Semicarbazone derivatives as promising therapeutic alternatives in leishmaniasis

Experimental Parasitology 201 (2019) 57–66

Contents lists available at ScienceDirect

Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr

Semicarbazone derivatives as promising therapeutic alternatives in leishmaniasis

T

Aline Cavalcanti de Queiroza,b,d,1, Marina Amaral Alvesb,c,1, Eliezer Jesus Barreirob,c,2, Lídia Moreira Limab,c,2, Magna Suzana Alexandre-Moreiraa,b,∗,2 a

Laboratório de Farmacologia e Imunologia- Universidade Federal de Alagoas, Laboratório de Farmacologia e Imunidade, Instituto de Ciências Biológicas e da Saúde, Av. Lourival Melo Mota, s/n, Tabuleiro do Martins, CEP:57072-900, Maceió – AL, Brazil b Instituto Nacional de Ciência e Tecnologia de Fármacos e Medicamentos (INCT-INOFAR)3. Universidade Federal do Rio de Janeiro, Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio®)4 CCS, Cidade Universitária, P.O. Box 68024, ZIP, 21941-971, Rio de Janeiro-RJ, Brazil c Programa de Pós-graduação em Química- Instituto de Química- UFRJ, Brazil d Universidade Federal de Alagoas, Campus Arapiraca, Av. Manoel Severino Barbosa, Bom Sucesso, CEP: 57309-005, Arapiraca-AL, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Neglected diseases Leishmaniasis Leishmania spp. Semicarbazone Apoptosis Autophagy

In the present study, we investigated the in vitro and in vivo leishmanicidal activity of synthetic compounds, containing a semicarbazone scaffold as a peptide mimetic framework. The leishmanicidal effect against amastigotes of Leishmania amazonensis was also evaluated at concentration of 100 μM–0.01 nM. The derivatives 2e, 2f, 2g and 1g, beyond the standards miltefosine and pentamidine, significantly diminished the number of L. amazonensis amastigotes in macrophages. These derivatives were also active against amastigotes of L. braziliensis. As 2g presented potent leishmanicidal activity against the amastigotes of L. amazonensis in macrophages, we also investigated the in vivo leishmanicidal activity of this compound against L. amazonensis. Approximately 105 L. amazonensis promastigotes were subcutaneously inoculated into the dermis of the right ear of BALB/c mice, which were subsequently treated with 2g (p.o. or i.p.), miltefosine (p.o.) or glucantime (i.p.) at 30 μmol/kg/day x 28 days. Thus, a similar reduction in the lesion size was observed after the administration of 2g through oral (63.7 ± 10.1%) and intraperitoneal (61.8 ± 3.7%) routes. A larger effect was observed after treatment with miltefosine (97.7 ± 0.4%), and glucantime did not exhibit activity at the dose administered. With respect to the ear parasite load, 2g diminished the number of parasites by p.o. (30.5 ± 5.1%) and i.p. (33.3 ± 4.3%) administration. In addition, 2g induced in vitro apoptosis, autophagy and cell cycle alterations on L. amazonensis promastigotes. In summary, the derivative 2g might represent a lead candidate for antileishmanial drugs, as this compound displayed pronounced leishmanicidal activity.

1. Introduction Leishmaniasis is a major public health problem, causing morbidity and mortality in tropical and subtropical regions worldwide. The WHO estimated that approximately 0.9–1.6 million cases of leishmaniasis occur each year in 98 countries (Alvar et al., 2012). This disease is caused by a protozoan of the genus Leishmania, transmitted through the bite of the sand fly vector. This group of parasitosis is characterized by symptoms ranging from localized cutaneous lesions to mucocutaneous

tissue destruction and frequently fatal visceral formations (Chappuis et al., 2007). In relation to American tegumentary leishmaniasis, the species Leishmania braziliensis and Leishmania amazonensis possess the highest epidemiological and medical importance, because they can cause severe forms of cutaneous leishmaniasis (Silveira et al., 2009). L. braziliensis is the most common species in the New World and the most important causative agent of cutaneous and mucocutaneous leishmaniasis, while L.amazonensis may cause the disseminated or diffuse forms

∗

Corresponding author. Laboratório de Farmacologia e Imunidade, Setor de Fisiologia e Farmacologia, Instituto de Ciências Biológicas e da Saúde, Universidade Federal de Alagoas, Av. Lourival Melo Mota, s/n, Cidade Universitária, Maceió, AL, CEP, 57072-900, Brazil. E-mail addresses: [email protected], [email protected] (M.S. Alexandre-Moreira). 1 These authors contributed equally to this work. 2 These authors also contributed equally to this work. 3 http://www.inct-inofar.ccs.ufrj.br/ 4 http://www.lassbio.icb.ufrj.br/ https://doi.org/10.1016/j.exppara.2019.04.003 Received 23 August 2018; Received in revised form 9 March 2019; Accepted 12 April 2019 Available online 17 April 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

BA788) were obtained from Dr. Valéria de Matos Borges (Gonçalo Moniz Research Center, Fiocruz-BA). The parasites were maintained in vitro in Schneider's medium, supplemented with 10% FBS and 2% human urine at 27 °C in a BOD incubator.

of the disease (Zauli-Nascimento et al., 2010; Carvalho et al., 2019). The current therapy for this infection is associated with severe side effects, long-term treatment and high costs (Sundar and Chakravarty, 2013). Therefore, new, efficient, cheap and safe alternatives for the treatment of leishmaniasis are greatly needed and several compounds, including synthetic, natural products extracted from plants and marine sources have been studied, showing different degrees of efficacy in the treatment of this disease (Tiuman et al., 2011; Sen and Chatterjee, 2011; Tempone et al., 2011). Among the synthetic class of compounds described as potential leishmanicidal agents, semicarbazone is noteworthy. This scaffold can be considered a privileged structure, observed in the structures of several active compounds with different pharmacological activities, such as antimicrobials, pesticides, herbicides, hypnotics, anticonvulsants, anti-hypertensive drugs (Beraldo and Gambino, 2004), antiprotozoals (Greenbaum et al., 2004), antitumorals (Chorev and Goodman, 1993), antibacterials (Dobek et al., 1980) and antivirals (Lam et al., 1994). Recently, Alves et al. (2015) reported the design and synthesis of compounds containing a semicarbazone scaffold as a peptide mimetic framework. In the referredstudy, the cytotoxic activity against L. major (promastigote and amastigote forms) and T. cruzi (epimastigote form) was evaluated, and the results further demonstrated an adequate in silico drug-likeness profile and enhanced chemical and plasma stability for compound LASSBio-1483.Specifically, the present study describes the activity of semicarbazone derivatives (1a-h and 2a-h, Fig. 1) on intracellular amastigotes of L. amazonensis and L. braziliensis to characterize the underlying mechanism and in vivo activity of semicarbazone 2g (LASSBio-1483) using a cutaneous leishmaniasis BALB/c mice model infected with L. amazonensis.

2.3. J774.A1 murine macrophage culture The adherent-phenotype murine macrophage line, J774.A1, was cultured in Dulbecco's Modified Eagle's medium (DMEM, Sigma) supplemented with 10% FBS at 37 °C in 95% humidity and 5% CO2. 2.4. In vitro activity against amastigote forms of Leishmania spp Inicially, it was realized a screnning test against intracellular amastigotes of L. amazonensis at 30 μM. After, the most activies compounds were selected to study the concentration response curve against the amastigote stages of L. amazonensis and L. braziliensis. To assess the activity of the test compounds against the amastigote stages of L. amazonensis and L. braziliensis, a cell model of infection was generated on coverglass (Nunes et al., 2005). The murine macrophages (J774.A1 cell line) were prepared in 24-well vessels (Corning) at 2 × 105 adherent cells/well, subsequently infected with 2 × 106 promastigotes on glass coverslips and placed in 1 mL of culture. The cells were cultured in the presence or absence of test compounds (1a-h and 2a-h) or reference drugs (pentamidine or miltefosine) at 0.01–100 μM, and maintained for 24 h at 37 °C, 5% CO2. After 24 h, the coverslips were washed, stained with Giemsa-MayGrünwald, and the intracellular amastigotes were counted in 100 macrophages. The data obtained from in vitro experiments were expressed as the means ± S.E.M. of duplicate cultures of representative assays. Significant differences between the treated and control groups were evaluated using ANOVA and Dunnett hoc tests. Differences with a p value < 0.05 or lower were considered significant.

2. Material and methods 2.1. Chemistry

2.5. Analysis of phospholipid externalization

The semicarbazone derivatives were synthesized again according to Alves et al. (2015). 8 The semicarbazone compounds obtained were subjected to in vitro and in vivo activity assays.

In addition, double staining with annexin V-PE and 7-AAD was performed to measure the effects of pentamidine or compound 2g (100 and 10 μM) on the plasma membrane of Leishmania promastigotes. The expression of phospholipids in the outer membrane of treated and untreated L. amazonensis promastigotes was monitored after labeling with annexin V-PE, and staining with 7-AAD was used to measure the permeability of the plasma membrane. L. amazonensis promastigotes were grown to 1 × 105 cells/ml and subsequently treated with pentamidine or compound 2g (100 and 10 μM) and incubated at 26 °C. After 48 h, 1 mL of the culture was pelleted and resuspended in 1 mL of PBS buffer supplemented with 1% FBS, followed by the addition of annexin V-PE and 7-AAD. The cells were incubated for 30 min in the dark prior to analysis by flow cytometry using a Muse® Cell Analyzer and Muse™ 1400 Analysis software.

2.2. Parasite culture Promastigotes of L. amazonensis (MHOM/BR/77/LTB0016) were obtained from Dr. Eduardo Caio Torres dos Santos (Oswaldo Cruz Institute - Fiocruz). Promastigotes of L. braziliensis (MHOM/BR/87/

2.6. Estimation of mitochondrial transmembrane electric potential To determine changes in the mitochondrial membrane potential, we used the Muse®MitoPotential Kit according to the manufacturer's instructions. The Muse®MitoPotential Kit utilizes MitoPotential reagent to detect changes in the mitochondrial membrane potential. A dead cell marker was also used as an indicator of cell membrane structural integrity, as this marker is excluded from live, healthy cells and early apoptotic cells. Quantitative data (percentages and concentrations) was generated for 4 populations of cells: live, depolarized, depolarized/dead and dead cells. L. amazonensis promastigotes were grown to 1 × 105 cells/ml and subsequently treated with pentamidine or compound 2g (100 and 10 μM) and incubated at 26 °C. After 48 h, 1 mL of the culture was pelleted and resuspended in 1 mL of PBS buffer supplemented with 1% FBS, followed by the addition of Muse®

Fig. 1. Structures of the semicarbazone derivatives 1a-h and 2a-h. 58

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

MitoPotential reagent and 7-AAD. The cells were incubated for 30 min in the dark prior to analysis using flow cytometry using a Muse® Cell Analyzer and Muse™ 1400 Analysis software. 2.7. Determination of caspase-like proteases To determine the percentage of caspase-positive cells, we used the Muse®Multicaspase Kit in accordance with the manufacturer's instructions. L. amazonensis promastigotes were grown to 1 × 105 cells/ml and subsequently treated with pentamidine or compound 2g (100 and 10 μM) and incubated at 26 °C. After 48 h, 1 mL of the culture was pelleted and resuspended in 1 mL of PBS buffer supplemented with 1% FBS. Subsequently, Muse® Multicaspase Reagent and 7-AAD were added to the tubes. Data acquisition was performed using a Muse® Cell Analyzer, followed by analysis using Muse®1400 Analysis software. 2.8. Determination of presence of autophagic LC3 Fig. 2. Effects of 30 μM of Pentamidine, Miltefosine, LASSBio-1064 and semicarbazone derivatives (1a-h and 2a-h) against amastigote forms of L. amazonensis.

To determine the percentage of caspase-positive cells, we used a Muse® Autophagy LC3 antibody-based kit, according to the manufacturer's instructions. L. amazonensis promastigotes were grown to 1 × 105 cells/ml and subsequently treated with pentamidine or compound 2g (100 and 10 μM) and incubated at 26 °C. After 48 h, 1 mL of the culture was pelleted and resuspended in 1 mL of PBS buffer supplemented with 1% FBS. The cells were permeabilized, and subsequently the Fluor®555-conjugated anti-LC3 Alexa antibody was added. Data acquisition was performed using a Muse® Cell Analyzer, followed by analysis using Muse™ 1400 Analysis software.

(LASSBio-1201), 1d (LASSBio-1203), 1e (LASSBio-1206), 1f (LASSBio1210), 1g (LASSBio-1302), 1h (LASSBio-1303), 2a (LASSBio-1487), 2b (LASSBio-1701), 2c (LASSBio-1490), 2d (LASSBio-1489),2e (LASSBio1486),2f (LASSBio-1488), 2g (LASSBio-1483) and 2h (LASSBio-1699) were assessed against amastigotes (intramacrophage parasite) of L. amazonensis. The standard drugs (miltefosine and pentamidine) and semicarbazone compounds were first evaluated at a screening concentration of 30 μM. As depicted in Fig. 2, only compounds 1g, 2a, 2e, 2f and 2g diminished the number of intracellular amastigotes. Therefore, these compounds were selected to study the concentration response curve. As demonstrated in Table 1, compound 2a presented an IC50 value > 100 μM, while the other selected compounds were more active, particularly semicarbazone 2g (LASSBio-1483), which was more potent than miltefosine and pentamidine. Derivatives 1g, 2e, 2f and 2g and the standard drugs miltefosine and pentamidine decreased the number of intracellular amastigotes of L. amazonensis per macrophage, with IC50values of 23.8 ± 0.3,16.7 ± 2.3, 39.8 ± 8.2, 3.5 ± 0.6, 22.0 ± 1.8 and 32.8 ± 4.6 μM, respectively (Table 1). The maximum cytotoxic effects of 71.6 ± 1.6, 81.5 ± 1.6, 66.1 ± 3.4, 93.2 ± 2.0, 59.1 ± 5.6 and 58.1 ± 4.9% were observed for compounds 1g, 2e, 2f and 2g, miltefosine and pentamidine, respectively (Table 1). In addition, it was verified that 2g presented selectivity index (SI) greater than 28.6, indicating high selectivity against intramacrophage parasite of L. amazonensis. Notably, 2g (LASSBio-1483) was not only more potent, but this compound was also more effective than the standard drugs miltefosine and pentamidine. Thus, the leishmanicidal activity against L. braziliensis was also determined. As shown in Table 1, compound 2g (LASSBio1483, IC50 = 31.7 ± 5.8 μM) was equipotent to pentamidine (IC50 = 32.1 ± 1.1 μM) and more potent than miltefosine (IC50 = 78.4 ± 4.7 μM). Moreover, compounds 2a (IC50 = 2.7 ± 0.3 μM) and 2e (IC50 = 4.2 ± 1.1 μM) were the most potent against amastigotes of L. braziliensis. Likewise, compound 2a presented high selectivity against this Leishmania specie, with SI > 37.0. However, 2e (SI equal to 8.5) was less selective than 2a against L. braziliensis.

2.9. Cell cycle progression analysis Promastigotes of L. amazonensis (1 × 105 cells) were treated with pentamidine and 2g at 100 or 10 μM for 48 h. Subsequently, the cells were fixed in chilled 70% ethanol for 3 h. After washing the cells in PBS, the pellet was resuspended in 7-AAD and incubated in the dark for 30 min. Data acquisition was performed using a Muse® Cell Analyzer, followed by analysis using Muse® 1400 Analysis software. 2.10. In vivo activity against L. amazonensis This study (protocol number 2013.02) was approved by the Ethics Committee for Animal Experimentation of the Federal University of Alagoas (Brazil). All animals received humane care in compliance with the ‘Principles of laboratory animal care’ according to the National Society for Medical Research and the ‘Guide for the care and use of laboratory animals’ of the National Academy of Sciences (Washington, DC). A total of 105 stationary promastigotes (5 days of culture in Schneider's medium) of L. amazonensis were subcutaneously inoculated into the dermis of the right ear of 6-week-old female BALB/c mice weighing ca. 20 g and subsequently treated with compound 2g (i.p. or p.o.), miltefosine (p.o.) or meglumine antimoniate (i.p.) at 30 μmol/kg x 28 days. The lesion size was measured using a paquimeter (Pereira et al., 2010). The parasite loads of infected ears and draining lymph nodes were determined using a quantitative limiting-dilution assay (Taswell, 1986). Toxicity was also evaluated after measuring alterations in spleen weight and biochemistry dosages in the plasma according to the manufacturer's instructions (Doles, BRA). The data were expressed as the means ± S.E.M., and significant differences between the treated and vehicle groups were evaluated using ANOVA and Dunnett hoc tests.

3.2. Phospholipid externalization and cellular membrane integrity 3. Results The promising compound 2g selected to further study its mechanismof action, because it was the most active derivative against L. amazonensis, and presented high activity agains amastigotes of L. braziliensis (Table 1) and L. major (Alves et al., 2015). First, this compound was subjected to flow cytometric assay to evaluate its potential for

3.1. In vitro leishmanicidal activity The in vitro evaluations of the anti-leishmanial activity of semicarbazone compounds1a (LASSBio-1200), 1b (LASSBio-1205), 1c 59

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

Table 1 Determination of the cytotoxicity of semicarbazone derivatives against amastigote forms of L. amazonensis and L. braziliensis. Substance

Amastigotes of L. amazonensis a

Miltefosine Pentamidine LASSBio 1064 1g (LASSBio-1302) 2a (LASSBio-1487) 2e (LASSBio-1486) 2f (LASSBio-1488) 2g (LASSBio-1483)

32.8 ± 4.6 > 100 23.8 ± 0.3 > 100 μM 16.7 ± 2.3 39.8 ± 8.2 3.5 ± 0.6

IC50 ± S.E.M.) (μM)

Maximum cytotoxicity (% ± S.E.M.)

22.0 58.1 NA 71.6 35.6 81.5 66.1 93.2

59.1 ± 5.6*** > 3.0 – > 4.2 – 2.1 > 2.5 > 28.6

± 1.8 ± 4.9*** ± ± ± ± ±

1.6*** 0.5** 1.6*** 3.4*** 2.0***

Amastigotes of L. braziliensis b

Selectivity index (SI) > 4.5 32.1 ± 1.1 > 100 89.7 ± 5.8 2.7 ± 0.3 4.2 ± 1.1 81.5 ± 5.9 31.7 ± 5.8

c

IC50 a ± S.E.M.) (μM)

Maximum cytotoxicityb (% ± S.E.M.)

Selectivity indexc (SI)

78.4 84.0 NA 52.9 74.6 74.6 56.7 65.8

61.7 ± 2.2** > 3.1 – > 1.1 > 37.0 8.5 > 1.2 > 3.1

> 1.3

± 4.7 ± 1.5** ± ± ± ± ±

2.4** 2.8** 8.8** 2.4** 1.2**

The data are reported as the means ± S.E.M. The asterisks denote the levels of significance compared with the control groups. Differences with ***P < 0.001 were considered significant with respect to the DMSO 0.1% group. NA: The compound is not active at 100 μM.a: IC50 is the concentration required for 50% inhibition of the growth of amastigote forms, calculated using the linear regression analysis from the Kc values at the employed concentrations (100, 10, 1, 0.1 and 0.01 μM).b: the maximum cytotoxicity is the percentage of death observed at 100 μM, the major concentration tested in this study.c:selectivity index against parasite in relation hostcell.

Fig. 3. Analysis of L. amazonensis promastigotes death using flow cytometry after treatment with pentamidine and 2g for 48h. (A) Phospholipid externalization; (B) Plasma membrane permeabilization by necrosis; (C) Measurement of the mitochondrial membrane potential; (D) Determination of the presence of active caspase-like proteases; (E) Determination of the presence of autophagic LC3; (F)Cell cycle analysis. The values represent the means ± SEMs of three samples.

parasites did not suffer cell membrane damage and likely did not undergo necrosis. The degree of 7-AAD-stained in medium and DMSO 0.1% controls groups of cells were 0.05 and 0.13%, respectively. These results of untreated cells controls were not significantly higher than that of unmarked 7-AAD cells (0.02%), presenting p > 0.05 (data not shown), confirming that the cell membranes remained intact.

induction of apoptosis or necrosis in Leishmania amazonensis parasites. Accordingly, the promastigote forms of L. amazonensis was treated with 2g or pentamidine at the concentrations of 100 μM and 10 μM for 48 h. To further characterize parasite death, the promastogotes were costained with Annexin V and 7-AAD. and then analyzed by flow cytometry. The promastigotes that treated with compound 2g at 100 μM and 10 μM indicated 52.42 and 70.91% of cells with surface binding of annexin V after 48 h, respectively. While only 1.75 and 2.55% of apoptosis was detected in the medium or DMSO 0.1% controls (untreated cells), pentamidine at 100 μM and 10 μM showed 64.06 and 63.95% of apoptosis after 48 h incubation, respectively (Fig. 3A). Therefore, the results also demonstrated that compound 2g did not induce unregulated necrosis. Treatment with 2g at 100 and 10 μM for 48 h did not alter the percentage of cells only labeled with 7-AAD (0.03 and 0.05%, respectively), such as shown Fig. 3B, demonstrating that the

3.3. Depolarization of mitochondrial membrane potential The compound 2g also presented effect on mitochondrial of the parasite. In promastigotes of L. amazoenensis, data indicate that the compound 2g caused a sustained decrease on mitochondrial transmembrane potential (Fig. 3C). The incubation with 2g at 100 or 10 μM exhibited a higher number of cells (85.67 and 73.02%, respectively) with low mitochondrial transmembrane potential compared to medium 60

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

4. Discussion

(28.67%) or DMSO 0.1% (28.06%) controls. Pentamidine also induced an inhibition in mitochondrial transmembrane potential by 83.76 and 36.35% at 100 and 10 μM, respectively.

Semicarbazones present a wide range of biological activities, including antiprotozoal effects. Generally, the mechanisms of these compounds involve enzyme inhibition through complex formation with endogenous metals, redox reactions, DNA interactions or DNA synthesis inhibition (Beraldo and Gambino, 2004). The leishmanicidal activity of the semicarbazones examined in the present study is consistent with that observed in a recent report showing the leishmanicidal activity of semicarbazones and vanadium complexes against the promastigotes and amastigotes of L. panamensis and L. chagasi with IC50 values ranging from 1.33 to 33.39 μM and high parasite/mammalian cell selectivity (Benítez et al., 2011). Greenbaum et al. (2004) suggested that the mechanisms of semicarbazone are complex and might involve multiple targets, such as enzymes, redox reactions, DNA binding or DNA synthesis inhibition. Previous studies on semicarbazones and thiosemicarbazones derivatives have proposed the use of these compounds for the treatment of leishmaniasis, trypanosomiasis and malaria, reflecting the ability of these compounds to inhibit the cysteine proteases of protozoan parasites (i.e., Leishmania sp., Trypanosoma sp. and Plasmodium sp.) (Cohen et al., 2002). Recently, Alves et al. described a series of semicarbazones (1a-h and 2a-h) designed as new peptide mimetic derivatives enclosing frameworks for recognized by trypanosomatids proteases (Alves et al., 2015). Among the studied compounds, the semicarbazone 2g (LASSBio-1483) showed dual in vitro trypanosomicidal and leishmanicidal activities with potency similar to the standards drugs nifurtimox and pentamidine. Considering these results, we examined the leishmanicidal activity of semicarbazones 1a-h and 2a-h against L. amazonensis and L. braziliensis in BALB/c mice infected with L. amazonensis to characterize the effects of compound 2g (LASSBio-1483) and obtain information about the mechanism and toxicity of this compound. The comparison between series 1a-h and 2a-h revealed the absence of leishmanicidal activity for compounds 1a-h, bearing a 1,3-benzodioxole ring as a substituent of semicarbazone framework, except compound 1g (LASSBio-1302). In contrast, a greater number of active compounds were observed in series 2a-h, presenting the 4-chlorophenyl subunit as a substituent of semicarbazone moiety. From this initial study, compounds 2g, 2a, 2e, 2f and 2g were selected to determine the concentration-response curves associated with the effects of these compounds. As expected, compound 2g (LASSBio-1483) showed enhanced leishmanicidal activity against amastigotes of L. amazonensis, which were six- and nine-fold more potent than miltefosine and pentamidine, respectively. The leishmanicidal activity of 2g against amastigotes of L. braziliensis was also enhanced, showing equal potency to pentamidine and two-fold more potency than miltefosine. Surprisingly, compounds 2a and 2e presented significant leishmanicidal activity against amastigotes of L. braziliensis, with potency eighteen- and twenty-nine fold superior to miltefosine, respectively. Compound 1g was the only active compound in series 1a-h with the same substituent associated with imine (N=CH) function presented in the same semicarbazone framework as compound 2g (LASSBio-1483). Indeed, the 5-nitrofuran substituent is a common subunit observed in the structure of antibacterial, antifungal and antiprotozoal prototypes or drugs, such as nitrofurazone and nifurtimox (Pires et al., 2001; Muelas-Serano et al., 2002). Nitroheterocyclics, such as those presented in 1g and 2g, have significant biological redox activity leading to the potential cytocidal production of ROS (Kovacic and Becvar, 2000; Kovacic et al., 2005). Likewise, the increase in ROS leads to damage in mitochondrial membrane potential which is accompanied by cell stress and death (Vishwakarma et al., 2016), corroborating with the fact that 2g induced mitochondrial dysfunction (Fig. 3C). This effect of the compound on the mitochondrial function of L. amazonensis may active mitophagy, an autophagy like selective process which may be occurring in an attempt to degrade the mitochondria (Antinarelli et al., 2018).

3.4. Caspase-like proteases detection The mechanism of apoptosis-like cell death that was induced in promastigotes of Leishmania amazonensis by 2g was also evaluated by caspase-like proteases detection (Fig. 3D). This another marker of apoptosis was unaffected at the lower concentration tested of 2g or pentamidine. At 100 μM, compound 2g also increased the percentage of caspase-positive L. amazonensis promastigotes (64.15%), such as pentamidine (40.22%). 3.5. Determination of presence of autophagic LC3 In the present study, the Alexa Fluor®555-conjugated anti-LC3 antibody was used to evaluate potential autophagic programmed cell death in promastigotes treated with 2g at 100 and 10 μM for 48 h using flow cytometry analysis (Fig. 3E). At 100 μM, compound 2g exacerbated the autophagy process (more than 50%). 3.6. Cell cycle analysis Furthermore, the cell cycle distribution was analyzed by flow cytometry after Leishmania treatment with 2g at 100 and 10 μM for 48 h. To evaluate changes in the promastigote cell cycle induced by treatment with 2G or pentamidine, the parasites were stained with 7-AAD and analyzed using flow cytometry. As depicted in Fig. 3F, 2g induced significant changes in the cell cycle of promastigotes of L. amazonensis at 10 μM. After 48 h of incubation, the majority of treated parasite cells with 2g at 10 μM were arrested on G0/G1 phase of cell cycle (42.85%), opposite to what occurs in untreated controls medium (29.86%) and DMSO 0.1% (27.73%). Fig. 3F also shows that pentamidine at 100 and 10 μMdecreased the proportion to 16.36% and 15.15% of cells in the G2/M phase, respectively, compared with not treated cells in medium (23.50%) and DMSO 0.1% (24.30%) experimental groups. 3.7. Leishmanicidal activity in BALB/c mice infected with L. amazonensis The in vivo leishmanicidal activity was investigated, using BALB/c mice infected with L. amazonensis in accordance with the methodology of Pereira et al. (2010). This murine model was chosen for the in vivo assays due to the variety of clinical forms of leishmaniasis that this species can cause in the New World (Granato et al., 2018). Moreover, in the case of mice infected with L. amazonensis, BALB/c mice develop progressive lesions that tend to be more ulcerating compared with L.braziliensis (Sacks and Melby, 2015). In this model, the infected animals were treated with 30 μmol/kg/ day of compound 2g (LASSBio-1483) and the drug standards miltefosine and glucantime for 28 days. The cutaneous lesions of the external ear were similarly reduced through the oral (63.7 ± 10.1%) and intraperitoneal (61.8 ± 3.7%) administration of compound 2g (LASSBio1483) (Fig. 4). A better effect was observed after treatment with miltefosine (97.7 ± 0.4%), while glucantime did not exhibit activity. Regarding the ear parasite load (Fig. 5), compound 2g reduced the number of parasites by p.o. (30.5 ± 5.1%) and i.p. (33.3 ± 4.3%) administration. Only miltefosine treatment decreased the parasite load from draining lymph nodes. Concerning toxicological issues, LASSBio-1483 (2g) did not induce alterations in the plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and urea (Fig. 6) of animals treated with 30 μmol/kg/dayof compound 2g for 28 days. 61

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

Fig. 4. In vivo efficacy of 2g, miltefosine and Glucantime treatments (30 μmol/kd/day x 28 days) in BALB/c mice infected with L. amazonensis.A) Progression of the lesion thickness of infected ears.Lesion sizes were monitored weekly. The values are presented as the mean lesion sizes of five mice in each group, and the bars represent the standard error of the mean. B) Macroscopic evaluation of the lesions in untreated and treated mice at 28 days post-infection.

Interestingly, 2g also induced apoptosis-like cell death in L. amazonensis promastigotes at 100 and 10 μM after 48 h (the treatment promoted surface binding of annexin V and loss of mitochondrial transmembrane potential) (Fig. 3A and C). The term apoptosis describes the biochemical processes and morphological features leading to controlled cellular self-destruction (Jiménez-Ruiz et al., 2010), and this type of cell death is now considered a prerogative of unicellular organisms, including the trypanosomatids of the genera Leishmania spp. Once apoptosis is triggered, a cascade of events common to mammalian apoptosis occurs, such as the exposition of phospholipids in the outer leaflet of the plasma membrane (Sudhandiran and Shaha, 2003). Moreover, mitochondrial dysfunction is also one of the hallmarks of apoptosis frequently associated with changes in mitochondrial membrane potential, a key indicator of mitochondrial function that might either reflect a consequence or an early requirement for apoptosis (Ly et al., 2003). In trypanosomatids, both an increase and a decrease in respiration and both hyperpolarization and the loss of mitochondrial membrane potential might be associated with apoptosis, demonstrating the importance of the maintenance of proper mitochondrial

transmembrane potential in these parasites (Mehta and Shaha, 2004). Together with mitochondrial injury, caspase-like activation occurs in 2g (LASSBio-1483)-mediated apoptosis (Fig. 3D). Caspases are the primary proteases activated during mammalian apoptosis, mediating the cleavage of a variety of proteins, ultimately leading to cell death (Li and Yuan, 2008). However, the mechanisms concerning life or death decisions in protozoan parasites remain imperfectly understood (Meslin et al., 2011). Comparisons with higher eukaryotes suggest that caspaselike enzymes could be involved in death pathways, however Leishmania does not encode caspase(s) (Carmona-Gutierrez et al., 2010). Moreover, the caspase-like activity might be performed by some other unrelated protease that remains unidentified in Leishmania (Ivens et al., 2005). Caspase-3/7-like activation can be induced by a number of stimuli, despite the absence of genes encoding caspases within the L. major genome. However, the trigger for the activation of these caspases is not clear in the Leishmania system, but might involve the leakage of cytochrome c from mitochondria and the activation of a caspase-9-like enzyme to activate caspase-3/7, or alternatively, the caspase-8-like or caspase-12 enzyme activation could induce caspase-3/7 activation 62

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

antiproliferative activity of this semicarbazone derivative likely reflects an exacerbated autophagic process (Fig. 3E). The visualization of autophagosomes in dying cells suggests that autophagy is a nonapoptotic form of programmed cell death (Levine and Yuan, 2005). Macroautophagy, generally known as autophagy (Klionsky et al., 2012), is an intracellular catabolic mechanism for the degradation of long-lived proteins and organelles and the recycling of their constituents. Despite the plethora of pro-survival roles for autophagy, under certain circumstances cells also execute a specific regulated cell death subtype known as autophagic cell death. Indeed, autophagic cell death represents a potential protozoan-regulated cell death modality. The cellular apparatus required for autophagy is widely conserved among parasitic protozoa (Duszenko et al., 2011). Consistent with other organisms, parasitic protozoa undergo autophagy as a response to nutrient starvation (Williams et al., 2012). Autophagy also directly influences parasite virulence through cellular remodeling during life cycle differentiation in Leishmania spp. and the maintenance of mitochondrial function in L. major (Williams et al., 2006; Bera et al., 2003). On the other hand, some studies suggest that autophagy can precede or even activate apoptosis, by causing the depletion of apoptosis endogenous inhibitors or the activation of caspases (Mariño et al., 2014). In addiction, the mitochondria can represent a nexus at which autophagy and apoptosis pathways may interact (Levine and Yuan, 2005; Williams et al., 2012).Similar to the semicarbazone 2g (LASSBio-1483), several compounds have also been demonstrated to cause alterations in Leishmania associated with autophagy and apoptosis. For example, L. chagasi and L. amazonensis promastigotes treated with yangambin also showed the ultrastructural features of both apoptosis and autophagy (Monte-Neto et al., 2011). L. amazonensis parasites treated with amiodarone presented alterations in the mitochondrial structure and function, which culminate in cell death by apoptosis and autophagy (DeMacedo-Silva et al., 2011). Antimicrobial peptides also induced the death of L. donovani promastigotes with the appearance of vacuoles stained with monodansylcadaverine, a biochemical marker of autophagy. Moreover, the aziridine-2,3-dicarboxylate-based cysteine cathepsin inhibitor induced cell death in L. major promastigotes, showing the accumulation of debris in autophagy-related lysosome-like vacuoles at an early phase of parasite death, however this death eventually occurred through an apoptosis-like death mechanism (Schurigt et al., 2010). Moreover, such as shown in Fig. 2F, g at 10 μM for 48 h induced a significant increase in the proportion of L. amazonensis promastigotes in the G0/G1 phase, a stage in which the cells only possess one copy of DNA, and induced a marked reduction in DNA replication, with a decrease in the number of cells in S phase, compared with control cells or cells exposed only to vehicle (i.e., DMSO). This result suggests that cells did not undergo DNA synthesis and mitosis in the presence of 2g. Likewise, the cell cycle arrest at the G0/G1 phase have been reported to correlate with apoptosis-like cell death (Proto et al., 2013). Consistent with our data, several studies in the literature have already pointed the induction of similar cell cycle arrest in response to leishmanicidal compounds. For example, it was reported that copper salisylaldoxime and trans-dibenzalacetone also induced apoptosis and cell cycle arrest in L. donovani at G0/G1 phase (Singh et al., 2017; Chauhan et al., 2018). Recently, it was also demonstrated that VOSalophen (a vanadium complex) and AMQ-j (a 4-hydrazinoquinoline derivative) induced autophagy and apoptosis-related processes in L. amazonensis, arrest of the cell cycle in G0/G1 (Machado et al., 2017; Antinarelli et al., 2018). Regarding the exacerbation of autophagy and indiction of apoptosis, our hypothesis is that this compound can act in different ways. First, the compound 2g, designed as new inhibitor of cysteine cathepsin (lysossomal enzyme), leads activation of autophagic-like pathway in L. amazonensis promastigotes, acting through alterations in the mitochondrial function, which increase the production of ROS, culminating in the activation of caspases, cell cycle arrest at G0/G1 phase,

Fig. 5. Parasite burden throughout the course of 2g, miltefosine and Glucantime treatments (30 μmol/kd/day x 28 days) in BALB/c mice infected with L. amazonensis. (A) Log10 of the parasite loads in the infected ears. (B) Log10 of the parasite loads of the draining lymph node. The parasite loads of infected ears and draining lymph nodes were determined using a quantitative limiting-dilution assay. The values represent the mean parasites loads of five mice in each group, and the bars represent the standard error of the mean. *P < 0.05, **P < 0.01 vs. control.

(Chen et al., 2001; Eichler et al., 2006). Thus, the results in this study also suggested that compound 2g induced mitochondrial dysfunction leading to caspase-induced apoptosis cell death in L. amazonensis promastigotes. Another form of regulated cell death is autophagic cell death. In classical apoptosis, or type I programmed cell death, the early collapse of cytoskeletal elements and preservation of organelles until late in the process is observed. In contrast, in autophagy, or type II programmed cell death, the early degradation of organelles and preservation of cytoskeletal elements until late stages is observed (Lockshin and Zakeri, 2004). Treatment with 2g at 100 μM alters the autophagy induction ratio of promastigotes (more than 50%), indicating that the 63

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

Fig. 6. In vivo effect of 2g, miltefosine and Glucantime treatments (30 μmol/kd/day x 28 days) on serum ALT (A), AST (B), creatinine (C) and urea (D) levels in BALB/c mice infected with L. amazonensis. The lesion sizes were monitored weekly. The values represent the mean lesion sizes of five mice in each group, and the bars represent the standard error of the mean. *P < 0.05, **P < 0.01 vs. control.

loads, however, the in vivo leishmanicidal activity of 2g (30 μmol/kg/ day x 28 days, i.p and p.o.) was higher than Glucantime (30 μmol/kg/ day x 28 days, i.p). This pentavalent antimonial is used as a first-line drug for the treatment of leishmaniasis (Goto and Lindoso, 2010), and in the present model, glucantime showed an effect against lesion size but did not decrease parasitemia at the same dose. The lack of a clinical or parasitological response to glucantime in L. amazonensis-infected BALB/c mice has previously been reported (Gonçalves et al., 2005), and a higher dose of this antimonial is necessary to obtain a similar effect of semicarbazone 2g (LASSBio-1483) or miltefosine. Intriguingly, it was observed that 2g induced an in vivo analogous effect in both routes of adminitration used in this study. However, as previously demonstrated by Alves et al. (2015), 2g (LASSBIO-1483) exhibited high chemical stability in buffer solution in pH equal to2 (that simulate gastric juice) or in pH equal to 7.4 (value that mimic serum content), great plasma stability and oral bioavailability (%F) equal to 39%. Taken together, these data suggest that 2g is absorbed by the gastrointestinal tract, but it is metabolized into a pharmacologically active metabolite within the body through first-pass effect. Notably, the first-pass effect may be present in any administration route (except intra-arterial administration), but it is considerably more significant for the oral route (Talevi and Bellera, 2018). This hypothesis explain how the oral treatment was as effective as intraperitoneal administration,

and apoptotic cell death. Second, the biological redox activity of 2g leads to the potential cytocidal production of ROS, a possible mechanisms of this nitroheterocyclic semicarbazone against Leishmania sp. (Greenbaum et al., 2004; Kovacic and Becvar, 2000; Kovacic et al., 2005), which can explain some of the phenotypes observed in 2g treated parasites, especially depolarization of mitochondrial membrane potential, augmentation of autophagy, and membrane phospholipids exposure, because all of them have been reported for Leishmania species as a consequence of ROS action (Alzate et al., 2007; Chowdhury et al., 2014; Williams et al., 2012). Considering the leishmanicidal activity of semicarbazone 2g (LASSBio-1483) against the amastigotes of L. braziliensis and L. amazonensis and the enhanced plasma stability and drug-likeness profile of this compound (Alves et al., 2015), in vivo leishmanicidal activity against L. amazonensis was investigated. The results revealed a significant effect of 2g in the reduction of ear lesions caused by L. amazonensis in BALB/c mice (Fig. 4). The effectiveness was apparent not only as reduced swelling and ulceration in treated animals but also as an important reduction in local parasite burden in infected ears, although no reduction in the parasitemia of the draining lymph node was observed (Fig. 5). Comparisons with standard drugs revealed that miltefosine (30 μmol/kg/day x 28 days, p.o.) uniquely reduced both ear and cervical lymph node parasite 64

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

but other pharmacokinetic studies are needed to understand these findings. Moreover, such as the present study, Palit et al. (2012) demonstrated that PP10, a 4-aminoquinaldine analogue, also presented similiar leishmanicidal activity in both intraperitoneal and oral therapy. The biochemical analysis of the serum from animals treated with LASSBio-1483 (30 μmol/kg/day x 28 days, i.p and p.o.) showed no alteration in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and urea production, indicating that semicarbazone 2g does not have hepato- or renal toxicity. Under the same conditions, glucantime (30 μmol/kg/day x 28 days, i.p) reduced the levels of ALT and AST (Fig. 6). In summary, the effectiveness of new semicarbazone derivatives against the amastigotes of L. amazonensis and L. braziliensis at non-toxic concentrations to the host cell and the in vivo leishmanicidal activity of compound 2g (LASSBio-1483), by i.p. or p.o. administration, in cutaneous leishmaniasis in BALB/c mice infected with L. amazonensis, with no indicative of renal or hepatic toxicity supports further studies with this compound as an additional option to available chemotherapies. These data also highlight perspectives to explore the potential of LASSBio-1483 (2g) as another option for the chemotherapy of leishmaniasis, encouraging the extension of these studies for the treatment of visceral leishmaniasis.

Vanadium polypyridyl compounds as potential antiparasitic and antitumoral agents: new achievements. J. Inorg. Biochem. 105, 303–312. Bera, A., Singh, S., Nagaraj, R., Vaidya, T., 2003. Induction of autophagic cell death in Leishmania donovani by antimicrobial peptides. Mol. Biochem. Parasitol. 127, 23–35. Beraldo, H., Gambino, D., 2004. The wide pharmacological versatility of semicarbazones, thiosemicarbazones and their metal complexes. Mini Rev. Med. Chem. 4, 31–39. Carmona-Gutierrez, D., Eisenberg, T., Buttner, S., Meisinger, C., Kroemer, G., Madeo, F., 2010. Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ. 17, 763–773. Carvalho, S.H., Frézard, F., Pereira, N.P., Moura, A.S., Ramos, L.M.Q.C., Carvalho, G.B., et al., 2019. American tegumentary leishmaniasis in Brazil: a critical review of the current therapeutic approach with systemic meglumine antimoniate and short-term possibilities for an alternative treatment. Trop. Med. Int. Health. https://doi.org/10. 1111/tmi.13210. Chappuis, F., Sundar, S., Hailu, A., Ghalib, H., Rijal, S., Peeling, R.W., et al., 2007. Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat. Rev. Microbiol. 5, 873–882. Chauhan, I.S., Rao, G.S., Shankar, J., Chauhan, L.K.S., Kapadia, G.J., Singh, N., 2018. Chemoprevention of Leishmaniasis: in-vitro antiparasitic activity of dibenzalacetone, a synthetic curcumin analog leads to apoptotic cell death in Leishmania donovani. Parasitol. Int. 67, 627–636. Chen, C.Y., Juo, P., Liou, J.S., Li, C.Q., Yu, Q., Blenis, J., et al., 2001. The recruitment of Fas-associated death domain/caspase-8 in Ras-induced apoptosis. Cell Growth Differ. 12, 297–306. Chorev, M., Goodman, M., 1993. A dozen years of retro-inverso peptidomimetics. Acc. Chem. Res. 26, 266–273. Chowdhury, S., Mukherjee, T., Chowdhury, S.R., Sengupta, S., Mukhopadhyay, S., Jaisankar, P., et al., 2014. Disuccinyl betulin triggers metacaspase-dependent endonuclease G-mediated cell death in unicellular protozoan parasite Leishmania donovani. Antimicrob. Agents Chemother. 58, 2186–2201. Cohen, F.E., Du, X., Guo, C., McKerrow, J.H., 2002. Thio Semicarbazone and Semicarbozone Inhibitors of Cysteine Proteases and Methods of Their Use 7495023 B2 Database: US Patent US. De-Macedo-Silva, S.T., de-Oliveira-Silva, T.L., Urbina, J.A., de-Souza, W., Rodrigues, J.C., 2011. Antiproliferative, ultrastructural, and physiological effects of amiodarone on promastigote and amastigote forms of Leishmania amazonensis. Mol. Biol. Int. 2011, 876021. Dobek, A.S., Klayman, D.L., Dickson Jr., E.T., Scovill, J.P., Tramont, E.C., 1980. Inhibition of clinically significant bacterial organisms in vitro by 2-acetylpyridine thiosemicarbazones. Antimicrob. Agents Chemother. 18, 27–36. Duszenko, M., Ginger, M.L., Brennand, A., Gualdrón-López, M., Colombo, M.I., Coombs, G.H., et al., 2011. Autophagy in protists. Autophagy 7, 127–158. Eichler, T., Ma, Q., Kelly, C., Mishra, J., Parikh, S., Ransom, R.F., et al., 2006. Single and combination toxic metal exposures induce apoptosis in cultured murine podocytes exclusively via the extrinsic caspase 8 pathway. Toxicol. Sci. 90, 392–399. Greenbaum, D.C., Mackey, Z., Hansell, E., Doyle, P., Gut, J., Caffrey, C.R., et al., 2004. Synthesis and structure-activity relationships of parasiticidal thiosemicarbazone cysteine protease inhibitors against Plasmodium falciparum, Trypanosoma brucei, and Trypanosoma cruzi. J. Med. Chem. 47, 3212–3219. Gonçalves, G.S., Fernandes, A.P., Souza, R.C., Cardoso, J.E., de-Oliveira-Silva, F., Maciel, F.C., et al., 2005. Activity of a paromomycin hydrophilic formulation for topical treatment of infections by Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. Acta Trop. 93, 161–167. Granato, J.T., Santos, J.A., Calixto, S.L., Silva, N.P., Martins, J.S., Silva, A.D., et al., 2018. Novel steroid derivatives: synthesis, antileishmanial activity, mechanism of action, and in silico physicochemical and pharmacokinetics studies. Biomed. Pharmacother. 106, 1082–1090. Goto, H., Lindoso, J.A., 2010. Current diagnosis and treatment of cutaneous and mucocutaneous leishmaniasis. Expert Rev. Anti Infect. Ther. 8, 419–433. Ivens, A.C., Peacock, C.S., Worthey, E.A., Murphy, L., Aggarwal, G., Berriman, M., et al., 2005. The genome of the kinetoplastid parasite, Leishmania major. Science 309, 436–442. Jiménez-Ruiz, A., Alzate, J.F., MacLeod, E.T., Lüder, C.G.K., Fasel, N., Hurd, H., 2010. Apoptotic markers in protozoan parasites. Parasites Vectors 3, 104. Klionsky, D.J., Abdalla, F.C., Abeliovich, H., Abraham, R.T., Acevedo-Arozena, A., Adeli, K., et al., 2012. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544. Kovacic, P., Becvar, L.E., 2000. Mode of action of anti-infective agents: focus on oxidative stress and electron transfer. Curr. Pharmaceut. Des. 6, 143–167. Kovacic, P., Pozos, R.S., Somanathan, R., Shangari, N., O'Brien, P., 2005. Mechanism of mitochondrial uncouplers, inhibitors, and toxins: focus on electron transfer, free radicals, and structure-activity relationships. J. Curr. Med. Chem. 12, 2601–2623. Lam, P.Y.S., Jadhav, P.K., Eyermann, C.J., Hodge, C.N., Ru, Y., Bacheler, L.T., et al., 1994. Rational design of potent, bioavailable.nonpeptide cyclic ureas as HIV protease inhibitors. Science 263, 380–384. Levine, B., Yuan, J., 2005. Autophagy in cell death: an innocent convict? J. Clin. Investig. 115, 2679–2688. Li, J., Yuan, J., 2008. Caspases in apoptosis and beyond. Oncogene 27, 6194–6206. Lockshin, R.A., Zakeri, Z., 2004. Apoptosis, autophagy, and more. Int. J. Biochem. Cell Biol. 36, 2405–2419. Ly, J.D., Grubb, D.R., Lawen, A., 2003. The mitochondrial membrane potential (deltapsi (m)) in apoptosis; an update. Apoptosis 8, 115–128. Machado, P.A., Morais, J.O.F., Carvalho, G.S.G., Lima, W.P., Macedo, G.C., Britta, E.A., et al., 2017. VOSalophen: a vanadium complex with a stilbene derivative— induction of apoptosis, autophagy, and efficiency in experimental cutaneous leishmaniasis. J. Biol. Inorg. Chem. 22, 929–939.

Funding This work was supported by the INCT-INOFAR (573.564/2008–6), CNPq (479822/2013–1), CNPq (404344/2012–7), FAPERJ and FAPEAL (PRONEM, 20110722-006-0018-0010). Author Contributions Conceived and designed the experiments: ACQ, EJB, LML and MSAM. Performed the experiments: ACQ and MAA. Analyzed the data: ACQ, MAA, LML and MSAM. Contributed reagents/materials/analysis tools: EJB, LML and MSAM. Drafted the manuscript: ACQ, LML and MAA. Declarations of interest none. Acknowledgments The authors would like to thank Dr. Eduardo Caio Torres dos Santos for kindly providing the promastigotes of L. amazonensis (MHOM/BR/ 77/LTB0016), Dr. Valéria de Matos Borges at Gonçalo Moniz Research for providing L. braziliensis(MHOM/BR/87/BA788). The authors would also like to thank the CAPES, CNPq, INCT-INOFAR, MCTC, FINEP, FAPEAL and FAPERJ. Moreover, the authors would like to thank several colleagues working at the UFAL and UFRJ for constructive criticism of and assistance with this project. References Alvar, J., Vélez, I.D., Bern, C., Herrero, M., Desjeux, P., Cano, J., et al., 2012. Control Team.Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7, e35671. Alves, M.A., Queiroz, A.C., Alexandre-Moreira, M.A., Varela, J., Cerecetto, H., González, M., et al., 2015. Design, synthesis and in vitro trypanocidal and leishmanicidal activities of novel semicarbazone derivatives. Eur. J. Med. Chem. 100, 24–33. Alzate, J.F., Arias, A.A., Moreno-Mateos, D., Alvarez-Barrientos, A., Jiménez-Ruiz, A., 2007. Mitochondrial superoxide mediates heatinduced apoptotic-like death in Leishmania infantum. Mol. Biochem. Parasitol. 152, 192–202. Antinarelli, L.M.R., Souza, I.O., Capriles, P.V.Z., Gameiro, J., Britta, E.A., Nakamura, C.V., et al., 2018. Antileishmanial activity of a 4-hydrazinoquinoline derivative: induction of autophagy and apoptosis-related processes and effectiveness in experimental cutaneous leishmaniasis. Exp. Parasitol. 195, 78–86. Benítez, J., Becco, L., Correia, I., Leal, S.M., Guiset, H., Pessoa, J.C., et al., 2011.

65

Experimental Parasitology 201 (2019) 57–66

A. Cavalcanti de Queiroz, et al.

Silveira, F.T., Lainson, R., Castro-Gomes, C.M., Laurenti, M.D., Corbett, C.E., 2009. Immunopathogenic competences of Leishmania (V.) braziliensis and L. (L.) amazonensis in American cutaneous leishmaniasis. Parasite Immunol. 31, 423–431. Singh, M.K., Bhaumik, S.K., Karmakar, S., Paul, J., Sawoo, S., Majumder, H.K., et al., 2017. Copper salisylaldoxime (CuSAL) imparts protective efficacy against visceral leishmaniasis by targeting Leishmania donovani topoisomerase IB. Exp. Parasitol. 175, 8–20. Sundar, S., Chakravarty, J., 2013. Leishmaniasis: an update of current pharmacotherapy. Expert Opin. Pharmacother. 14, 53–63. Sudhandiran, G., Shaha, C., 2003. Antimonial-induced increase in intracellular Ca2+ through non-selective cation channels in the host and the parasite is responsible for apoptosis of intracellular Leishmania donovani amastigotes. J. Biol. Chem. 278, 25120–25132. Talevi, A., Bellera, C.L., 2018. Drug metabolism. In: Talevi, A., Quiroga, P.A. (Eds.), ADME Processes in Pharmaceutical Sciences: Dosage, Design, and Pharmacotherapy Success. Springer, Gewerbestrasse, pp. 72. Taswell, C., 1986. Limiting dilution assays for the separation, characterization and quantification of biologically active particles and their clonal progeny. In: Pretlow, T.C., Pretlow, T.P. (Eds.), Cell Separation: Methods and Selected Applications. Academic Press, New York, pp. 109–145. Tempone, A.G., Martins-de-Oliveira, C., Berlinck, R.G., 2011. Current approaches to discover marine antileishmanial natural products. Planta Med. 77, 572–585. Tiuman, T.S., Santos, A.O., Ueda-Nakamura, T., Filho, B.P., Nakamura, C.V., 2011. Recent advances in leishmaniasis treatment. Int. J. Infect. Dis. 15, 525–532. Vishwakarma, P., Parmar, N., Yadav, P.K., Chandrakar, P., Kar, S., 2016. 15dProstaglandin J2 induced reactive oxygen species-mediated apoptosis during experimental visceral leishmaniasis. J. Mol. Med. 94, 695–710. Williams, R.A.M., Smith, T.K., Cull, B., Mottram, J.C., Coombs, G.H., 2012. ATG5 is essential for ATG8-dependent autophagy and mitochondrial homeostasis in Leishmania. PLoS Pathog. 8, e1002695. Williams, R.A.M., Tetley, L., Mottram, J.C., Coombs, G.H., 2006. Cysteine peptidases CPA and CPB are vital for autophagy and differentiation in Leishmania mexicana. Mol. Microbiol. 61, 655–674. Zauli-Nascimento, R.C., Miguel, D.C., Yokoyama-Yasunaka, J.K.U., Pereira, L.I.A., Oliveira, M.A.P., Ribeiro-Dias, F., et al., 2010. In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B. Trop. Med. Int. Health 15, 68–76.

Mariño, G., Niso-Santano, M., Baehrecke, E.H., Kroemer, G., 2014. Self-consumption: the interplay of autophagy and apoptosis. Nat. Rev. Mol. Cell Biol. 15, 81–94. Mehta, A., Shaha, C., 2004. Apoptotic death in Leishmania donovani promastigotes in response to respiratory chain inhibition: complex II inhibition results in increased pentamidine cytotoxicity. J. Biol. Chem. 279, 11798–11813. Meslin, B., Zalila, H., Fasel, N., Picot, S., Bienvenu, A.L., 2011. Are protozoan metacaspases potential parasite killers? Parasites Vectors 4, 26. Monte-Neto, R.L., Sousa, L.M., Dias, C.S., Barbosa-Filho, J.M., Oliveira, M.R., Figueiredo, R.C., 2011. Morphological and physiological changes in Leishmania promastigotes induced by yangambin, a lignan obtained from Ocotea duckei. Exp. Parasitol. 127, 215–221. Muelas-Serano, S., Perez-Serrano, J., Gomez-Barroi, A., Aran, V.J., Rodriguez-Caabeiro, F., 2002. Ultrastructural alterations induced by nifurtimox and another nitro derivative on epimastigotes of Trypanosoma cruzi. Parasitol. Res. 88, 97–101. Nunes, M.P., Cysne-Finkelstein, L., Monteiro, B.C., De-Souza, D.M., Gomes, N.A., DosReis, G.A., 2005. CD40 signaling induces reciprocal outcomes in Leishmania-infected macrophages; roles of host genotype and cytokine milieu. Microb. Infect. 7, 78–85. Palit, P., Hazra, A., Maity, A., Vijayan, R.S.K., Manoharan, P., Banerjee, S., et al., 2012. Discovery of safe and orally effective 4-aminoquinaldine analogues as apoptotic inducers with activity against experimental visceral leishmaniasis. Antimicrob. Agents Chemother. 56, 432–445. Pereira, J.C., Carregaro, V., Costa, D.L., da-Silva, J.S., Cunha, F.Q., Franco, D.W., 2010. Antileishmanial activity of ruthenium(II)tetraammine nitrosyl complexes. Eur. J. Med. Chem. 45, 4180–4187. Proto, W.R., Coombs, G.H., Mottram, J.C., 2013. Cell death in parasitic protozoa: regulated or incidental? Nat. Rev. Microbiol. 11, 58–66. Pires, J.R., Saito, C., Gomes, S.L., Giesbrecht, A.M., Amaral, A.T., 2001. Investigation of 5nitrofuran derivatives: synthesis, antibacterial activity, and quantitative structureactivity relationships. J. Med. Chem. 44, 3673–3681. Sacks, D.L., Melby, P.C., 2015. Animal models for the analysis of immune responses to leishmaniasis. Curr. Protoc. Im. 108, 19.2.1–19.2.24. Schurigt, U., Schad, C., Glowa, C., Baum, U., Thomale, K., Schnitzer, J.K., et al., 2010. Aziridine-2,3-dicarboxylate-based cysteine cathepsin inhibitors induce cell death in Leishmania major associated with accumulation of debris in autophagy-related lysosome-like vacuoles. Antimicrob. Agents Chemother. 54, 5028–5041. Sen, R., Chatterjee, M., 2011. Plant derived therapeutics for the treatment of Leishmaniasis. Phytomedicine 18, 1056–1069.

66