In vitro and in vivo activities of 2,3-diarylsubstituted quinoxaline derivatives against Leishmania amazonensis

Article abstract of DOI:10.1128/AAC.02582-15

Leishmaniasis is endemic in 98 countries and territories worldwide. The therapies available for leishmaniasis have serious side effects, thus prompting the search for new therapies. The present study investigated the antileishmanial activities of 2,3-diaryl-substituted quinoxaline derivatives against Leishmania amazonensis. The antiproliferative activities of 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and 2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) against promastigotes and intracellular amastigotes were assessed, and the cytotoxicities of LSPN329 and LSPN331 were determined. Morphological and ultrastructural alterations were examined by electron microscopy, and biochemical alterations, reflected by the mitochondrial membrane potential (ΔΨm), mitochondrial superoxide anion (O₂^.-) concentration, the intracellular ATP concentration, cell volume, the level of phosphatidylserine exposure on the cell membrane, cell membrane integrity, and lipid inclusions, were evaluated. In vivo antileishmanial activity was evaluated in a murine cutaneous leishmaniasis model. Compounds LSPN329 and LSPN331 showed significant selectivity for promastigotes and intracellular amastigotes and low cytotoxicity. In promastigotes, ultrastructural alterations were observed, including an increase in lipid inclusions, concentric membranes, and intense mitochondrial swelling, which were associated with hyperpolarization of ΔΨm, an increase in the O₂^.- concentration, decreased intracellular ATP levels, and a decrease in cell volume. Phosphatidylserine exposure and DNA fragmentation were not observed. The cellular membrane remained intact after treatment. Thus, the multifactorial response that was responsible for the cellular collapse of promastigotes was based on intense mitochondrial alterations. BALB/c mice treated with LSPN329 or LSPN331 showed a significant decrease in lesion thickness in the infected footpad. Therefore, the antileishmanial activity and mitochondrial mechanism of action of LSPN329 and LSPN331 and the decrease in lesion thickness in vivo brought about by LSPN329 and LSPN331 make them potential candidates for new drug development for the treatment of leishmaniasis.

Full text of DOI:10.1128/AAC.02582-15

AAC Accepted Manuscript Posted Online 21 March 2016
Antimicrob. Agents Chemother. doi:10.1128/AAC.02582-15
Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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In vitro and in vivo activity of 2,3-diarylsubstituted quinoxaline
derivatives in Leishmania amazonensis
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Vanessa Kaplum , Juliana Cogo , Diego Pereira Sangi , Tânia Ueda-Nakamura ,
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Arlene Gonçalves Corrêa , Celso Vataru Nakamura
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Programa de Pós Graduação em Ciências Farmacêuticas, Universidade Estadual de
Maringá, Avenida Colombo 5790, 87020-900 Maringá, PR, Brazil.
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Instituto de Ciências Exatas, Universidade Federal Fluminense, 27213-145 Volta
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Redonda, RJ, Brazil.
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Laboratório de Síntese de Produtos Naturais, Departamento de Química, Universidade
Federal de São Carlos, Rodovia Washington Luis Km 235, 13565-905 São Carlos, SP,
Brazil.
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Corresponding author. Phone: +55 44 3011 5012; E-mail: cvnakamura@uem.br
Running title: Dysfunction in Leishmania sp. induced by quinoxalines.
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Keywords: Leishmania amazonensis, quinoxaline derivatives, antileishmanial activity,
mechanism of action, in vivo activity.
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Abstract
Leishmaniasis is endemic in 98 countries and territories worldwide. The
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therapies available for leishmaniasis have serious side effects, thus prompting the search
for new therapies. The present study investigated the antileishmanial activity of 2,3-
diarylsubstituted quinoxaline derivatives against Leishmania amazonensis.
Antiproliferative activity against promastigotes and intracellular amastigotes was
assessed, and the cytotoxicity of 6,7-dichloro-2,3-diphenylquinoxaline (LSPN329) and
2,3-di-(4-methoxyphenyl)-quinoxaline (LSPN331) was determined. Morphological and
ultrastructural alterations were examined by electron microscopy, and biochemical
alterations were evaluated, reflected by mitochondrial membrane potential (ΔΨm),
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mitochondrial superoxide anion (O •−), intracellular ATP, cell volume,
phosphatidylserine exposure on the cell membrane, cell membrane integrity, and lipid
inclusions. In vivo antileishmanial activity was evaluated in a murine cutaneous
leishmaniasis model. Compounds LSPN329 and LSPN331 showed significant
selectivity for promastigotes and intracellular amastigotes and low cytotoxicity. In
promastigotes, ultrastructural alterations were observed, including an increase in lipid
inclusions, concentric membranes and intense mitochondrial swelling, which were
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associated with hyperpolarization of ΔΨm, increase in O •−, decreased intracellular
ATP levels, and a decrease in cell volume. Phosphatidylserine exposure and DNA
fragmentation were not observed. The cellular membrane remained intact after
treatment. Thus, the multifactorial response that was responsible for the cellular
collapse of promastigotes was based on intense mitochondrial alterations. BALB/c
treated with LSPN329 or LSPN331 showed a significant decrease of lesion thickness in
the infected footpad. Therefore, the antileishmanial activity, mitochondrial mechanism
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of action and decrease of lesion thickness in vivo of LSPN329 and LSPN331 make
them potential candidates for new drug development for the treatment of leishmaniasis.
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1. Introduction
Neglected tropical diseases largely affect economically and socially
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marginalized populations in developing countries (1). Leishmaniasis is endemic in 98
countries and territories worldwide, with approximately 350 million people at risk of
infection and 12 million people currently infected (2).
Leishmaniasis is caused by over 20 species of the protozoa Leishmania sp,
which are divided into two developmental forms: promastigotes and amastigotes (3).
Promastigotes are characterized by elongation and a free flagellum. It is transmitted
during blood feeding by the sandflies Lutzomyia sp in the New World and Phlebotomus
sp in the Old World (4). Amastigotes are characterized by a rounded body with a short
flagellum, located inside parasitophorous vacuoles of macrophages (5, 6).
The clinical manifestations of leishmaniasis can be classified primarily as
cutaneous, mucocutaneous, and visceral (7). Cutaneous leishmaniasis predominates,
with 1.5 million new cases per year (2). Lesions appear as papules that may progress to
nodules and even severe forms characterized by ulcerated lesions (8, 9). In Brazil, the
most relevant species are L. amazonensis, L. guyanensis, L. braziliensis, and L. chagasi,
there was approximately 30,000 cases reported only in 2010 (2, 10).
The first-line treatment of cutaneous leishmaniasis is pentavalent antimonials,
represented by meglumine antimoniate (Glucantime) and sodium stibogluconate
(Pentostam; (11)). Among the second-line drugs are miltefosine (the first drug that can
be orally administered), pentamidine, and amphotericin B (liposomal or free), despite its
high toxicity (12).
Quinoxaline derivatives have promising biological properties because of their
privileged scaffold (13–15). Several biological effects have been reported, including
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anticancer (14, 16, 17), antimicrobial (18–22), antiviral (23), antiinflammatory, and
antioxidant (24, 25).
The antiprotozoal activity of quinoxalines has been reported, especially their
antitrypanosomatid activity, such as for 1,4-di-N-oxide quinoxaline (26), 3-
trifluoromethylquinoxaline N,N’-dioxides (27), and 3-aminoquinoxaline-2-carbonitrile
1,4-dioxides to antimony (III) (28). The antileishmanial activity of quinoxaline
derivatives has been reported for 4-alkapolynylpyrrolo[1,2-α]quinoxalines (29, 30) and
1,4-di-N-oxide quinoxaline (13, 26, 31, 32). We recently reported the activity of 2,3-
disubstituted quinoxaline derivatives against Trypanosoma cruzi and L. amazonensis
(33, 34).
The currently available therapeutic options for the treatment of cutaneous
leishmaniasis have serious side effects, a long treatment duration, and variability in
efficacy (35). The search for new therapeutic options is important, and quinoxaline
derivatives are among the promising compounds. The aim of the present study was to
investigate the antileishmanial activity of 2,3-diarylsubstituted quinoxaline derivatives,
characterize the biochemical changes induced by these compounds in promastigotes,
and to evaluate the in vivo antileishmanial activity in a murine cutaneous leishmaniasis
model.
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2. Materials and Methods
2.1. Chemicals
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The following chemicals were used: actinomycin D, amphotericin B, antimycin
A (AA), fetal bovine serum (FBS), carbonylcyanide m-chlorophenylhydrazone (CCCP),
Cell Titer-Glo® Luminescent Cell Viability Assay, dimethylsulfoxide (DMSO), 4-
amino-5-methylamino-2’,7’-difluoroflurescein
rhodamine 123 (Rh 123), Roswell Park Memorial Institute Medium (RPMI 1640; Gibco
Invitrogen, Grand Island, NY, USA), 3-[4,5-dimethylthiazol-2-yl]-2,5-
diacetate
(DAF-FM
diacetate),
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diphenyltetrazolium bromide (MTT), 3,8-phenanthridinediamine-5-(6-triphenyl-
phosphoniumhexyl)-5,6-dihydro-6-phenyl (MitoSOX Red kit), propidium iodide (PI),
and the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) kit.
All of the other reagents were of analytical grade. All synthetic compounds are known
and were identified by the comparison of their spectroscopic data with the reported in
literature (34, 36–39) and showed purity rates above to 99% by gas chromatography.
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2.2. Synthesis and preparation of quinoxaline LSPN329 and LSPN331
Compound LSPN329 and LSPN331 were synthesized as previously described
by Cogo et al. (2015).Stock solutions of LSPN329 and LSPN331 were aseptically
prepared in DMSO (the concentration did not exceed 1% in the experiments) and
diluted in culture medium. The concentrations of LSPN329 and LSPN331 that were
used in the assays were based on the IC50 and IC90 values.
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2.3. Animals
Male BALB/c mice (20 – 25 g) between 4 and 6 weeks of age were obtained
from Universidade Estadual de Maringá (UEM,PR, Brazil). Animals were maintained at
room temperature (22 ± 2°C), and constant 12 h day/night cycle. Food and water were
given ad libitum. The Institutional Ethics Committee of Universidade Estadual de
Maringá approved all procedures adopted in this study (Protocol 029/2014).
2.4. Parasites and cell culture
Leishmania amazonensis promastigotes (strain WHOM/BR/75/JOSEFA) were
maintained at 25°C in Warren’s medium (brain-heart infusion plus hemin and folic acid;
pH 7.2) supplemented with 10% heat-inactivated FBS. J774-A1 macrophages were
maintained at 37°C under a 5% CO
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atmosphere in RPMI medium (pH 7.2)
supplemented with 10% heat-inactivated FBS.
2.5. Activity against promastigotes of L. amazonensis
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Promastigotes of L. amazonensis (1  10 cells/ml) were treated with LSPN329
and LSPN331 (1, 10, 50, and 100 µM) for 72 h at 25°C. The positive controls were
amphotericin B and miltefosine. After incubation, promastigotes were counted in a
Neubauer hemocytometer. The concentration that inhibited cell growth by 50% (IC50
was determined by regression analysis of the data.
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2.6. Activity against intracellular amastigotes of L. amazonensis
Peritoneal macrophages from BALB/c mice were harvested by washing with
cold phosphate-buffered saline (PBS) supplemented with 3% FBS, centrifuged, and
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resuspended in RPMI medium supplemented with 10% FBS (5  10 cells/ml). The
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macrophages were then adhered to coverslips placed in 24-well tissue culture plates for
2 h at 37°C under a 5% CO
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atmosphere. Adherent macrophages were infected with
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metacyclic promastigotes (3.5  10 cells/ml) for 4 h at 34°C. Non-interiorized parasites
were then removed by washing, and the infected culture was incubated with different
concentrations of LSPN329 and LSPN331 (5, 10, 20, and 25 µM) for 48 h at 34ºC.
After incubation, the coverslips were washed, fixed with methanol, and stained with
Giemsa. The percentage of infected macrophages was determined by counting by light
microscopy at least 200 cells in duplicate. Parasite load was assessed by the association
index (the percentage of infected cells multiplied by the number of intracellular
amastigotes per macrophage, divided by the total number of macrophages), which was
used to calculate the IC50, determined by regression analysis of the data.
2.7. Cytotoxicity assay in macrophage cells
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J774-A1 macrophages (5  10 cells/ml) were plated with RPMI-1640 medium
supplemented with 10% FBS and incubated for 24 h at 37°C under a 5% CO
atmosphere. The macrophages were then treated with LSPN329 and LSPN331 (10,
100, 500, and 1000 µM) and incubated for 48 h at 37°C under a 5% CO atmosphere.
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The macrophages were then washed in PBS. MTT (50 µl; 2 mg/ml) was added,
followed by incubation for 4 h at 37°C. Formazan crystals were solubilized in DMSO,
and absorbance was read at 492 nm in a microplate reader (BioTek Power Wave XS
spectrophotometer). The concentration that inhibited absorbance in 50% compared with
the negative control (CC50) was determined by regression analysis of the data.
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2.8. Hemolytic assay
Human blood (type A+) was collected, defibrinated, and washed with a saline
solution of glucose. Red blood cells were inoculated in 96-well plates at 3% in
glycosylated saline with different concentrations of LSPN329 and LSPN331 (25-200
µM). The plates were incubated for 2 h at 37°C, and the supernatant was read at 540
nm. To calculate the percentage of hemolysis, 1% Triton X-100 was used as a positive
control, and 1% DMSO was used as a negative control.
2.9.
Scanning electron microscopy of promastigotes of L. amazonensis
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Promastigotes of L. amazonensis (10 cells/ml) were treated with the IC50 of
LSPN329 (5.3 µM) or LSPN331 (30.0 µM). After 48 h incubation, the cells were fixed
with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 25°C for 2 h.
The promastigotes were then placed on glass coverslips that contained poly-L-lysine and
incubated for 1 h. After incubation, the coverslips were dehydrated in an ethanol
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gradient, critical-point-dried in CO , coated with gold, and observed in a Shimadzu SS-
550 scanning electron microscope.
2.10. Transmission electron microscopy of promastigotes of L. amazonensis
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Promastigotes of L. amazonensis (10 cells/ml) were treated with the IC50 of
LSPN329 (5.3 µM) or LSPN331 (30.0 µM). After 48 h incubation, the cells were fixed
with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 25°C for 2 h.
The promastigotes were then post-fixed with 1% osmium tetroxide and 0.8% potassium
ferricyanide for 60 min at room temperature and protected from light. The cells were
washed with 0.1 M sodium cacodylate buffer and dehydrated in increasing
concentrations of acetone. The material was embedded in increasing concentrations of
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Epon resin and polymerized at 60°C for 72 h. Thin sections were contrasted with uranyl
acetate and lead citrate and observed in a JEM 1400 JEOL transmission electron
microscope.
2.11. Mitochondrial membrane potential (ΔΨm) assay of promastigotes of L.
amazonensis
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Promastigotes of L. amazonensis (5  10 cells/ml) were treated with LSPN329
(1, 5, 10, and 20 µM) or LSPN331 (20, 40, 75, and 100 µM). The positive control was
CCCP (100 µM). After 24 h incubation, the promastigotes were centrifuged, washed
and resuspended in 0.9% saline solution, and then stained with marker Rh 123 (13.1
µM) for 15 min. After resuspension, 10,000 events were acquired using a FACSCalibur
flow cytometer (Becton-Dickinson, Rutherford, NJ, USA) and analyzed using
CellQuest software (Joseph Trotter, The Scripps Research Institute, La Jolla, CA,
USA). Alterations in Rh 123 fluorescence were quantified using an index of variation
(IV) obtained from the equation (M
T
- M
C C T
) / M , in which M is the median
fluorescence for the treated parasites, and M
C
is the median fluorescence for the control
parasites. Negative IV values correspond to depolarization, and positive IV values
correspond to hyperpolarization of the mitochondrial membrane.
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2.12. Detection of mitochondrial-derived superoxide anion (O ) in promastigotes of
L. amazonensis
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Promastigotes of L. amazonensis (2  10 cells/ml) were washed with Krebs-
Henseleit (KH) solution buffer (pH 7.3; 15 mM NaHCO
0.7 mM Na HPO , and 1.5 mM NaH PO ) and then marked with 5 µM MITOSox Red
for 10 min at 25°C. Marked promastigotes were treated with LSPN329 (1, 5, 10, and 20
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µM) or LSPN331 (20, 40, 75, and 100 µM). The positive control was antimycin A (10
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µM). Mitochondrial-derived superoxide anion (O ) was measured at 0, 1, 2, 3, and 4 h.
The fluorescence intensity was quantified using a fluorescence microplate reader
(VICTOR X3, PerkinElmer) at excitation and emission wavelengths of 510 and 580 nm,
respectively.
2.13. Intracellular ATP determination in promastigotes of L. amazonensis
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Promastigotes of L. amazonensis (1  10 cells/ml) were treated with LSPN329
(1, 5, 10, and 20 µM) or LSPN331 (20, 40, 75, and 100 µM). The positive control was
CCCP (100 µM). After 24 h incubation at 25°C, the promastigotes were centrifuged,
washed, resuspended in PBS. In a white 96-well plate was added an equal volume of
CellTiter-Glo reagent (50 µl) and an aliquot of each sample (50 µl), mixed and
incubated for 10 min. The luminescence intensity was quantified using a luminescence
microplate reader (VICTOR X3, PerkinElmer).
2.14. Cell volume determination in promastigotes of L. amazonensis
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Promastigotes of L. amazonensis (5  10 cells/ml) were treated with LSPN329
(1, 5, 10, and 20 µM) or LSPN331 (20, 40, 75, and 100 µM). The positive control was
actinomycin D (50 µM). After 24 h incubation at 25°C, the promastigotes were
centrifuged and washed and resuspended in PBS. After resuspension, 10,000 events
were acquired using a FACSCalibur flow cytometer (Becton-Dickinson, Rutherford,
NJ, USA) and analyzed by CellQuest software (Joseph Trotter, The Scripps Research
Institute, La Jolla, CA, USA).
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2.15. Phosphatidylserine externalization and cellular membrane integrity assay of
promastigotes of L. amazonensis
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Promastigotes of L. amazonensis (5  10 cells/ml) were treated with LSPN329
(1, 5, 10, and 20 µM) or LSPN331 (20, 40, 75, and 100 µM). The positive control was
CCCP (100 µM). After 24 h incubation at 25°C, the promastigotes were centrifuged,
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washed, resuspended with binding buffer (140 mM NaCl, 5 mM CaCl , and 10 mM
HEPES-Na, pH 7.4), and then stained with 5 µl of marker FITC-conjugated annexin-V
for 15 min. Binding buffer and PI (3 µM) were then added. Afterward, 10,000 events
were acquired using a FACSCalibur flow cytometer (Becton-Dickinson, Rutherford,
NJ, USA), and the data were analyzed using CellQuest software (Joseph Trotter, The
Scripps Research Institute, La Jolla, CA, USA).
2.16. Nile red accumulation in promastigotes of L. amazonensis
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Promastigotes of L. amazonensis (5  10 cells/ml) were treated with LSPN329
(1, 5, 10, and 20 µM) or LSPN331 (20, 40, 75, and 100 µM). After 24 h incubation at
25°C, the promastigotes were centrifuged, washed, resuspended in PBS, and labeled
with 10 µl of Nile Red solution (1000 µg/ml) for 30 min at 25°C. The fluorescence
intensity was quantified using a fluorescence microplate reader (VICTOR X3,
PerkinElmer) at excitation and emission wavelengths of 485 and 535 nm, respectively.
2.17. Scanning electron microscopy of intracellular amastigotes of L. amazonensis
Peritoneal macrophages that adhered to the surface of small glass coverslips
were incubated with L. amazonensis promastigotes for 4 h at 34°C. For the first
scanning electron microscopy (SEM) evaluation, the samples were immediately treated
with the IC50 of LSPN329 (16.3 µM) or LSPN331 (19.6 µM). After 24 and 48 h
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treatment, infected macrophages with intracellular amastigotes were fixed with 2.5 %
glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C for 12 h. The
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coverslips were then dehydrated in an ethanol gradient and critical-point-dried in CO .
The slides were placed in an appropriate medium, and infected macrophages were
fractured with adhesive tape, coated with gold, and observed in a Shimadzu SS-550
scanning electron microscope.
2.18. In vivo antileishmanial assay
Male BALB/c mice were subcutaneously inoculated in the left hind-footpad with
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1  10 infective promastigotes of L. amazonensis (late-log phase of growth). After 8
weeks, the animals were randomly separated in 5 groups of 5 animals each, as follows:
infected control, 5 mg/Kg/day of LSPN329 administered intralesionally, 5 mg/Kg/day
of LSPN331 administered intralesionally, 20 mg/Kg/day miltefosine administered
orally. Treatment was administered daily for 3 weeks. Stock solutions of LSPN329 and
LSPN331 were prepared daily in oil after previous solubilization in DMSO (final
concentration of 0.1%). Volume injected in the left hind-footpad was 20 µl, daily.
Infected control was administrated with the same number of vehicle injections (oil and
DMSO). Lesion development was monitored weekly by measuring the thickness of the
infected footpad with a dial caliper (DIGIMESS® 150 mm), subtracting the
contralateral uninfected footpad.
2.19. Statistical analysis
The data shown in the tables and graphs are expressed as the mean ± standard
deviation of at least three independent experiments. The data were analyzed using one-
or two-way analysis of variance (ANOVA) followed by Dunnett’s or Bonferroni’s post
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hoc test. Values of p ≤ 0.05 were considered statistically significant. The statistical
analysis was performed using Prism 5 software (GraphPad, San Diego, CA, USA). The
selectivity index (SI) was calculated as SI = CC50 / IC50, where CC50 is the
concentration is able to reduce macrophage viability by 50%, and IC50 is the
concentration inhibits cell growth in 50% of the promastigotes or intracellular
amastigotes.
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3.
Results
3.1. Synthesis of quinoxalines LSPN329 and LSPN331
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LSPN329 (6,7-dichloro-2,3-diphenylquinoxaline) and LSPN331 (2,3-di-[4-
methoxyphenyl] quinoxaline) were synthesized as described in Figure 1 (34) through
the condensation of 1,2-diarylethanediones with o-phenylenediamine by employing
ultrasound irradiation as the energy source (37). The diarylethanediones were prepared
by Friedel-Crafts acylation followed by SeO
(36).
2
oxidation under microwave irradiation
3.2. Antileishmanial activity and cytotoxicity
The 2,3-diarylsubstituted quinoxalines LSPN329 and LSPN331 showed
antileishmanial activity against the promastigote and amastigote forms of the parasite
(Fig. 2). The activity against promastigotes was dose-dependent for both compounds,
especially the activity of LSPN329, with an IC50 of 5.3 µM. Likewise, the intracellular
activity against amastigotes was dose-dependent, with an IC50 of 16.3 and 19.3 µM for
LSPN329 and LSPN331, respectively (Table 1).
The preliminary safety assessment of LSPN329 and LSPN331 was performed
using the cytotoxicity assay in J774-A1 macrophages, which showed a CC50 of 203.0
and 589.6 µM, respectively. Thus, the relationship between cytotoxicity and
antileishmanial activity resulted in the SI. In promastigote forms, LSPN329 was 38.5-
times more selective for the parasite than for macrophages. For intracellular
amastigotes, LSPN331 had a better SI (30.6; Table 1). Another toxicity test was
conducted to determine hemolytic activity, which was < 0.5% for both compounds at
the highest concentration tested (200 µM; data not shown).
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3.3. Scanning and transmission electron microscopy of promastigotes of L.
amazonensis
Morphological changes were evaluated by SEM. The control group exhibited
typical features of the parasite, with an elongated cell body and free flagellum (Fig. 2A).
In contrast, parasites that were treated with the IC50 of LSPN329 (5.3 µM) and
LSPN331 (30.0 µM) exhibited an altered shape, size reduction, and a rounding cell
body. Promastigotes that were treated with LSPN331 exhibited a wrinkled cell body
(Fig. 2B, C).
Ultrastructural alterations in promastigotes treated with the IC50 of LSPN329
(5.3 µM) included the presence of concentric membranes inside mitochondria, vesicles
within the flagellar pocket, and lipid inclusions (Fig. 3B, C). Treatment with the IC50
(30.0 µM) of LSPN331 revealed alterations in mitochondria, such as intense
mitochondrial swelling, concentric membrane structures inside, vesicles within the
flagellar pocket, and lipid inclusions (Fig. 3D, F). The control group exhibited no
ultrastructural alterations.
3.4. Mitochondrial membrane potential (ΔΨm) assay of promastigotes of L.
amazonensis
Based on mitochondrial ultrastructural alterations in promastigotes treated with
both compounds, we proceeded with the evaluation of the mitochondrial membrane
potential (ΔΨm) by flow cytometry using the cationic marker Rh 123. Promastigotes
that were treated with LSPN329 showed a dose-dependent increase in ΔΨm compared
with the control, with the greatest significance for the highest concentration tested (20
µM; Fig. 4A-D, Table 2). Compound LSPN331 caused a significant increase in ΔΨm at
concentrations of 75 and 100 µM (Fig. 4F-I, Table 2). Thus, the treatment of
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promastigotes with both compounds induced hyperpolarization of the mitochondrial
membrane. In contrast, the positive control, CCCP, induced depolarization of the
mitochondrial membrane (Fig. 4E and 4J, Table 2).
•−
3.5. Detection of mitochondrial-derived O
2
production in promastigotes of L.
amazonensis
Considering the ΔΨm results, the production of mitochondrial superoxide anion
•−
(O
2
) was evaluated in promastigotes treated with LSPN329 and LSPN331 using
MitoSOX Red reagent, which emits fluorescence when oxidized (oxMitoSOX).
Compound LSPN329 induced an increase in O •− at the higher concentrations (Fig.
5A). In promastigotes treated with different concentrations of LSPN331, O •− increased
2
2
for the two highest concentrations during the 4 h period of evaluation (Fig. 5B). The
positive control, AA, also induced an increase in fluorescence.
3.6. Intracellular ATP determination in promastigotes of L. amazonensis
2
Given the effect on ΔΨm and increase in O •−, intracellular ATP was evaluated
in promastigotes treated with LSPN329 and LSPN331 using CellTiter-Glo reagent.
Promastigotes treated with LSPN329 showed no changes in intracellular ATP levels
(Fig. 6A). On the other hand, promastigotes treated with different concentrations of
LSPN331 showed significant decrease in intracellular ATP levels in a dose-dependent
manner (Fig. 6B). The positive control, CCCP, also induced a decrease in luminescence.
3.7. Cell volume determination in promastigotes of L. amazonensis
Mitochondrial alterations are suggestive of cell death by apoptosis and may
cause changes in cell volume (40). Promastigotes treated with LSPN329 did not show a
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significant reduction of cell volume (Fig. 7A). Promastigotes treated with different
concentrations of LSPN331 showed a significant reduction of cell volume for the two
highest doses (Fig. 7B). The positive control, actinomycin D, reduced cell volume.
3.8. Phosphatidylserine externalization and cell membrane integrity in promastigotes
of L. amazonensis
•−
The association between the increase in ΔΨm and O
2
can induce cell death by
either apoptosis or necrosis (41). To identify such changes, promastigotes were treated
with different concentrations of LSPN329 and LSPN331 for 24 h, double-stained with
annexin-V and PI, and compared with the negative control. Flow cytometry revealed no
significant increase in promastigotes labeled with annexin-V in the upper and lower
right quadrants after treatment with LSPN329 or LSPN331 (Fig. 8). Similarly,
treatment with both compounds did not increase the number of promastigotes marked
by PI in the right and upper left quadrants (Fig. 8). The positive control, CCCP, showed
an increase in the number of labeled cells in the right and left upper quadrants (Fig. 8F).
3.9. Nile red accumulation in promastigotes of L. amazonensis
Mitochondrial dysfunction can lead to a build up of lipid inclusions in the
cytoplasm, a characteristic find in cells in apoptosis (42). Transmission electron
microscopy showed that promastigotes that were treated with 2,3-diarylsubstituted
quinoxalines exhibited an increase in the amount of lipid inclusions (Fig. 3C, F).
Another way to determine the presence of lipid inclusions in promastigotes was by its
labeling with Nile red. Promastigotes that were treated with LSPN329 showed a dose-
dependent increase in Nile red accumulation compared with the control, with a
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significant increase for the highest concentrations tested (Fig. 9A). LSPN331 caused a
significant increase in lipid inclusions at concentrations of 75 and 100 µM (Fig. 9B).
3.10. Scanning electron microscopy of intracellular amastigotes of L. amazonensis
After the fracture of peritoneal macrophages, intracellular amastigotes in the
control group showed typical features, such as a rounded cell body (Fig. 10A, D). In
contrast, parasites that were treated with the IC50 of LSPN329 (16.3 µM) and LSPN331
(19.3 µM) for 24 h exhibited alterations in size and shape, wrinkling, and extravasation
of cytoplasmic material (Fig. 10B, C). With 48 h treatment, alterations in the shape of
the cell body were observed, with the presence of residues within the parasitophorous
vacuole (Fig. 10E, F).
3.11. In vivo antileishmanial assay
The in vivo antileishmanial activity of LSPN329 and LSPN331 was evaluated in
a murine cutaneous leishmaniasis model. BALB/c mice were infected in the footpad
with L. amazonensis. Animals were treated intralesionally with 5 mg/Kg/day of
LSPN329 or LSPN331 every day for 3 weeks. Lesion development was monitored
weekly by measuring the footpad thickness. Treatment with LSPN329 and LSPN331
showed a significant decrease of lesion thickness compared to control (Fig. 11).
Miltefosine administered orally (20 mg/Kg/day) also decreased the lesion thickness
compared to control (Fig. 11).
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4.
Discussion
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The 2,3-diarylsubstituted quinoxalines showed significant antiproliferative
activity against promastigote and intracellular amastigote forms of L. amazonensis. In
promastigotes, the ultrastructural changes and biochemical results suggest the
occurrence of mitochondrial alterations associated with increases in lipid inclusions.
Furthermore, evaluation of the in vivo antileishmanial activity resulted in a significant
decrease of lesion thickness in a murine cutaneous leishmaniasis model.
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40
41
In the present study, the activity of LSPN329 and LSPN331 against L.
amazonensis promastigotes resulted in IC50 of 5.3 and 30.0 µM, respectively. For
intracellular amastigotes, the SIs were 12.5 for LSPN329 and 30.6 for LSPN331. These
are promising results, given that both compounds proved to be more selective to
intracellular amastigotes compared with amphotericin B, one of the drugs of choice for
leishmaniasis treatment (35). Previous studies reported the activity of quinoxaline
derivatives against evolutionary forms of Leishmania sp., further attesting to the
potential of these compounds (13, 30–32). On the other hand, the in vitro combination
effects of LSPN329 and LSPN331 with miltefosine indicates indifferent interaction in
promastigotes and intracellular amastigotes of L. amazonensis (data not shown). The
low cytotoxicity of quinoxaline derivatives have been reported previously by our group
on LLCMK
(34). The cytotoxicity data obtained for both compounds on J774-A1 macrophages and
LLCMK cells were lower than the amphotericin B and miltefosine.
2
cells, with CC50 of 471.8 µM for LSPN329 and 330.6 µM for LSPN331
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42
43
44
In addition to the in vitro cytotoxicity, hemolytic activity is also important to
investigate the safety of specific compounds (33), which was < 0.5% for LSPN329 and
LSPN331 at the highest concentration tested in our experiments. Treatment with
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amphotericin B at concentrations that were approximately four-times lower (50.8 µM)
caused 50% hemolysis of erythrocytes (43).
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53
The ultrastructural alterations observed in promastigotes treated with LSPN329
and LSPN331 were characterized by intense mitochondrial changes, mainly the
presence of concentric membranes inside the organelle, loss of the mitochondrial
matrix, and mitochondrial swelling. Considering that trypanosomatids have only one
mitochondrion, such an organelle may be considered a potential therapeutic target (44).
Several studies have reported mitochondrial dysfunction in trypanosomatids caused by
different classes of compounds (40, 45–47), including quinoxaline derivatives (27).
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In addition to ultrastructural changes, biochemical dysfunction was observed,
reflected by ΔΨm, which is an indicator of cellular damage (48). We also observed an
increase in marking by Rh 123 in promastigotes treated with LSPN329 and LSPN331,
indicating mitochondrial membrane hyperpolarization. Hyperpolarization is considered
to be less common than mitochondrial depolarization and is best described for
mammalian cells, arising mainly from dysfunctions in F0/F1 ATPase (48). A few
studies have reported mitochondrial hyperpolarization in promastigote forms, such as
with camptothecin (49), rotenone (50), conditional heat stress (19), and bis-pyridinium
derivatives (51).
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Reactive oxygen species are formed naturally in mitochondria, but
•−
mitochondrial hyperpolarization causes an increase in O
2
production in mammalian
cells (52). However, in promastigotes of Leishmania sp., the occurrence of
•−
hyperpolarization and increase in O
2
production may not be directly related (19, 50).
Treatment with bis-pyridinium derivatives resulted in concomitant hyperpolarization
•−
and an increase in O
2
(51). Likewise, mitochondrial membrane hyperpolarization and
2
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•
−
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a significant increase in O
2
concentration was found in promastigotes treated with 2,3-
diarylsubstituted quinoxalines. Other works have also presented mitochondrial damage
•−
associated with the increase of O
2
only in the highest concentrations tested and
concluded that mitochondrial dysfunction was the main cause of cell death (47, 53).
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Intracellular ATP levels are susceptible to alterations when there is
mitochondrial damage, such as, mitochondrial membrane potential changes and increase
in reactive oxygen species (51, 54). Promastigotes treated with LSPN331 showed
significant decrease in intracellular ATP levels in a dose-dependent manner. However,
promastigotes treated with LSPN329 showed no changes in the intracellular ATP
levels.
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Ultrastructural changes in the mitochondria of the parasite, together with
changes in ΔΨm (both depolarization and hyperpolarization), decrease in intracellular
ATP levels, and the accumulation of reactive oxygen species are considered signs of
apoptosis (55).
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Thus, the signs of apoptotic cell death that were evaluated included cell volume,
DNA fragmentation, and phosphatidylserine exposure on the cell membrane (56). The
cell volume of promastigotes treated with LSPN329 reduced slightly. However,
promastigotes treated with LSPN331 showed a significant reduction of cell volume.
Alteration in cell volume was also revealed by SEM. Just as with 2,3-diarylsubstituted
quinoxalines, the 3-chloro-7-methoxy-2-(methylsulfonyl)quinoxaline (LSPN337)
caused changes in cell volume in epimastigotes of T. cruzi only in higher concentrations
(33).
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In the DNA fragmentation assay using TUNEL staining, no DNA fragmentation
was found in promastigotes that were treated with LSPN329 or LSPN331 for 72 h (data
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not shown). To confirm the absence of type I programmed cell death or apoptosis, we
evaluated the exposure of phosphatidylserine at the cell membrane using annexin-V,
and however, the promastigotes were not marked. Promastigotes that were treated with
the 2,3-diarylsubstituted quinoxalines showed no characteristic marking of apoptosis-
like death.
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The association between hyperpolarization and increased reactive oxygen
species generation may also result in the initiation and occurrence of necrotic cell death
(41). The lack of integrity of the cell membrane is one of the signs of cell death by
necrosis (57).
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Scanning electron microscopy of promastigotes treated with LSPN329 and
LSPN331 showed alterations in shape, such as size reduction and cell body rounding.
However, no cell membrane alterations were observed. Thus, absence of PI labeling in
treated cells indicating that the cell membrane was intact after treatment. This was also
seen with T. cruzi epimastigotes treated with LSPN337 (33).
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Transmission electron microscopy showed that promastigotes treated with
LSPN329 and LSPN331 exhibited an increase in the amount of lipid inclusions. This
was confirmed by Nile red staining, in which a dose-dependent increase in the amount
of lipid inclusions was observed, in addition to the random distribution of droplets
throughout the cytoplasm of the cells. The increase in lipid inclusions may be related to
changes in phospholipids and sterols in promastigotes of L. amazonensis (58). Lipid
inclusions in the cytoplasm can be caused by mitochondrial dysfunction (42).
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Despite mitochondrial damage and alterations, neither DNA fragmentation nor
phosphatidylserine on the cell membrane was observed, thus ruling out cell death by
apoptosis. Given the cell membrane integrity, cell death was also not caused by
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necrosis. Thus, the treatment of L. amazonensis promastigotes with LSPN329 and
LSPN331 caused a multifactorial response that culminated in cellular collapse,
characterized by intense mitochondrial changes. Similar results were observed with the
treatment of promastigotes with the iron chelator 2,2-dipyridyl and 4-amino bis-
pyridinium derivatives (51, 59).
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Scanning electron microscopy studies of amastigotes of Leishmania sp.
generally have evaluated parasites that are external to the host cell (60, 61). In the
present study, morphological changes were evaluated in intracellular amastigotes that
were present in parasitophorous vacuoles of peritoneal macrophages. The changes were
characterized by wrinkling, the extravasation of cytoplasmic material, and alterations in
the size and shape of the cell body. Transmission electron microscopy will be
performed to corroborate the changes observed by SEM.
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32
33
Based on in vitro selectivity of 2,3-diarylsubstituted quinoxaline derivatives,
especially in intracellular amastigotes, the in vivo activity was evaluated in a murine
cutaneous leishmaniasis model. BALB/c mice treated intralesionally with LSPN329 or
LSPN331 showed a significant decrease of lesion thickness compared to control. Oral
miltefosine also decreased the thickness of the lesion in the footpad.
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35
36
37
Although the mechanism of action of 2,3-diarylsubstituted quinoxalines in
promastigotes of L. amazonensis has not been fully elucidated, intense mitochondrial
changes were observed, together with an increase in lipid inclusions that resulted in a
multifactorial response that may be responsible for cellular collapse.
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39
The considerable selectivity of 2,3-diarylsubstituted quinoxalines for
intracellular amastigotes of L. amazonensis in vitro, and significant decrease of lesion
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41
thickness in a murine cutaneous leishmaniasis model suggests that such compounds
may be therapeutic candidates for leishmaniasis.
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43
5.
Conclusion
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46
47
48
49
50
The 2,3-diarylsubstituted quinoxalines were selective for promastigotes and
intracellular amastigotes of L. amazonensis. In promastigote forms, the multifactorial
response that was responsible for cellular collapse was based on intense mitochondrial
alterations. Because of their in vitro antiprotozoal activity, significant decrease of lesion
thickness in a murine cutaneous leishmaniasis model and, mitochondrial mechanism of
action, such compounds may be therapeutic candidates for drug development for the
treatment of leishmaniasis.
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6.
Acknowledgments
This work was supported by Conselho Nacional de Desenvolvimento Científico
e Tecnológico (CNPq), Capacitação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), Financiadora de Estudos e Projetos (FINEP), Fundação de Amparo a
Pesquisa do Estado de São Paulo (FAPESP grants 13/50680-8 and 13/07600-3),
GlaxoSmithKline Trust in Science Project, and Programa de Pós-Graduação em
Ciências Farmacêuticas.
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