Hydroxamic acid derivatives: a promising scaffold for rational compound optimization in Chagas disease

Article abstract of DOI:10.3109/14756366.2015.1077330

This work describes the antitrypanocidal activity of two hydroxamic acid derivatives containing o-ethoxy (HAD1) and p-ethoxy (HAD2) as substituent in the aromatic ring linked to the isoxazoline ring. HAD1 and HAD2 induced a significant reduction in the nu

Full text of DOI:10.3109/14756366.2015.1077330

This article was downloaded by: [New York University]
On: 05 September 2015, At: 13:10
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place,
London, SW1P 1WG
Journal of Enzyme Inhibition and Medicinal Chemistry
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/ienz20
Click for updates
Hydroxamic acid derivatives: a promising scaffold for
rational compound optimization in Chagas disease
ab
c
d
Dayanne da Rocha de Menezes , Claudia Magalhães Calvet , Giseli Capaci Rodrigues ,
c
e
f
Mirian Claudia de Souza Pereira , Igor Rodrigues Almeida , Alcino Palermo de Aguiar ,
g
a
Claudiu T. Supuran & Alane Beatriz Vermelho
a
BIOINOVAR – Biotechnology Laboratories: Biocatalysis, Bioproducts and Bioenergy, Institute
of Microbiology Paulo de Góes, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil,
b
Faculdade São Francisco de Barreiras (FASB), Barreiras, BA, Brazil,
c
Laboratório de Ultra-estrutura Celular, Instituto Oswaldo Cruz – Fiocruz, Rio de Janeiro,
RJ, Brazil,
d
Escola de Ciência e Tecnologia e Programa de Pós-Graduação em Ensino das Ciências,
Universidade do Grande Rio, Duque de Caxias, RJ, Brazil,
e
Departamento de Produtos Naturais e Alimentos, Faculdade de Farmácia, UFRJ, Rio de
Janeiro, RJ, Brazil,
f
Laboratório de Síntese Orgânica, Departamento de Química, Instituto Militar de
Engenharia, Rio de Janeiro, Brazil, and
g
Laboratorio di Chimica Bioinorganica, Università degli Studi di Firenze, Polo Scientifico,
Sesto Fiorentino (Florence), Italy
Published online: 01 Sep 2015.
To cite this article: Dayanne da Rocha de Menezes, Claudia Magalhães Calvet, Giseli Capaci Rodrigues, Mirian Claudia de
Souza Pereira, Igor Rodrigues Almeida, Alcino Palermo de Aguiar, Claudiu T. Supuran & Alane Beatriz Vermelho (2015):
Hydroxamic acid derivatives: a promising scaffold for rational compound optimization in Chagas disease, Journal of Enzyme
Inhibition and Medicinal Chemistry
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1077330
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained
in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no
representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the
Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and
are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and
should be independently verified with primary sources of information. Taylor and Francis shall not be liable for
any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever
or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of
the Content.
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any

form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://
www.tandfonline.com/page/terms-and-conditions

http://informahealthcare.com/enz
ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1077330
!
RESEARCH ARTICLE
Hydroxamic acid derivatives: a promising scaffold for rational
compound optimization in Chagas disease
1
Dayanne da Rocha de Menezes , Claudia Magalh a˜ es Calvet , Giseli Capaci Rodrigues ,
,2
3
4
3
Mirian Claudia de Souza Pereira , Igor Rodrigues Almeida , Alcino Palermo de Aguiar , Claudiu T. Supuran , and
5
6
7
1
Alane Beatriz Vermelho
1
BIOINOVAR – Biotechnology Laboratories: Biocatalysis, Bioproducts and Bioenergy, Institute of Microbiology Paulo de G o´ es, Federal University of
2
3
Rio de Janeiro, Rio de Janeiro, Brazil, Faculdade S a˜ o Francisco de Barreiras (FASB), Barreiras, BA, Brazil, Laborat o´ rio de Ultra-estrutura Celular,
4
Instituto Oswaldo Cruz – Fiocruz, Rio de Janeiro, RJ, Brazil, Escola de Ci eˆ ncia e Tecnologia e Programa de P o´ s-Gradua c¸ a˜ o em Ensino das Ci eˆ ncias,
5
Universidade do Grande Rio, Duque de Caxias, RJ, Brazil, Departamento de Produtos Naturais e Alimentos, Faculdade de Farm a´ cia, UFRJ, Rio de
6
Janeiro, RJ, Brazil, Laborat o´ rio de S ´ı ntese Org aˆ nica, Departamento de Qu ´ı mica, Instituto Militar de Engenharia, Rio de Janeiro, Brazil, and
7
Laboratorio di Chimica Bioinorganica, Universit a` degli Studi di Firenze, Polo Scientifico, Sesto Fiorentino (Florence), Italy
Abstract
Keywords
This work describes the antitrypanocidal activity of two hydroxamic acid derivatives containing Chagas disease, hydroxamic acid derivatives,
o-ethoxy (HAD1) and p-ethoxy (HAD2) as substituent in the aromatic ring linked to the
isoxazoline ring. HAD1 and HAD2 induced a significant reduction in the number of intracellular
parasites and consequently showed activity on the multiplication of the parasite. Treatment of
cardiomyocytes and macrophages with the compounds revealed no significant loss in cell
viability. Ultrastructural alterations after treatment of cardiomyocytes or macrophages infected
by Trypanosoma cruzi with the IC50 value of HAD1 revealed alterations to amastigotes, showing
initial damage seen as swelling of the kinetoplast. This gave a good indication of the ability of
the drug to permeate through the host cell membrane as well as its selectivity to the parasite
target. Both compounds HAD1 and 2 were able to reduce the cysteine peptidases and decrease
the activity of metallopeptidases
Trypanosoma cruzi
History
Received 3 July 2015
Revised 24 July 2015
Accepted 25 July 2015
Published online 31 August 2015
7
Introduction
transmission in endemic areas all of which alert the need to
implement strategies to control the transmission and burden of
Chagas disease, a zoonosis caused by the protozoan Trypanosoma
cruzi, is currently a global public health challenge. The disease
has two distinct clinical phases: acute, mostly asymptomatic with
patent parasitemia, and, chronic, which can be asymptomatic
(indeterminate stage) or symptomatic (determinate stage), show-
ing severe cardiomyopathy, megacolon, or megaoesophagous .
8
this infectious disease .
The current chemotherapies for clinical practice are benzni-
dazole (BZ) and nifurtimox. The medications are predominantly
active during the acute phase of the disease. Resistance to
benznidazole (BZ) has been observed with some parasite strains .
Additionally, adverse side effects of these drugs, such as digestive
and dermatological intolerance, have been implicated in the
10
treatment compliance . Also it is important to highlight that the
9
1
2
This disease affects 7–8 million people mostly in Latin American
and it is currently the leading cause of heart failure in South
3
American countries . However, there is a new epidemiological
scenario showing a growing number of infected individuals in
T. cruzi polyparasitism and co-infection with other pathogens may
11
interfere with the failure of the present therapeutic interventions .
4
non-endemic countries that is drawing attention to this silent and
12
The failure of promising drugs undergoing clinical trials and
neglected disease. There are a large number of immigrants with
,6
Chagas disease in the United States and European countries and
also there is the emerging infection by the congenital and oral
13
also the unsuccessful stem cell therapy reinforce the need for
new strategies in the treatment and management of Chagas’
5
14
disease . These strategies should include an analysis of the
1
advantage of combination therapy and eventual benefits from
5
1
6
Address for correspondence: Alane Beatriz Vermelho, BIOINOVAR – vaccine development , as well as the identification of new drug
Biotechnology Laboratories: Biocatalysis, Bioproducts and Bioenergy, targets and biomarkers to monitor the treatment response and cure
Institute of Microbiology Paulo de G o´ es, Federal University of Rio de
prediction. Thus, the lack of effective drugs for the treatment of
Janeiro, Av. Carlos Chagas Filho, 373, 21941-902 Rio de Janeiro, Brazil.
this worldwide, emerging disease encourages initiatives on the
Tel: +55-21- 39386743. Fax: +55-21-2560-8344. E-mail: abvermelho@
rational drug development of new trypanocidal drugs.
micro.ufrj.br
Given the key role of peptidases in biological systems, these
Mirian Claudia de Souza Pereira Laborat o´ rio de Ultra-estrutura Celular,
Instituto Oswaldo Cruz Fiocruz, Av. Brasil 4365, Manguinhos 21040-
00, Rio de Janeiro, RJ, Brazil. E-mail: mirianioc@gmail.com
enzymes have been considered a potential therapeutic target
1
7,18
9
against infectious agents
. Different classes of peptidases

2
D. d. R. de Menezes et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
participate in important biological events in the T. cruzi life cycle, aldehydes and 60 mmol of NH OH.HCl contained in 20 mL of
2
including processes of invasion, differentiation, proliferation as solvent. The reaction medium was maintained for 2 h at a
1
9
ꢀ
well as mechanisms of escape from host immune responses . temperature of 27 C under constant agitation. After which, the
Thus, peptidase inhibitors are a potential to design trypanocidal respective aldoximes were reacted with trichloroisocyanuric acid
drugs. For example, K777, a peptidase inhibitor of the major (10 mmol) and triethylamine (20 mmol) for 30 min under stirring
cysteine proteinase of T. cruzi known as cruzain or cruzipain, at room temperature to produce the corresponding hydroximoyl
became a potent candidate to advance into clinical trials in view chlorides. Then, dipolarophile (ethyl acrylate) was added to the
2
0
of its effective activity against different strains of T. cruzi , solutions and, after 24 h, the 4,5-dihydroisoxazol derivatives were
including its efficiency in protecting against cardiac damage in a generated by 1,3-cycloaddiction reactions. Finally, a solution
2
canine experimental model and in response to multi-drug containing NH OH (20 mmol) was added to the 4,5-dihydroisox-
1
2
2
2
therapy . Aspartic peptidase from T. cruzi has also been azol derivatives formed and kept at reflux for 24 h. The respective
proposed as a drug target since treatment of epimastigotes with hydroxamic acid derivatives generated were recrystallized from
pepstatin A, an aspartic peptidase inhibitor, inhibited parasite hot water and filtered. The yields were 40% and 50%, respectively.
proliferation, induced morphological changes with detachment of
2
3
flagellum, and increased expression of gp63 . Moreover, matrix Parasites
metallopeptidase-9 homologues detected in T. cruzi may be a
Trypomastigote forms of T. cruzi, Y strain, were isolated from the
potential therapeutic target, since they could be able to induce
a reduction in the expression of matrix metallopeptidase-9
blood of infected mice at the peak of parasitemia (7 d after
infection). To increase the yield of T. cruzi trypomastigotes per
mouse and reduce the number of animals used in the procedure,
mice were immunosuppressed with intraperitoneal (ip) adminis-
tration of cyclophosphamide 500 mg/kg on the 4th day after
infection. To isolate the parasites, blood was collected by cardiac
puncture with 3.8% sodium citrate as an anticoagulant and
centrifuged at 1200 rpm for 26 min to separate them from the
blood cells. The supernatant containing the parasites was then
harvested and centrifuged at 4500 rpm for 14 min to pellet the
trypomastigotes. The parasites were resuspended in culture
medium DMEM-F12 and the concentration of parasites was
assessed using a hemocytometer. The amastigote forms were
obtained and analyzed through infection of mammalian cultures
with trypomastigotes.
2
4,25
(MMP-9) during interaction with host cells .
A series of novel 4,5-dihydroisoxazoles incorporating hydro-
xamate moieties were synthesized and evaluated against Chagas
disease. These hydroxamic acid derivatives (HAD) proved to be
very effective against T. cruzi. The assays carried out revealed
molecules with low cytotoxicity and efficient action against T.
2
6
cruzi carbonic anhydrase (TcCA) and metallopeptidase .
Carbonic anhydrases (CA, EC 4.2.1.1) are metalloenzymes
which catalyze the conversion of CO2 to the bicarbonate ion
and protons. CAs are involved in numerous physiological and
pathological processes including microbial diseases caused by
2
7
fungi and bacteria . The identification of a gene encoding
a-carbonic anhydrase in T. cruzi (TcCa) raised interest in the
application of this enzyme as a potent therapeutic target in Chagas
26,28
disease
.
Primary culture
Thus, this study aimed to investigate the detailed trypanocidal
action of the two derivatives of this new series, since they showed
Murine peritoneal macrophages were obtained from Swiss
Webster mice (18–20 g). Cold Dulbecco’s modified Eagle
medium (DMEM; Sigma, St. Louis, MO) was injected into the
peritoneal cavity and then removed by fluid aspiration with a
syringe. Peritoneal exudate cells were centrifuged at 400 ꢁ g for
2
6
promising results against TcCA , and their trypanocidal activity
has not been investigated until now. Our results showed that these
hydroxamic acid derivatives (HAD1 and 2) demonstrated a good
activity against intracellular amastigotes, the clinical form found
in the chronic phase. An inhibitory effect against parasite
peptidases was also observed. Altogether, our data suggest that
the hydroxamates scaffold can be considered as interesting
candidates to move forward towards the development of more
effective, selective and efficient Chagas disease drugs.
1
0 min and the pellet resuspended in culture medium supple-
mented with 10% fetal bovine serum (FBS) followed by
determination of cell number/mL using a Neubauer chamber.
5
5
The cells were plated at a density of 1 ꢁ 10 and 4 ꢁ 10 cells per
well in black, clear bottom 96-well (Greiner Bio-One, #655090,
Frickenhausen, Germany) and 24-well tissue culture plates,
ꢀ
Materials and methods
respectively, and maintained at 37 C with 5% CO
Primary culture of cardiac muscle cells was obtained as
2
atmosphere.
Chemicals
29
previously described . Briefly, the heart was removed from 18-d
Medium constituents were obtained from Oxoid Ltd. (Hampshire, old mouse embryos and fragmented ventricles were dissociated
England). Reagents used in electrophoresis were purchased from with trypsin and collagenase. Isolated cells were resuspended in
Amersham Life Science (Little Chalfont, England). Gelatin and DMEM-F12 (Sigma, St. Louis, MO) supplemented with 7% FBS,
casein were purchased from Merck (Darmstadt, Germany). The 2% chicken embryo extract, 2.5 mM of CaCl , 1 mM L-glutamine
2
4
metallopeptidase (1,10-PHE – 1,10-phenanthroline), the cysteine and antibiotics. The cells were plated at a density of 5 ꢁ 10 and
5
peptidase (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)bu- 1.2 ꢁ 10 cells per well in 96-well and 24-well tissue culture
ꢀ
tane [E-64]) inhibitors and all reagents used in the synthesis of plates, respectively, and maintained at 37 C at 5% CO
atmos-
2
the 4,5-dihydroisoxazole derivatives were obtained from Sigma- phere. All procedures with animals were performed according to
Aldrich (St. Louis, MO). All other reagents were of analytical the Oswaldo Cruz Foundation committee of ethics for the use of
grade.
experimental animals (LW-37/13).
Synthesis of 3-(2-ethoxyphenyl)-N-hydroxy-4,5-dihydroi-
soxazole-5-carboxamide (HAD1) and 3-(4-ethoxyphenyl)-
N-hydroxy-4,5-dihydroisoxazole-5-carboxamide (HAD2)
Cell cytotoxicity assay
Primary cardiomyocyte and macrophage cultures were used to
evaluate the cytotoxic effect of the compounds. After 24 h of
The hydroxamic acid derivatives were synthesized as described culture, the cells were treated with the synthetic derivatives of
2
by Rodrigues et al. Briefly, the 2-ethoxy and 4-ethoxy-phenyl- 4,5-dihydroisoxazoles incorporating hydroxamate moieties
6
2
aldoxime were synthesized from 20 mmol of the respective (HAD1 and HAD2) synthesized as described . A wide range of
6

DOI: 10.3109/14756366.2015.1077330
Hydroxamic acid derivatives
3
concentrations, ranging from 0.22 to 500 mg/mL, were tested Transmission electron microcopy (TEM)
to evaluate toxicity to mammalian cells. The cell viability
Primary cultures of cardiomyocyte and macrophage were
infected with Y strain of bloodstream trypomastigotes in a
analysis (CC50 value ¼ the cytotoxic concentration correspond-
ing to 50% reduction of cell viability) was performed after 3 d
of treatment with HAD1 and HAD2 compounds using
resazurin 0.005% (4 h at 37 C) as the metabolic function
indicator, followed by fluorescence intensity measurements at
1
0:1 host cell–parasite ratio. After 24 h of infection, free
trypomastigotes were removed from the culture supernatant
and, after washing with Ringer’s solution (154 mM NaCl, 56 mM
KCl, 17 mM Na HPO pH 7.0), the cells were treated for 24 h
ꢀ
2
4,
5
30 nm and 630 nm in a Molecular Devices Flexstation 3
or 48 h with the IC50 concentration (30 mM) of the HAD1
compound. Subsequently, the cultures were fixed for 1 h at 4 C
Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale,
CA). Negative controls were performed with the vehicle
medium with or without DMSO (ꢂ1%) and untreated cell
cultures under similar conditions.
ꢀ
with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH
ꢀ
7
.2, and followed by post-fixation for 1 h at 4 C with 1% OsO4
in the same buffer. The samples were dehydrated in serial-
graded acetone batches impregnated and embedded in Epon 812.
Ultra-thin sections, contrasted with uranyl acetate and lead
Trypanocidal assay
The effect of the HAD1 and HAD2 hydroxamic derivatives on citrate, were observed in a JEOL transmission electron micro-
T. cruzi was evaluated using bloodstream trypomastigotes of scope (JEM/1011).
the Y strain. Parasites were isolated from infected Swiss Webster
mice at the peak of parasitemia (7 d post-infection).
6
Analysis of peptidase activity
Trypomastigotes (3 ꢁ 10 parasites/well) were incubated for
ꢀ
2
4 h at 37 C in the presence or absence of serial dilutions of The inhibitory effect on the cellular proteolytic activity was
HAD1 or HAD2 compounds (0.04–90 mM). After treatment, the evaluated on 10% SDS–PAGE with 0.1% gelatin incorporated
resazurin 0.005% was added to the parasites for 4 h at 37 C and as substrate . Trypanosoma cruzi epimastigote cultures were
ꢀ
30
ꢀ
the fluorescence emission measurements were registered at harvested by centrifugation at 1500 ꢁ g for 15 min at 4 C, and
7
ꢀ
5
30 nm and 630 nm in a Molecular Devices Flexstation 3 Multi- 1 ꢁ 10 cells were then lysed at 0 C by the addition of 100 mL
Mode Microplate Reader (Molecular Devices, Sunnyvale, CA). of sodium dodecyl sulfate–polyacrylamide gel electrophoresis
The inhibitory concentration (IC ), the drug concentration that (SDS–PAGE) sample buffer. The gels were loaded with 25 mL
5
0
7
reduces 50% of trypomastigotes viability, was determined by of parasite extract per slot (equivalent to 1 ꢁ 10 cells) and
fitting the dose–response curve to inhibitory activity compared after electrophoresis at a constant voltage of 170 V for 2 h at
with the untreated control. A similar curve for treatment with 4 C; then they were soaked for 1 h in 2.5% Triton X-100 under
ꢀ
ꢀ
benznidazole (BZ) was also carried out in a parallel assay. constant agitation. The gel was incubated for 48 h at 37 C in
Untreated trypomastigotes and cell-free media were used as 50 mM sodium phosphate buffer, pH 5.5, with 100 mM of the
negative controls.
HAD1 and HAD2. In order to determine the class of
The efficacy of the compounds was also evaluated, in terms of peptidases, 10 mM E-64 (a cysteine-peptidase inhibitor),
eliminating the intracellular parasites, by incubating 24 h-infected 10 mM 1,10-phenanthroline (a non-specific metallopeptidase
mammalian cells primary cultures (macrophages and cardiomyo- inhibitor) and 2 mM DTT (a cysteine-peptidase activator) were
ꢀ
26
cytes) for 72 h at 37 C with a serial dilution (3.3–90 mM) of also tested .
HAD1 and HAD2 compounds, followed by fixation with Bouin
solution fixative. The percentage of infection was determined by
Statistical analysis
quantification of a minimum of 200 cells in randomly selected
microscopic fields using a Zeiss Axioplan microscope (Zeiss, Student’s t-test was used to determine the significance of
Rochester, NY). The experimental assays were performed at least differences between the means in the amastigote proliferation
three times in duplicate.
assays. A p value ꢂ 0.05 was considered significant.
Figure 1. Hydroxamic acid derivatives
(HAD) with potential action against Chagas
disease. Carbonic anhydrases human iso-
forms hCA I and II and T. cruzi enzyme
2
6
TcCA .

4
D. d. R. de Menezes et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Result and discussion
This led us to analyze the efficacy of the two analogs containing
only o-ethoxy (HAD1) and p-ethoxy (HAD2) moieties as
substituents in the aromatic ring (Figure 1) against trypomasti-
Chemistry
Previous studies have demonstrated promising antitrypanocidal gotes and amastigotes.
2
activities of hydroxamic acid derivatives – HAD , acting as
6
effective T. cruzi carbonic anhydrase (TcCA) inhibitors and Effect of the hydroxamic acid derivatives (HAD1-2)
ineffective as human CA inhibitors. Also, a lead compound against T. cruzi
containing a p-benzyloxy as a substituent on the aromatic ring
We first analyzed the effect of the synthetic drugs (HAD1 and 2)
on the viability of the bloodstream trypomastigotes (Y strain). Our
findings demonstrated that even the highest concentration
(HAD-ph, Figure 1) besides its inhibitory activity against TcCA
also showed promising features as an antitrypanosomal agent .
2
6
(90 mM) of both compounds (HAD1 and 2) was not effective
against the trypomastigote forms of T. cruzi, while the IC50 values
for benznidazole (BZ), reference drug, was 12.24 mM ± 3.42
(Figure 2). In fact, the synthetic hydroxamic derivatives tested
here (HAD1 and 2) were not as effective as their analog (HAD-
2
6
ph) investigated earlier by Rodrigues et al.
This drug
Figure 2. Effect of hydroxamic acid derivatives (HAD1-2) on blood-
stream trypomastigotes (Y strain) compared with BZ activity.
ꢀ
Trypomastigotes were treated at 37 C for 24 h with different concentra-
tions of the compounds. HAD1 and HAD2 compounds were inactive
against the classical invasive form of T. cruzi. In contrast, the reference
drug, BZ, showed an IC50 value of 12.2 mM.
Figure 4. Analyses of the effect of HAD1 and 2 against intracellular
amastigotes in cardiomyocyte culture. (A) Treatment with both synthetic
compounds, HAD1 and 2, resulted in a dose-dependent inhibition of
amastigote proliferation in cardiomyocytes. The lower IC50 value was
achieved by HAD1 (30 mM). A significant decrease in the total
intracellular amastigotes was observed after HAD1 (ꢃ30 mM) and
HAD2 (90 mM) treatment (B). *Statistically significant, p ꢂ 0.05.
Table 1. Antitrypanocidal effect of hydroxamic acid derivatives (HAD).
Evolutive forms of Trypanosoma cruzi
Trypomastigotes
IC50 (mM)
Amastigotes
IC50 (mM)y
Compound
Figure 3. (A) Dose-dependent cardiomyocyte viability curve detected by HAD1
resazurin in response to HAD1 treatment, resulting in CC50 HAD2
value ¼ 329.8 mM. (B) HAD1 and 2 cytotoxicity were also assessed in
NE*
NE*
30 ± 10.7
66 ± 19.3
macrophages in the same fashion, resulting in similar viability curves and *Not effective in the highest concentration (90 mM) evaluated.
a CC50 value of 349 mM and 221 mM, respectively.
yIC50 values in T. cruzi-infected cardiomyocytes.

DOI: 10.3109/14756366.2015.1077330
Hydroxamic acid derivatives
5
demonstrated a potent activity (greater than BZ) against all ring (HAD-ph) for trypanocidal activity, either by a steric effect or
2
evolutive forms of T. cruzi, reaching an IC50 value from 7 to by increasing the liposolubility of the molecule .
6
51 mM for different strains of the parasite. The absence of a third
aromatic ring in the HAD1-2 structures decreased their inhibitory
activity against TcCA and trypanocidal activity trypomastigote
Screening of cytotoxicity in mammalian cells
forms. Additionally, it was possible to observe that the ortho or Prior to analyze the effect of the synthetic compounds on
para ethoxy positions of the substituent on the aromatic ring intracellular amastigotes, we established the non-toxicity concen-
linked to the heterocyclic ring did not influence the trypanocidal trations of the compounds to mammalian cells. The cellular
activity. This fact highlighted the importance of the p-benzyloxy toxicity assay was estimated on cardiomyocyte and macrophage
as the substituent on the aromatic ring linked to the heterocyclic cultures using resazurin as a viability indicator. Low toxicity was
Figure 5. Light microscopy of cardiomyocytes treated with HAD1 and 2 compounds. (A) Untreated cells displaying high number of intracellular
amastigotes; (B) benznidazole treatment at 15 mM eliminated the parasites. HAD1 treatment resulted in an expressive reduction of amastigote
proliferation (C, 90 mM; D, 30 mM). Addition of HAD2 in T. cruzi-infected cardiomyocyte cultures inhibited amastigote proliferation at high
concentrations (90 mM, E), but lower concentrations did not produce the same effect (30 mM, F). Bar ¼ 10 mM.

6
D. d. R. de Menezes et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
seen for both compounds tested. Treatment of cardiomyocytes
with the HAD1 compound revealed no significant loss in viability
up to 166 mM, resulting in a CC50 value ¼ 329.8 mM (Figure 3A).
Also against macrophages, HAD1 and 2 did not demonstrate any
cytotoxicity effect, reaching a CC50 value of 349 mM and 221 mM,
respectively (Figure 3). The lack of toxicity in both cell types was
clearly demonstrated by light microscopy, where even the highest
concentration of HAD1 (90 mM) did not alter the morphology
and viability of the macrophages (data not shown). The absence of
p-benzyloxy as the substituent on the aromatic ring decreased the
antitrypanosomal activity as well as the cellular cytotoxicity,
which could be influenced by lower liposolubility of the HAD1
and 2 molecules than the HAD-ph prototype.
Efficacy of compounds against intracellular amastigotes
Considering the previous observations that TcCA inhibitors affect
2
parasite growth and that both compounds were effective against
8
2
6
epimastigotes , we hypothesized that these derivatives could
have a potential activity on the multiplication of the parasite.
Therefore, we investigated the effectiveness of the drugs in
clearing the infection. The activity against intracellular amasti-
gotes was assessed by treating T. cruzi-infected cardiomyocyte
cultures with the HAD1 and 2 compounds. Both drugs induced a
significant reduction in the number of intracellular parasites in
concentrations of 30 mM and 90 mM, but HAD1 was two-fold
more effective than HAD2 at 30 mM (Figure 4). The IC50 values, a
concentration that reduces 50% of intracellular parasites, in
T. cruzi-infected cardiomyocytes, were 30 ± 10.7 mM and
6
6 ± 19.3 mM for HAD1 and 2, respectively. This indicated that
although these molecules have a lower liposolubility than the lead
compound they are able to act against the intracellular forms of
the parasite (amastigotes). Furthermore, it was observed that the dependent inhibition of amastigote proliferation, achieving an IC50 value
Figure 6. HAD1 compound effect on T. cruzi infection in macrophages.
(
A) Treatment of infected macrophages with HAD1 resulted in a dose-
intracellular activity was influenced by the ethoxy substituent of 75 mM. (B) A significant decrease in the total number of intracellular
amastigotes was detected with 90 mM of HAD1. *Statistically significant,
p ꢂ 0.05.
position in the aromatic ring from the two hydroxamic acids, an
important observation for future strategy of rational drug design.
Table 1 summarizes the results of the antitrypanocidal effect of
hydroxamic acid derivatives
Furthermore, the treatment of T. cruzi-infected cardiomyocyte
cultures with 90 mM of HAD1 reduced the number of intracellular
amastigotes 7-fold when compared with untreated cultures
(Figure 4). This phenomenon was clearly observed by light
microscopy (Figure 5), which demonstrates a low level of
infection in the highest concentration (90 mM) of the HAD1 and
of rational design of drugs has been encouraged in the search for
31
new active drugs for the treatment of neglected diseases . Lead
optimization of compounds involving structure–activity relation-
ships (SAR) may improve compound potency and physicochem-
ical properties. However, the synthesis of more active compounds
is a challenge and slight structural changes can reduce the
compound activity. Therefore, our findings can guide the design
of new molecules since the presence of p-benzyloxy instead of
ethoxy impacts the activity against T. cruzi.
2 compounds compared with control. It is important to highlight
that treatment of the T. cruzi-infected cardiomyocyte cultures with
BZ at 15 mM eliminated all the intracellular parasites.
The fact that HAD1 displayed the highest selectivity index
Ultrastructural analysis of the effect of HAD1 inhibitor
against intracellular parasites
(
SI ꢃ 11) and the lowest IC prompted us to analyze its activity
5
0
against T. cruzi-infected macrophages, also an important cell
involved in the infection by this parasite. Although the IC50 value To analyze possible ultrastructural alterations induced by the
was higher in macrophages (75 mM), the treatment with 90 mM of synthetic compounds, transmission electron microscopy of
HAD1 also induced a 5.5-fold reduction of intracellular parasites T. cruzi-infected cardiomyocytes and macrophages after HAD1
(Figures 6 and 7). At the highest concentration (90 mM), the treatment was performed (Figure 8). Treatment of cardiomyocytes
macrophages demonstrated normal morphology and spreading, or macrophages infected by T. cruzi with the IC50 value of HAD1
showing the low toxicity of the drug (Figure 7). Although the revealed initial damage in the amastigotes, visualized by swelling
drugs have shown activity against T. cruzi, they were not as of the kinetoplast. One important fact is that both cells,
efficient in the elimination of parasites as benznidazole, which macrophages and cardiomyocytes, did not undergo structural
revealed a reduction of 99% of intracellular amastigotes with change with this treatment, demonstrating no cytotoxic effect of
1
5 mM. the compounds for mammalian cells. Thus, our ultrastructural
Curiously, HAD1 and 2 were less effective than the HAD-ph analysis associated with the cytotoxicity findings provided a good
from the series of hydroxamic acid derivatives investigated here. indication of the ability of the drug to permeate through the host
The substitution of the p-benzyloxy (HAD-ph) with o-ethoxy and cell membrane and its selectivity to the parasite target. The
p-ethoxy in HAD1 and HAD2 reduced the efficacy of these ultrastructural analysis of the effect of nonazoleCYP51 inhibitors
compounds to eliminate the intracellular amastigotes in mamma- against intracellular amastigotes showed blebbling of cellular and
lian cells by three-fold and seven-fold, respectively. The strategy nuclear membranes and autophagic vesicles, in parallel with the

DOI: 10.3109/14756366.2015.1077330
Hydroxamic acid derivatives
7
Figure 7. Light microscopy images of T. cruzi-infected macrophage cultures treated with HAD1. The cultures were treated 24 h after the establishment
of T. cruzi infection. (A) General aspect of macrophages cultures after 72 h of infection by T. cruzi. (B) Clearance of intracellular parasites after 72 h
treatment with 15 mM of benznidazole. Note the effectiveness of HAD1 compound against amastigotes after 72 h of treatment with 90 mM (C) and
1
0 mM (D).
Effect of HAD1 and 2 on the peptidase activity of T. cruzi
accumulation of intermediate metabolites from the ergosterol
3
2
biosynthesis pathway . Most morphological studies of drug
treatment emphasize the direct effect of compounds on the
isolated parasites, mainly T. cruzi epimastigotes. Ultrastructural
changes were also evidenced in T. cruzi treated with serine and
Cell-associated peptidases from T. cruzi displayed two distinct
proteolytic classes: cysteine-(60 kDa) and metallopeptidase
(
1
70 and 80 kDa) activities inhibited, respectively, by E-64 and
,10-phenanthroline, as shown in Figure 9. Both compounds
3
3,34
cysteine protease inhibitors
. A Kunitz-type serine protease
HAD1 and 2 were able to reduce the cysteine peptidases and
decrease the activity of metallopeptidases. Cruzain/cruzipain is a
cysteine peptidase required for the survival of T. cruzi. This
enzyme, present in all life stages of the trypanosome, has been the
focus of several studies and patents and is implicated in parasite
cellular entry and digestion of immunoglobulin, immune system
inhibitor from Caribbean Sea anemone Stichodactyla helianthus
induced the formation of concentric membrane structures and
endoplasmic reticulum surrounding vesicles in the cytoplasm of
epimastigotes, suggesting the autophagic cell death as its
3
3
mechanism of action . Cysteine protease inhibitors (morpholi-
necarbonyl–phenylalanine–homophenylalanine–vinyl sulphone
phenyl and morpholineureaphenylalanine–homophenylalanine–
fluoromethyl ketone) induced severe alterations in the Golgi
complex with chagasin accumulation in enlarged cisternae,
suggesting a disturbance in the transport within the ER–Golgi
1
9,41
evasion, and other cellular mechanisms
.
In conclusion, this work demonstrated that hydroxamates are a
promising class of compound with anti-T. cruzi activity. Our data
showed that hydroxamates HAD1 and 2 were effective against the
replicative forms of the parasite, but not against the infective
forms. This could be an indication that its mechanism of action
involves the inhibition of parasite peptidases and carbonic
anhydrase, essential enzymes for the parasite life cycle. HAD1
and 2 partially inhibited the cysteine and metallopeptidases of
T. cruzi under the conditions evaluated. Furthermore, previous
studies have already demonstrated the selectivity of these
compounds against carbonic anhydrase of Trypanosoma cruzi
3
4
system . Multiple intracellular compartment damage has been
3
5–37
reported with other active drugs against T. cruzi
.
Mitochondrial swelling and the presence of concentric structures
were frequently noticed in parasites treated with lysophospholipid
3
8
39
and naphthoquinone derivatives . Remarkable
analogues
changes were also induced in the kinetoplast–mitochondrial
system, microtubule, and reservosomes organization after treat-
ment of T. cruzi with DNA-intercalating drugs and sterol
biosynthesis inhibitors, leading to kinetoplast collapse and cell
(TcAC). In our assays, we observed that the position of the ethoxy
substituent on the aromatic ring linked to the heterocyclic one
strongly influenced the biological activity. HAD1, which has an
3
6,37,40
death
.

8
D. d. R. de Menezes et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 8. Transmission electron microscopy of mammalian cells infected by T. cruzi and treated with HAD1 compound. Untreated controls showing
normal general amastigote structure and regular kinetoplasts (k) from cardiomyocyte (A) and macrophage (B) infected cultures. Forty-eight hours
treatment of HAD1 at IC50 concentration (30 mM) induced swelling of kinetoplasts (black arrows) both in cardiomyocyte (B) and in macrophage
(
D)-infected cultures.
ortho-ethoxy, was 2-fold more effective than HAD2, which has a
para-ethoxy moiety, against the intracellular amastigotes. The
absence of p-benzyloxy moiety, present in the lead molecule
HAD-ph, decreased the lipophilicity of these compounds, but the
HAD1 and 2 were still able to be effective against the replicative
forms of the parasite. This fact demonstrates that this moiety is
important for trypanocidal activity, but not essential. Moreover,
new structural changes in the scaffold seem to be important for
investigating the trypanocidal activity and the discovery of a more
potent agent for the treatment of Chagas disease.
Conclusions
This work demonstrated a promising class of hydroxamates with
anti-T. cruzi activity. The hydroxamates were effective against
replicative forms of the parasite, but not against infective,
trypomastigote forms. Ultrastructural analysis of amastigotes,
the replicative, forms showed swelling of the kinetoplast after
treatment with HAD1. Both compounds reduce the cysteine and
Figure 9. Zymography of T. cruzi cellular peptidases after treatment with
the HAD1 and HAD2 inhibitors. CTL, control – without treatment; E-64,
cysteine peptidase inhibitor; DTT, cysteine peptidase activator; 1,10-
PHE, metallopeptidase inhibitor.

DOI: 10.3109/14756366.2015.1077330
Hydroxamic acid derivatives
9
metallopeptidase activities. No cytotoxic effect of the compounds 17. Ndao M, Beaulieu C, Black WC, et al. Reversible cysteine protease
inhibitors show promise for a Chagas disease cure. Antimicrob
Agents Chemother 2014;58:1167–78.
8. Branquinha MH, Marinho FA, Sangenito LS, et al. Calpains:
potential targets for alternative chemotherapeutic intervention
against human pathogenic trypanosomatids. Curr Med Chem 2013;
for mammalian cells was observed. HAD1 and HAD2 could be an
important scaffold to future rational drug design for anti-chagasic
chemotherapy.
1
1
2
0:3174–85.
9. Laverri e` re M, Cazzulo JJ, Alvarez VE. Antagonic activities of
Trypanosoma cruzi metacaspases affect the balance between cell
proliferation, death and differentiation. Cell Death Differ 2012;19:
Acknowledgements
The authors thank Liliane Mesquita, Alanderson Nogueiraand Maria do
Socorro Silva for their technical support in the cardiomyocyte and
macrophage cultures.
1
0. Cazzulo JJ. Proteinases of Trypanosoma cruzi: patential targets for
358–69.
2
2
the chemotherapy of Changas desease. Curr Top Med Chem 2002;2:
1
261–71.
1. Barr SC, Warner KL, Kornreic BG, et al. A cysteine protease
inhibitor protects dogs from cardiac damage during infection by
Trypanosoma cruzi. Antimicrob Agents Chemother 2005;49:
5160–2.
Declaration of interest
This work was supported by grants from Funda c¸ a˜ o Oswaldo Cruz
(
FIOCRUZ), Conselho Nacional de Desenvolvimento Cient ´ı fico e
2
2
2. McKerrow JH, Doyle PS, Engel JC, et al. Two approaches to
discovering and developing new drugs for Chagas disease. Mem Inst
Oswaldo Cruz 2009;104:263–9.
3. Santos LO, Garcia-Gomes AS, Catanho M, et al. Aspartic
peptidases of human pathogenic trypanosomatids: perspectives
and trends for chemotherapy. Curr Med Chem 2013;20:
Tecnol o´ gico (MCTI-CNPq), Programa Estrat e´ gico de Apoio a`
Pesquisa em Sa u´ de (PAPES) VI), Funda c¸ a˜ o de Amparo a`
Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordena c¸ a˜ o
de Aperfei c¸ oamento Pessoal de N ´ı vel Superior (CAPES) and
Instituto Militar de Engenharia (IME),
3
116–33.
2
2
4. Nogueira de Melo AC, de Souza EP, Elias CGR, et al. Detection of
matrix metallopeptidase-9-like proteins in Trypanosoma cruzi. Exp
Parasitol 2010;125:256–63.
5. Nogueira de Melo AC, Meirelles MNSL, Porrozzi R, et al. Reduced
activity of matrix metalloproteinase-9 in Trypanosoma cruzi-
infected mouse embryo hepatocyte cell. Hepatol Res 2004;28:
References
1.
2.
3.
4.
Rassi A, Marcondes RJ. American trypanosomiasis (Chagas
disease). Infect Dis Clin North Am 2012;26:275–91.
WHO. Chagas disease-factsheet. Wkly Epidemiol Rec 2012;87:
5
19–22.
Bocchi EA. Heart failure in South America. Curr Cardiol Rev 2013;
:147–56.
4
9–56.
2
6. Rodrigues GC, Feij o´ DF, Bozza MT, et al. Design, synthesis,
and evaluation of hydroxamic acid derivatives as promising agents
for the management of Chagas disease. J Med Chem 2014;57:
9
Strasen J, Williams T, Ertl G, et al. Epidemiology of chagas disease
in Europe: many calculations, little knowledge. Clin Res Cardiol
2
98–308.
2
014;103:1–10.
2
2
7. McKenna R, Supuran CT. Carbonic anhydrase inhibitors drug
design. Subcell Biochem 2014;75:291–323.
8. Pan P, Vermelho AB, Capaci Rodrigues G, et al. Cloning,
characterization, and sulfonamide and thiol inhibition studies of an
a-carbonic anhydrase from Trypanosoma cruzi, the causative agent
of Chagas disease. J Med Chem 2013;56:1761–71.
5.
6.
7.
8.
9.
Jackson Y, Herrera V, Gascon J. Economic crisis and increased
immigrant mobility: new challenges in managing Chagas disease in
Europe. Bull World Health Organ 2014;92:771–2.
Carlier Y. Globalization of Chagas disease (American trypanosom-
iasis): the situation in Europe and Belgium. Bull Mem Acad R Med
Belg 2011;166:347–55.
S a´ nchez LV, Ram ´ı rez JD. Congenital and oral transmission of
American trypanosomiasis: an overview of physiopathogenic
aspects. Parasitology 2012;140:1–13.
Montgomery SP, Starr MC, Cantey PT, et al. Neglected parasitic
infections in the United States: Chagas disease. Am J Trop Med Hyg
2
3
9. Meirelles MNSL, de Ara u´ jo-Jorge TC, Miranda CF, et al.
Interaction of Trypanosoma cruzi with heart muscle cells:
ultrastructural and cytochemical analysis of endocytic vacuole
formation and effect upon myogenesis in vitro. Eur J Cell Biol
1
986;41:198–206.
0. Heussen C, Dowdle EB. Electrophoretic analysis of plasmino-
gen activators in polyacrylamide gels containing sodium dodecyl
sulfate and copolymerized substrates. Anal Biochem 1980;102:
2
014;90:814–18.
Mej ´ı a-Jaramillo AM, Fern a´ ndez G, Montilla M, et al. Trypanosoma
cruzi strains resistant to benznidazole occurring in Colombia.
Biomedica 2012;32:196–205.
1
96–202.
3
3
1. Kellici T, Ntountaniotis D, Vrontaki E, et al. Rational drug design
paradigms: the Odyssey for designing better drugs. Comb Chem
High Throughput Screen 2015;18:238–56.
1
0. Pinazo MJ, Guerrero L, Posada E, et al. Benznidazole-related
adverse drug reactions and their relationship with serum drug
concentrations in patients with chronic Chagas disease. Antimicrob
Agents Chemother 2012;57:390–5.
1. Perez CJ, Lymbery AJ, Thompson RCA. Chagas dis-
ease: the challenge of polyparasitism? Trends Parasitol 2014;30:
2. Doyle PS, Chen C-K, Johnston JB, et al. A nonazole
CYP51 inhibitor cures Chagas’ disease in a mouse model
1
1
1
1
1
of acute infection. Antimicrob Agents Chemother 2010;54:
2480–8.
1
76–82.
2. Molina I, G o´ mez i Prat J, Salvador F, et al. Randomized trial of 33. De Almeida Nogueira NP, Morgado-Dı
´
az JA, Menna-Barreto RFS,
posaconazole and benznidazole for chronic Chagas’ disease. N Engl
J Med 2014;370:1899–908.
3. De Carvalho KAT, Abdelwahid E, Ferreira RJ, et al. Preclinical stem
et al. Effects of a marine serine protease inhibitor on viability and
morphology of Trypanosoma cruzi, the agent of Chagas disease.
Acta Trop 2013;128:27–35.
cell therapy in Chagas disease: perspectives for future research. 34. Engel JC, Doyle PS, Hsieh I, Mckerrow JH. Cysteine protease
World J Transplant 2013;3:119–26.
4. Bahia MT, Diniz L de F, Mosqueira VCF. Therapeutical approaches
inhibitors cure an experimental Trypanosoma cruzi infection. J Exp
Med 1998;188:725–34.
under investigation for treatment of Chagas disease. Expert Opin 35. Veiga-Santos P, Barrias ES, Santos JFC, et al. Effects of amiodarone
Investig Drugs 2014;23:1225–37.
5. Cencig S, Coltel N, Truyens C, Carlier Y. Evaluation of
and posaconazole on the growth and ultrastructure of Trypanosoma
cruzi. Int J Antimicrob Agents 2012;40:61–71.
benznidazole treatment combined with nifurtimox, posaconazole 36. Kessler RL, Soares MJ, Probst CM, Krieger MA. Trypanosoma
Õ
or AmBisome in mice infected with Trypanosoma cruzi strains.
Int J Antimicrob Agents 2012;40:527–32.
6. Dumonteil E, Bottazzi ME, Zhan B, et al. Accelerating
cruzi
response
to
sterol
biosynthesis
inhibitors:
morphophysiological alterations leading to cell death. PLoS One
2013;8:e55497.
1
the development of a therapeutic vaccine for human Chagas 37. Soeiro MNC, Werbovetz K, Boykin DW, et al. Novel amidines and
analogues as promising agents against intracellular parasites: a
systematic review. Parasitology 2013;140:1–23.
disease: rationale and prospects. Expert Rev Vaccines 2012;11:
043–55.
1

1
0
D. d. R. de Menezes et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
3
8. Santa-Rita RM, Barbosa HS, de Castro SL. Ultrastructural analysis 40. Batista DGJ, Pacheco MGO, Kumar A, et al. Biological,
of edelfosine-treated trypomastigotes and amastigotes of
Trypanosoma cruzi. Parasitol Res 2006;100:187–90.
9. Menna-Barreto RFS, Goncalves RLS, Costa EM, et al. The effects
ultrastructural effect and subcellular localization of aro-
matic diamidines in Trypanosoma cruzi. Parasitology 2010;137:
251–9.
3
on Trypanosoma cruzi of novel synthetic naphthoquinones are 41. Sajid M, Robertson SA, Brinen LS, McKerrow JH. Cruzain: the path
mediated by mitochondrial dysfunction. Free Radic Biol Med 2009;
7:644–53.
from target validation to the clinic. Adv Exp Med Biol 2011;712:
100–15.
4