEt, dried over Na2SO4, filtered and the
solvent was removed under reduced pressure.
4.1.2.1 H2N-Tz-Tz-COOEt (7): Compound 7 was prepared following the general procedure for
NH-Boc deprotection from 5b. Yield 100%. 1H NMR ((CD3)2CO, 400 MHz): δ 1.35 (t, 3H, J
= 7.19 Hz), 4.32 (q, 2H, J = 7.19 Hz), 6.85 (s, 2H), 7.61 (s, 1H), 8.0 (s, 1H), 10.54 (s, 1H).
13C-NMR ((CD3)2CO, 100 MHz): δ 14.6, 61.4, 116.0, 123.3, 143.1, 144.2, 158.1, 159.7,
161.8, 169.7.
4.1.2.2 H2N-Tz-Tz-Tz-COOEt (11, Supp. Fig. S8): Compound 11 was prepared following the
general procedure for NH-Boc deprotection from 11b. White solid. Yield 90 %. M.p. 295 ºC
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decomp. H NMR (DMSO-d6, 400 MHz): δ 1.29 (t, 3H, J = 7.12 Hz), 4.28 (q, 2H, J = 7.12
Hz), 7.69 (s, 1H), 8.11 (s, 1H), 8.36 (s, 1H), 11.58 (s, 1H), 12.51 (s, 1H). 13C NMR (DMSO-
d6, 100 MHZ): δ 14.2, 61.7, 115.5, 121.4, 123.3, 141.2, 142.2, 142.9, 157.7, 158.0, 159.2,
159.5, 161.0, 168.5. HRMS m/z calcd for C14H12N6NaO4S3 ([M+Na]+) 446,9980, found
446.9977. IR KBr ν(cm-1): 3350, 3211, 3118, 1720, 1680, 1369, 1288, 1242, 1159.
4.1.3. General procedure for ethyl ester hydrolysis: An aqueous KOH (10% w/v) solution was
added to an ester solution in THF. The reaction mixture was stirred at room temperature until
reagent disappearance was confirmed by TLC. HCl 1 M was added until pH 4 and the solution
was extracted with EtOAc. The organic layer was dried over MgSO4 and concentrated in
vacuo to afford the acid.
4.1.3.1 H3C-Tz-Tz-COOH (6a, Supp. Fig. S4): Compound 6a was prepared following the
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general procedure for ethyl ester hydrolysis from 5a. Yield 100%. H NMR (DMSO-d6, 400
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MHz): δ 2.77 (s, 3H), 8.09 (s, 1H), 8.59 (s, 1H), 12.44 (s, 1H), 12.95 (s, 1H). 13C-NMR
(DMSO-d6, 100 MHz): δ 19.3, 123.4, 127.6, 142.7, 147.2, 158.1, 159.7, 162.8, 167.2. HRMS
m/z calcd for C9H7N3NaO3S2 ([M+Na]+) 291.9821, found 291.9820. IR film ν(cm-1) 3362,
3159, 3040, 2613, 2459, 1750, 1674, 1518, 1388, 1269, 1240, 1157.
4.1.3.2 BocHN-Tz-Tz-COOH (6c): Compound 6c was prepared following the general
procedure for ethyl ester hydrolysis from 5c. White solid. Yield 100%. 1H NMR (CDCl3, 400
MHz): d 1.50 (s, 9H), 8.03 (s, 1H), 8.26 (s, 1H), 11.84 (s, 1H), 12.21 (bp, 2H). 13C NMR
(CDCl3, 100 MHz): δ 28.7, 83.0, 121.1, 123.4, 142.9, 143.1, 153.4, 158.1, 159.7, 161.1, 162.6,
172.2.
4.1.3.3 AcHN-Tz-Tz-COOH (9, Supp. Fig. S7): Compound 9 was prepared following the
general procedure for ethyl ester hydrolysis from 8. White solid. Yield 100%. M.p. 278 ºC
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decomp. H NMR (DMSO-d6, 400 MHz): δ 1.29 (s, 3H), 7.15 (s, 1H), 7.42 (s, 1H), 11.5 (s,
1H), 11.59 (bp, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 22.4, 120.9, 122.6, 142.1, 142.3,
157.8, 158.1, 159.4; 162.4; 169.2; 170.6. HRMS m/z calcd for C120H8N4NaO4S2 ([M+Na]+)
334.9879, found 334.9844. IR KBr ν(cm-1): 3442, 3180, 3109, 2974, 1669, 1550, 1371,
1298, 1222, 1122.
4.1.3.4 BocHN-Tz-Tz-Tz-COOH (13, Supp. Fig. S10): Compound 13 was prepared following
the general procedure for ethyl ester hydrolysis from 10b. White solid. Yield 100 %. 1H NMR
(DMSO-d6, 400 MHz): δ 1.51 (s, 9Η), 8.04 (s, 1H), 8.25 (s, 1H), 8.37 (s, 1H), 11.83 (s, 1H),
12.19(s, 1H), 12.43 (s, 1H). 13C-NMR (DMSO-d6, 100 MHz): δ 27.9, 79.1, 120.6, 121.3,
122.8,142.2, 142.3, 157.7, 157.9, 159.3, 159.4, 160.4, 162.3, 162.8. HRMS m/z calcd for
C17H16N6NaO6S3 ([M+Na]+) 519.0186, found 519.0179. IR KBr ν(cm-1): 3358, 3190, 3113,
2980, 1720, 1678, 1288, 1269, 1246, 1157.
4.1.3.5 AcHN-Tz-Tz-Tz-COOH (14, Supp. Fig. S11): Compound 14 was prepared following
the general procedure for ethyl ester hydrolysis from 12. White solid. Yield 40 %. M.p. 290 ºC
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decomp. 1H NMR (DMSO-d6, 400 MHz): 2.06 (s, 3H), 7.92 (s, 1H), 8.16 (s, 1H), 8.26 (s, 1H),
12.33 (s, 1H), 12.36 (s, 1H), 12.6 (s, 1H). 13C NMR (DMSO-d6, 100 MHz): δ 22.5, 121.3,
122.8, 123.3, 141.9, 142.3, 158.0, 158.2, 159.4, 159.5, 162.3, 169.3. HRMS m/z calcd for
C14H10N6NaO5S3 ([M+Na]+) 460.9746, found 460.9767. IR KBr ν(cm-1): 3387, 3111, 1672,
1637, 1431m 1369, 1286, 1273, 1201, 1118.
4.1.4. BocHN-Tz-COOEt (4c): Thiazole 4b (0.439 g 2.5 mmol) was dissolved in a mixture of
CH2Cl2:THF (1:1) (10 mL). (Boc)2O (0.577 g, 2.65 mmol) and TEA (0.744 g, 7.36 mmol)
were added. The mixture was refluxed for 72 h and then concentrated under reduced pressure.
The residue was disolved in AcOEt, washed with HCl 5% v/v (3x 15 mL), dried over Na2SO4,
filtered and concentrated under reduced pressure. The residue was purified by silica flash
cromatography AcOEt:EP (3:7). White solid. Yield 74%. 1H NMR ((CD3)2CO, 400 MHz): δ
1.33 (t, 3H, J = 7.2 Hz), 1.54 (s, 9H), 4.30 (q, 2H, J = 7.2 Hz), 7.22 (s, 1H), 10.33 (s, 1H). 13C
NMR ((CD3)2CO, 100 MHz): δ 14.6, 28.2, 61.1, 82.2, 122.4, 143.0, 160.4, 161.8.
4.1.5 AcHN-Tz-Tz-COOEt (8, Supp. Fig. S6): To a solution of 5b (100 mg, 0.33 mmol) in dry
CH2Cl2 (5 mL) under a nitrogen atmosphere, once placed on an ice bath, an excess of pyridine
and acetic anhydre was added. Reaction mixture was stirred for 48 h. Then, H2O (15 mL) was
added and extracted in CH2Cl2 (3 x 15 mL). The organic layer was dried over Na2SO4, filtered
and concentrated under vaccum. The residue was purified by silica flash cromatography
AcOEt:EP (1:1). Pale yellow solid. Yield 90%. 1H NMR (CDCl3, 400 MHz): δ 1.41 (t, 3H, J
= 7.1 Hz), 2.36 (s, 3H), 4.42 (q, 2H, J = 7.1 Hz), 7.89 (s, 1H), 7.95 (s, 1H), 9.72 (s, 1H), 10.51
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(s, 1H). C NMR (CDCl3, 100 MHz): δ 14.4, 23.2, 61.5, 121.1, 122.7, 141.8, 141.9, 157.6,
158.1, 159.1, 161.7, 168.4. HRMS m/z calcd for C12H12N4NaO4S2 ([M+Na]+) 363.0192,
found 320.0190. IR KBr ν(cm-1): 3342, 3261, 3080, 1708, 1662, 1570, 1371, 1281, 1271,
1244, 1099. Mp: 203 ºC
4.1.6 AcHN-Tz-Tz-Tz-COOEt (12, Supp. Fig. S9): Compound 10b (100 mg, 0.18 mmol) was
disolved in dry CH2Cl2 (4 mL) under a nitrogen atmosphere on an ice bath and an excess of
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pyridine and Ac2O was added to the mixture. The reaction is stirrred 48 h and filtered. White
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solid. Yield 40%. M.p. 283 ºC decomp. H NMR (DMSO-d6, 400 MHZ): δ 1.23 (t, 3H, J =
7.2 Hz), 2.13 (s, 3H), 4.22 (q, 2H, J = 7.2 Hz), 8.06 (s, 1H), 8.26 (s, 1H), 8.32 (s, 1H), 12.39 (s,
1H), 12. 47 (bp, 2H). 13C NMR (DMSO-d6, 100 MHz): δ 14.2, 22.6, 61.0, 120.8, 121.5, 123.4,
141.1, 142.0, 142.3, 142.7, 158.1, 158.3, 159.5, 159.6, 161.1, 169.4. HRMS m/z calcd for
C16H14N6NaO5S3 ([M+Na]+) 489.0080, found 489.0071. IR KBr ν(cm-1): 3373, 3309, 3111,
1691, 1523, 1288, 1255, 1192, 825.
4.2. Biology
4.2.1 Materials
Chemical reagents were purchased from Sigma-Aldrich or ROCHE and were of analytical
grade. The media and the consumables for cell cultures were purchased from Invitrogen and
Greiner, respectively.
4.2.2 Viability assays for T. brucei and murine macrophages
The bloodstream form of T. b. brucei strain 427 was grown in HMI-9 medium complemented
with 10% (v/v) Fetal Bovine Serum (FBS; GIBCO ), 10 U/mL penicillin and 10 µg/mL
streptomycin. Cells were incubated aerobically in a humidified incubator containing 5% CO2
at 37 ºC. The cytotoxic effect of the distamycin analogues was evaluated as described by
Maiwald et al. [41]. Briefly, 200 µL of a cell suspension containing 5x105 parasites/mL in
exponential growth phase were seeded per well in a 96-well culture plate and 2 µL each
compound prepared at different concentrations in 100% v/v DMSO were added. For the
screening and EC50 assays the final concentrations of compounds tested were 5 and 30 µM,
and between 0.005 to 150 µM, respectively. Control wells included cells treated with 1% v/v
DMSO or 15 µM nifurtimox. After addition of the test and control compounds, the culture
plates were incubated at 37°C, 5% CO2 for 24 h. Then, 100 µL from each well was transferred
to a 2 mL tube or to a 96 U bottom well plate containing 200 µL of sterile phosphate-buffered
saline (PBS) 1% glucose (m/v) (pH 7.4), 2 µg/mL propidum iodide (PI) and analyzed by flow
cytometry using a CyAnTM ADP (Beckman Coulter) or an AccuriTM C6 (BD) devices with
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laser λex=488 nm and filter λem=613/20 nm for the CyAnTM and λem=540/85 for the AccuriTM
C6. The data were analyzed with the Summit (Dako) software or Accuri C6 software (BD).
The murine macrophages (cell line J774) were cultivated in DMEM medium supplemented
with 10% (v/v) FCS (GIBCO ), 10 U/mL penicillin and 10 µg/mL streptomycin, at 37ºC, 5%
CO2 in a humidified incubator. The EC50 against macrophages was assayed only for
compounds for which the corresponding EC50 towards parasites was previously determined.
Cell viability was determined at 6-point concentrations of compound tested in triplicate, using
the WST-1 reagent and the protocol described by Demoro et al. [42].
For all assays, cell viability was calculated as follows: viability (%) = 100 x (number of cells
for compound Y at concentration X/ number of cells in the DMSO-treated control).
All EC50 values were obtained from dose/response curves fitted to a sigmoidal Boltzmann
equation (errors calculated using errors propagation) or extrapolated from non-linear fitting
plots. The error is expressed as 2.S.D and estimated as 2σ (n-1).
4.2.3 Assays with redox reporter cell lines
Transgenic cell lines of bloodstream T. b. brucei strain 427 and Chinese hamster ovary cells
(CHO-K1) expressing the redox biosensor hGrx-roGFP2 [33] and rxYFP [34], respectively,
were employed to monitor, by a non-invasive manner, intracellular redox changes induced by
tests compounds. The generation and characterization of the cell lines will be described
elsewhere.
The T. b. brucei cell line hGrx-roGFP2 was grown as described above except that phleomycin
(0.2 µg/mL) and hygromycin (5 µg/mL) were added to select for the constitutive expression of
the tetracycline repressor protein and for the hGrx-roGFP2 gene, respectively, whereas the
expression of the last was induced by supplementing the medium with 1 µg/mL oxytetracyclin.
Exponentially growing parasites were resuspended in fresh medium added of oxytetracycline
at a density of 5x105 cells/mL, 200 µL of this cell suspension was seeded per well (96-well
culture microplate) and incubated aerobically with 5% CO2 in a humidified incubator at 37ºC
for 24 h. From this culture plate, a new plate containing 1x106 parasites/200 µL per well in
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culture medium without oxytetracylin was prepared. Next, 2 µL of the different compounds
were added in triplicates at final concentrations of 78, 156 and 310 nM for 10b, 1 mM for
menadione, 15 µM for nifurtimox, 1 mM for DTT or 1% v/v for DMSO. After 1 h incubation
(5% CO2 and 37ºC), 100 µL from each well were transferred to o a 96 U bottom well plate
containing 200 µL of sterile PBS 1% glucose (m/v) while the remaining parasites in the plate
were incubated for additional 30 min in the presence of 1 mM DTT.
Transgenic cells CHO-K1-rxYFP were cultured in DMEM medium supplemented with 10%
(v/v) FCS (GIBCO ), 10 U/mL penicillin and 10 µg/mL streptomycin, at 37ºC, 5% CO2 in a
humidified incubator. Forty eight h prior to the assay, zeocin (Invitrogen) was added at a final
concentration of 400 µg/mL. Cells were trypsinized for 5 min at 37ºC, centrifuged at 800
RPM for 5 min, resuspended in fresh medium and 200 µL/well of the cell suspension (8x105
cells/mL) was seeded on a 96-well plate. Then, 2 µL of the different compounds were added
in triplicates at final concentrations of 10, 5 and 1 µM for 10b, 1 mM for menadione,
hydrogen peroxide and DTT or 1% v/v for DMSO.
After 1 h incubation (5% CO2 and 37ºC), 100 µL from each well were transferred to a 96 U
bottom well plate containing 200 µL of sterile PBS 1% glucose (m/v) while the remaining
cells in the plate were incubated for additional 30 min in the presence of 1 mM DTT.
For both assays, PI was added at a final concentration of 2 µg/mL and then samples were
analyzed with an AccuriTM C6 flow cytometer using the following laser/filter pairs: λex=488
nm/λem=530/33 nm for hGrx-roGFP2 or rxYFP and λex=488 nm/λem=540/85 nm for PI. The
data were processed and analyzed with the Accuri C6 software.
4.2.4 Membrane permeability assay
The bloodstream form of T. b. brucei strain 427 was grown as previously described. Five
hundred thousand parasites/mL were seeded on a 96-well plate and 2 µL of the different
compounds were added in triplicates at final concentrations of 310, 155 and 77.5 nM for 10b,
0.001% v/v for Triton X-100, 15 µM for nifurtimox or 1 % v/v for DMSO. After incubation
for 0.5, 1, 2 and 4 h, 100 µL from each well were transferred to a 96 U bottom well plate
containing 200 µL of sterile PBS 1% glucose (m/v). PI was then added at a final concentration
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of 2 µg/mL and data acquisition was performed with an AccuriTM C6 device as described
above.
4.2.5 Intracellular iron
The bloodstream form of T. b. brucei strain 427 was grown as previously described. Two
million parasites/mL were seeded on a 96-well plate and 2 µL of the different compounds
were added in triplicates at final concentrations of 3.1 µM for 10b, 10 and 100 µM for
bipyridine or deferoxamine mesylate, or 1% v/v for DMSO. After 40 min incubation (5% CO2
and 37ºC), PhenGreen SK diacetate (Invitrogen) was added at a final concentration of 10 µM
and incubated for additional 20 min. Then, 100 µL from each well were transferred to a 96 U
bottom well plate containing 200 µL of sterile PBS 1% glucose (m/v). The samples were
analyzed using a CyAn™ ADP Flow Cytometer (Beckman Coulter, USA) with the following
laser/filter band-pass pair: λex=488 nm/λem= 530-40. Summit 4.3 software was used for data
acquisition and analysis.
4.2.6 Iron-chelators assays
The bloodstream form of T. b. brucei strain 427 was grown as previously described. One
million parasites/mL were seeded on a 96-well plate and 2 µL of the different compounds
were added in triplicates at final concentrations of 310 nM for 10b, 500, 250, 125 or 100 µM
for deferoxamine mesylate, 100 µM for bipyridine or 1% v/v for DMSO and incubated for 2
or 3 h at 37°C. Then, 100 µL from each well were transferred to a tube containing 200 µL of
sterile PBS 1% glucose (m/v) and added of 2 µg/mL PI. Samples were analyzed with a
AccuriTM C6 flow cytometer using the following laser/filter-band pass pair: λex=488
nm/λem=613-30 nm for PI. The data were processed and analyzed with the C6Accuri software.
4.2.7 Fluorescence microscopy assays
For assays involving mitochondrial staining, 1x106 bloodstream T. b. brucei (strain 427)/mL
were grown as previously described in the presence of 0.31 µM 10b for 30 min. Mitotracker®
Red CM-H2XRos (Invitrogen) was then added at a final concentration of 250 nM and
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