Dipeptidyl nitrile derivatives have cytostatic effects against Leishmania spp. promastigotes

Experimental Parasitology 200 (2019) 84–91

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Dipeptidyl nitrile derivatives have cytostatic effects against Leishmania spp. promastigotes

T

José C. Quilles Jr.a, Daiane Y. Tezukaa,b, Carla D. Lopesa,b, Fernanda L. Ribeiroa, Charles A. Laughtonc, Sérgio de Albuquerqued, Carlos A. Montanaria, Andrei Leitãoa,∗ a Medicinal Chemistry Group (NEQUIMED), São Carlos Institute of Chemistry (IQSC), University of São Paulo (USP), Av. Trabalhador São-carlense, 400, São Carlos, SP, 13.566-590, Brazil b Programa de Pós-graduação em Bioengenharia, University of São Paulo (USP), Av. Trabalhador São-carlense, 400, São Carlos, SP, Brazil c School of Pharmacy and Centre for Biomolecular Sciences, University of Nottingham, Nottingham, NG7 2RD, UK d Laboratório de Parasitologia, Falculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP-USP), Ribeirão Preto, SP, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Cytostatic activity Cell-based assays Dipeptidyl nitriles Leishmaniasis

Cysteine proteases are involved in critical cell processes to the protozoa from Leishmania genus, and their inhibition is a therapeutic alternative to treat the disease. In this work, derivatives of dipeptidyl nitriles acting as reversible covalent inhibitors of cysteine proteases were studied as cytostatic agents. The proteolytic activity inside the living and lysed parasite cells was quantified using a selective substrate for cysteine proteases (Z-FRMCA) from Leishmania amazonensis and L. infantum. The overall proteolytic activity of intact cells and even cell extracts was only marginally affected at high concentrations, with the observation of cytostatic activity and cell cycle arrest of promastigotes. However, the cytotoxic effects were only observed for infected J774 macrophages, which impaired further analysis of the amastigote infection. Therefore, the proteolytic inhibition in intact L. amazonensis and L. infantum promastigotes had no relationship to the cytostatic activity, which emphasizes that these dipeptidyl nitriles act through another mechanism of action.

1. Introduction Leishmaniasis is a disease caused by different species of the protozoa from genus Leishmania with various stages and morphologies during their life cycle (Kaye and Scott, 2011). Currently, about 350 million people are in endemic areas, becoming a major health concern all over the world (Trinconi et al., 2016). Natural transmission of leishmaniasis occurs when metacyclic promastigotes get in contact with the bite of an infected vector from genus Phlebotomus that feeds on mammalian blood (Sacks and Noben-Trauth, 2002). Pentavalent antimonials are the main drugs used to treat leishmaniasis (along with amphotericin B and some other drugs as a second line therapy) and commonly lead to harsh side effects in patients. New formulations of miltefosine (Vicent et al., 2014) and amphotericin B (Bruno et al., 2015) are now under development with the aim to reduce host cell toxicity, but they still have limited efficacy, often leading to the detection of resistant parasites (Trinconi et al., 2016). Drug discovery efforts usually rely on the study of modulators of

macromolecular targets deemed as validated for a disease (Almeida et al., 2015). Proteases are essential for many cellular processes linked to virulence and parasite survival being attractive targets for drug discovery programs. Among these proteins, cysteine peptidases (CPA, CPB and CPC) are the most familiar and characterized ones (Caroselli et al., 2012). CPB is expressed by Leishmania spp., and it is present in many isoforms during the life cycle of the parasite (Wyllie et al., 2012). Its main role in the infection process involves the evasion of the immune system by the cleavage of digestive enzymes inside of the parasitophorous vacuole of the host cell (Brandt et al., 2016). Many works report the importance of CPB as a virulence factor for Leishmania sp., in which CPB-deficient parasites presented reduced infection capability using in vitro and in vivo models (Denise et al., 2003; Casgrain et al., 2016). Besides the reduced virulence, the absence of CPB led to impairment of the intracellular parasite replication (Saraiva et al., 2006). Also, other roles are played by proteases during the parasitism (Williams et al., 2006). The decrease of protozoan growth is an approach under scrutiny,

∗

Corresponding author. E-mail addresses: [email protected] (J.C. Quilles), [email protected] (D.Y. Tezuka), [email protected] (C.D. Lopes), [email protected] (F.L. Ribeiro), [email protected] (C.A. Laughton), [email protected] (S. de Albuquerque), [email protected] (C.A. Montanari), [email protected] (A. Leitão). https://doi.org/10.1016/j.exppara.2019.04.001 Received 27 January 2018; Received in revised form 14 February 2019; Accepted 1 April 2019 Available online 05 April 2019 0014-4894/ © 2019 Elsevier Inc. All rights reserved.

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incubation for 4 h. After that, 100 μL of the solubilizing solution composed by SDS 70% (v/v), DMSO 29% and acetic acid 1% were added to the well and incubated for 90 min. The absorbance at 570 nm was quantified using Biotek Synergy HT plate reader. Assays were performed in quadruplicate using two independent experiments (N = 8) unless explicitly stated. Analysis of the results and significance tests (Dunnett and Tukey) were performed using GraphPad Prism 5 software.

with many in vitro assays being used to quantify the results (Freitas et al., 2015; Garcia-Garcia et al., 2016). These studies are relevant to evaluate the inhibitory potential of one of the most important characteristics of the disease - the high index of parasitic proliferation. Reversible covalent inhibitors of cysteine proteases bind to the active site of CPB enzyme (Siklos et al., 2015) which, in turn, may lead to cytostatic activity against the parasite. However, there are controversies about the CPB role in Leishmania spp. Despite the vast occurrence of CPB gene in different species, its expression or activity can be lower in promastigotes when compared with amastigotes (Coombs, 1982; Leon et al., 1994; Nasereddin et al., 2010). Moreover, compounds known to be cysteine protease irreversible covalent inhibitors, like K777, were inactive in axenically amastigotes but reduced the promastigote-amastigote differentiation and infection (Williams et al., 2006). Besides cysteine protease irreversible covalent inhibitors, there are many reactive centers (known as warheads) that work as reversible covalent inhibitors. Among them, dipeptidyl derivatives bearing the cysteine warhead present promising antiparasitic activity (Ndao et al., 2014; Burtoloso et al., 2017) and they are subject to further studies in this report. Hence, we assessed the parasiticidal effect, cytotoxic and cytostatic activities of a set of 18 known reversible covalent inhibitors of cysteine proteases derived from dipeptidyl nitriles (Avelar et al., 2015; Burtoloso et al., 2017) using promastigotes of Leishmania infantum and L. amazonensis. The same assay was performed using fibroblast cells, but now changing some conditions according to previously published assays (Cruz et al., 2017) to analyze the selectivity of these substances. Moreover, the assay for the J774 macrophage cells infected with amastigotes were also accessed and compared with the non-infected host macrophages.

2.4. Cytostatic assay Parasites were incubated with 10 μM of each inhibitor in 96-well plates. The number of cells was determined by flow cytometry every day for four days. In the fourth day, promastigotes were centrifuged at 500 RCF. The supernatant was removed, followed by the addition of 100 μL of fresh culture medium to evaluate the growth ability. The cell concentration was quantified after 48 h of the inhibitor withdrawal to check for residual activity of the compound inside the parasite. 2.5. Proteolytic activity: intact parasites The proteolytic activity inside the intact parasites was analyzed using the selective fluorogenic substrate Z-FR-MCA. After the incubation of parasites with inhibitors, all samples were transferred to a black 96-wells plate, and 10 μM (L. infantum) or 20 μM (L. amazonensis) of the substrate solution were added to each well followed by 3 h incubation. The fluorescence of the product was quantified to get the proteolytic activity. 2.6. Proteolytic activity: cell extract Promastigotes were cultivated until reaching the concentration of 1 × 107 cell/mL (about 72 h) to determine the enzymatic extract activity. Briefly, 10 mL of the parasites culture were centrifuged at 500 RCF, followed by the replacement with 10 mL of cold sodium phosphate buffer (10 mM, pH 8.0). Parasites were lysed using the sonic dismembrator (Fisher Scientific 500) in an ice bath using 10% amplitude pulses performed for 3 min, with intervals of 30 s. After cell dismembration, the extract was centrifuged at 500 RCF for 5 min at 4 °C to separate the cell debris. The supernatant was then centrifuged at 1200 RCF using the 10 kDa Amicon® membrane (Merck-Millipore) during 35 min at 4 °C. Proteins retained on the filter were quantified by Bradford method and diluted in sodium acetate butter (10 mM, pH 5.5). Inhibitors were added to the extract and incubated for 15 min. The proteolytic activity was quantified by the conversion of the selective fluorogenic substrate Z-FR-MCA using a plate reader (Biotek Synergy HT) at λexc 360/40 and λem 460/40 nm.

2. Materials and methods 2.1. Chemical compounds, reagents and cysteine protease probe All 18 dipeptidyl nitrile derivatives used in this study were obtained from our in-house collection, with purity of at least 95% according to HPLC-MS analyses. Reference cysteine protease inhibitors (E64 and MMTS) and the Z-FR-MCA (Z-Phe-Arg 7-amido-4-methylcoumarin) substrate were purchased from Sigma-Aldrich. DMSO was used to prepare stock solutions. Working solutions were prepared by further diluting the stock solution with medium to reach 0.5% (v/v) DMSO and sterile filtered. 2.2. Parasites Promastigotes of L. amazonensis (MHOM/BR/1973/M2269) and L. infantum (HU UFS17) were cultured for 72 h at 25 °C in 199 medium, pH 7.4, supplemented with 10% fetal bovine serum. Parasites were kept in the exponential growth phase for all experiments. Balb/C 3T3 clone A31 mouse fibroblast cells were used as reference cells to observe the selectivity of the tested compounds, being cultivated in DMEM medium (pH 7.2) supplemented with 10% fetal bovine serum at 37 °C, in 5% CO2 atmosphere with 90% humidity.

2.7. Analysis of the cell cycle Promastigotes were treated with 10 μM of each inhibitor and incubated for 24 or 72 h. After the incubation time, parasites were centrifuged at 500 RCF, and 100 μL of the cell cycle reagent (MerckMillipore) were added to the pellet in an ice bath. After 30 min incubation, the analysis was performed using the guavaSoft 2.7 cell cycle module of the Guava easyCyte 8HT Flow Cytometer.

2.3. Colorimetric method for cell viability 2.8. Leishmania infantum amastigote assay Logarithmic growth phase parasites were seeded in 96-well plates along with the compounds, 0.5% (v/v) DMSO solution (negative control), or amphotericin B (positive control). Viability assays were performed after 72 h of incubation by the MTT/PMS method. PMS is an electron transfer that induces a reduction of the MTT by the dehydrogenase mitochondrial enzymes producing the water-insoluble crystals of formazan. The MTT/PMS solution was prepared using 5 mg of MTT and 0.22 mg of PMS diluted in 1 mL of PBS. To access cell viability, 11.1 μL of this solution was pipetted into each well, following

L. infantum parasite culture stably expressing the mCherry gene was kindly provided by Prof. Dra. Ângela Kaysel Cruz from University of São Paulo-Ribeirão Preto and maintained in Schneider Medium supplemented with 10% of fetal bovine serum. To perform the L. infantum amastigote assay, J774 macrophages (1 × 105 cells/mL) were pipetted in a 96-well plate, resting for 2 h. Then, fluorescent promastigotes were added at the concentration of 1 × 106 parasites/mL, following the overnight infection. Promastigotes were removed by washing, with the 85

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inhibitors, even after the withdrawal of the substances (Fig. 2). With that experiment, we can assume that the action of the dipeptidyl nitriles has prolonged effect, only considering the amount of substance present inside the parasites after 96 h of incubation (Fig. 1a). Like before, Neq0551 and Neq0569 were the most effective compound to affect the cell proliferation of both parasites, while the difference in the selectivity between the species was more evident to Neq0413 and Neq0573 (almost five times more selective to L. amazonensis), Fig. 2. Proteolytic activity inside the intact cell was determined using a selective fluorescent substrate. Initially, different parasite concentrations were tested with the substrate (10 μM) to determine the best range of work, Fig. 3. It can be seen that the linear correlation was only achieved for L. infantum (Fig. 3A) probably due to the higher metabolism ratio for L. amazonensis (Fig. 3B). The use of 20 μM Z-FR-MCA was devised to check whether the concentration of the fluorophore could be limiting the metabolism in L. amazonensis. The hypothesis was confirmed with the obtention of a linear relationship for L. amazonensis (Fig. 3C). The next step was the assay of substances to quantify the proteolytic activity (Table 2). All substances were active against L. infantum at 10 μM (expect Neq0573) with proteolytic activity inhibition around 30% inside, while compounds were inactive for L. amazonensis. The growth inhibition after the removal of the compounds (Table 2) had no relationship to the inhibition of the proteolytic activity (IPA, Table 2). It indicates that the late (or residual) cell response to these substances may occur via a different process for each Leishmania species. The hydrolysis of the selective substrate by the enzymatic extract obtained from the lysis of the promastigote cells was measured to evaluate the cysteine protease inhibition by each compound (Fig. 4). In general, these inhibitors decreased the proteolytic activity of extracts from both leishmanias. Interestingly, L. amazonensis extracts kept the enzymatic activity closer to the control when compared with L. infantum. Neq0551, Neq0554, Neq0568 and Neq0569 showed a similar response to the reference compound (MMTS) for both species. Neq0544 and Neq0573 were less potent, but they kept the same profile, with highest L. infantum inhibition than L. amazonensis. The protein content of extracts from these two leishmanias was, on average, 0.8 mg/mL, according to the Bradford assay. It means that there is no bias toward the protein amount from the cell extract that could affect the inhibitory potential of these compounds. Instead, the quantity and activity of cysteine proteases expressed by these Leishmania species present in the protein pool were the sources of comparative analysis for the set of inhibitors. Flow cytometry was applied to evaluate any perturbation of the cell cycle after 72 h of incubation with the substances. Neq0544, Neq0551, and Neq0554 perturbed the cell cycle of promastigotes from L. infantum according to the statistical analysis (Fig. 5). The main change was the increment of the G1 phase and the reduction of the S phase for Neq0544 (Fig. 5). However, these substances did not alter the cell cycle profile for L. amazonensis (data not shown). The activity of a subset of compounds was analyzed by using the amastigote form of L. infantum. As seen in Fig. 6, all compounds were cytotoxic to the infected host macrophage cell for the whole set of concentrations tested. Hence, it led to negative effects of amastigotes due to the ratio between the number of macrophage nuclei and parasite nuclei. This unexpected result then led us to investigate the cytotoxicity for the most promising compounds against the host macrophage cell alone. As seen in Fig. 7, all dipeptidyl nitriles were inactive against the noninfected J774 cell in the same condition of Fig. 6.

Table 1 Evaluation of cysteine protease inhibitors parasiticidal effect and cytotoxicity against promastigotes of L. amazonensis and L. infantum and Balb/C 3T3 clone A31 mouse fibroblast cells after 72 h incubation. Substance [100 μM]

Neq0409 Neq0413 Neq0414 Neq0533 Neq0544 Neq0549 Neq0550 Neq0551 Neq0552 Neq0554 Neq0568 Neq0569 Neq0570 Neq0571 Neq0572 Neq0573 Neq0575 Neq0578 E-64

L. amazonensis

L. infantum

Fibroblast cells

Cell death (%)a

SD

Cell death (%)

SD

Cell death (%)

SD

NA NA NA 8.98 NA 16.67 NA NA NA NA NA 1.85 1.34 NA NA 36.51 13.49 6.34 5.45

– – – 0.55 – 3.68 – – – – – 3.95 9.31 – – 3.33 8.12 12.12 3.39

NA NA NA 6.28 NA 14.59 6.69 NA NA NA NA NA 7.11 1.21 ND 25.83 NA NA NA

– – – 3.80 – 5.61 5.40 – – – – – 7.95 1.63 – 3.11 – – –

* 5.61 3.47 12.86 42.02 14.52 NA 18.88 NA NA NA NA NA 16.42 19.52 34.40 21.14 21.83 NA

– 4.56 2.86 2.44 3.22 10.04 – 4.02 – – – – – 3.22 5.60 3.45 2.55 3.11 –

a Cell death was determined by the MTT/PMS colorimetric assay using 0.5% (v/v) DMSO solution as 100% of viability | NA: No cell death was detected (not active). ND: Not determined.

addition of compounds at serial concentrations (50 μM–0.78 μM). After 48 h incubation, the plate was washed, fixed with 4% formaldehyde and stained with DAPI (4,6-diamidine-2-phenylindole dihydrocloride, Sigma-Aldrich) (1:10.000). The number of intracellular amastigotes was automatically counted using the ImageXpress High Content Screening equipment (Molecular Devices, LLC.). The number of nuclear cells and nuclear parasites was converted into percentage inhibition considering the wells without treatment as 100% of infection. 3. Results The parasiticidal effect was analyzed after the incubation of the promastigote form of L. amazonensis and L. infantum parasites with inhibitors for 72 h. The MTT/PMS method was chosen to quantify the cell viability (Table 1). Substances had no significant parasiticidal effect for both species, neither the fibroblast cells. Therefore, these molecules do not lead to cell death. Leishmaniasis treatments present low efficacy to cure the disease. Cytostatic compounds could, in principle, be used to target other mechanisms involved in the cell replication process, like the inhibition of cysteine proteases. Based on the enzymatic inhibition by biochemical assays using a purified recombinant CPB form, all compounds with a wide inhibition range (micro to nanomolar, not shown) were selected to investigate their cytostatic effect. This assay was planned to study the replication of the promastigotes after replacing the supernatant solution with the compound by a fresh medium free of any inhibitor. To this end, the incubation period with inhibitors span 96 h, so these molecules could permeate the membrane to reach a constant intracellular concentration (Fig. 1A). After that, the medium was replaced by a fresh one without inhibitor, letting the cells grow for another 48 h; hence the cytostatic effect observed at the end of the experiment could only come from the intracellular concentration of the compound. All substances inhibited the parasitic growth at the end of the incubation time (Fig. 1B and C). We could observe higher cytostatic potency after 96 h, where Neq0551 and Neq0569 were the best ones for both species (Fig. 1B and C). After the incubation time, parasite growth was suppressed by the

4. Discussion The detection of the proteolytic activity inside Leishmania cells was previously standardized based on the metabolism of the selective substrate Z-FR-MCA by cysteine proteases expressed by the parasites. The 86

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Fig. 1. Cytostatic effect of cysteine protease inhibitors. The scheme of the assay (A). Growth curve of the promastigote form of L. amazonensis (B) and L. infantum (C) in the presence of cysteine protease inhibitors. The experiment was performed in duplicate with a standard error lower than 10%. CP: cysteine proteases.

cysteine proteases, with trypanosomicidal activity already described by our group (Avelar et al., 2015; Burtoloso et al., 2017). Reports for these molecules state them as nanomolar cruzain inhibitors (the orthologous cysteine protease from Trypanosoma cruzi), and they also inhibit cathepsin L (human) and CPB (Leishmania spp.) to the same extent. In a new approach to understand the antiparasitic profile of dipeptidyl nitriles, cytostatic assays were devised to detect and quantify the potency using the set of experiments reported here (Fig. 1). Dipeptidyl nitriles were effective to inhibit the cell growth, providing late (or residual) effect once the activity was observed even after 48 h of compound removal (Fig. 2). All-in-all, intact promastigotes from both species were sensitive to this set of compounds (Table 2). In general, the parasite responsible for the visceral form of the disease (L. infantum) is more resistant to different types of treatment. This is the same profile observed regarding the cytostatic assays performed here (Fig. 1). The later

condition established in the previous study was used to evaluate the bioactivity of dipeptidyl nitrile compounds (Caroselli et al., 2012). Our study points out that the inhibition of cysteine proteases by dipeptidyl nitriles was not effective to promote the cell death of L. amazonensis or L. infantum promastigotes (Table 1). Protease inhibitors have leishmanicidal activity for amastigotes according to some reports (Almeida et al., 2015), but usually these works demonstrate that the parasiticidal activity is not directly linked to the inhibition cysteine protease and may involve the inhibition of proteasome or another target (i.e. this could be an effect from a polypharmacological profile). Moreover, the inhibition of known the selective irreversible cysteine protease inhibitor E-64 is reduced over the number of passages for parasites kept in in vitro culture, which may reduce its bioactivity over time (Rebello et al., 2010). Dipeptidyl nitriles are known as reversible covalent inhibitors of 87

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understand the parasite virulence (Silva-Almeida et al., 2012) or toward the parasiticidal effect using protease inhibitors (Pereira et al., 2014), lacking further analyses of promastigote cytostatic activity. Few instances comprehend the study of irreversible covalent inhibitor ZLIII43A and derivatives against L. major (Selzer et al., 1997), and K777 in L. major and L. tropica promastigotes (Mahmoudzadeh-Niknam and McKerrow, 2004). These compounds worked as cytostatic up to 72 h in micromolar concentrations (5–100 μM), leading to cell death at the highest concentration. It is not possible to assure that the highest concentrations of K777 were able to fully abrogate cysteine protease activity from the data retrieved from the literature, but our preliminary studies are showing that promastigotes were alive despite the inactivation of these enzymes (not shown). Another striking observation is that alterations of the cell cycle profile were observed to L. infantum mainly using Neq0544 (Fig. 5) that also presented good percent inhibition of the proteolytic activity of both enzymatic extract (Fig. 4) and inside of the living cell (Table 2). Neq0551 also promoted the cell cycle perturbation and similar proteolytic activity inhibition to L. infantum, arresting it in the G1 phase (Fig. 5) in which the protein synthesis is critical in this step. Nonetheless, the inhibition of the proteolytic activity (IPA) by these compounds was low regarding the growth inhibition (GI, Table 2), which may point out to a polypharmacological behavior that needs to be studied. Some structural aspects of the dipeptidyl nitriles could be drawn from the set using the percentage of growth inhibition (Table 2). The cyclopropyl ring in position R1 (Neq0569) has a slight improvement in potency for both species in comparison with the non-substituted compound (Neq0568). It is a good outcome, once cyclopropyl substitution avoids a spurious nucleophilic attack on the nitrile moiety (reducing the promiscuous toxicity) along with the concomitant improvement of the compound half-life (Gagnon et al., 2007). Bulky substituents in position R3 improve the potency of L. infantum when comparing Neq0413 and Neq0544. Overall L. amazonensis is more sensitive than L. infantum for the series, with higher growth arrest ratio for the former species. No relationship could be established for the inhibition of proteolytic activity using intact cells (Table 2) due to the lack of potency of all compounds in L. amazonensis or low potency in L. infantum. There are many possible explanations for the lack of effectiveness when the intact parasite was used instead of the cell lysate, but the cell arrest observed

Fig. 2. Percentage of cell growth of the promastigote form of L. infantum (white) and L. amazonensis (gray) after 48 h of the inhibitor withdrawn from the extracellular medium. The experiment was performed in triplicate displayed as average and standard deviation. Statistical analysis was performed by the Dunnett test comparing the samples to negative control for P values < 0.05 (*).

one had a higher metabolic rate for the substrate (Fig. 3), but it is still not known how it could be related to parasite metabolism and survival. So far it is not possible to rule out the possibility that cysteine protease activity may play a much more important role for L. amazonensis than L. infantum regarding cell survival. Based on this idea, the analysis of the enzymatic extract from lysate cells was performed to access the inhibition of proteolysis without the interference of membrane permeation, which could affect the intracellular concentration of the compound. Overall, all substances had higher inhibition rate of proteolysis for L. infantum in comparison with L. amazonensis. Two fronts could be further explored to have an in-depth analysis of this phenomenon: (i) L. amazonensis can express higher amounts of active cysteine proteases than L. infantum; (ii) there is a higher turnover for the L. amazonensis enzymes. The conundrum here is that even though L. amazonensis proteolytic activity inhibition (IPA, Table 2) is lower than L. infantum for all compounds, the cytostatic activity observed for these molecules is higher for L. amazonensis than L. infantum (GI, Table 2). This result cannot be explained by the hypothetic mode of action regarding cysteine protease inhibition like CPA, CPB, CPC, and others. Most of the work on CPB activity is devoted to the host-pathogen interaction to

Fig. 3. Standardization assay to detect the total proteolytic activity using fluorimetry. Relative fluorescence units (RFU) were obtained using live promastigotes of L. infantum (a) and L. amazonensis (b, c). Concentrations of the substrate Z-FR-MCA employed in these experiments: 10 μM (a, b) and 20 μM (c). 88

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Table 2 Growth inhibition (GI) and inhibition of proteolytic activity (IPA) of L. amazonensis and L. infantum promastigotes 48 h after the removal of the inhibitors.

Code

Structure R1

L. amazonensis R2

L. infantum

R3

GI (%)a

IPA (%)b

GI (%)a

IPA (%)b

Neq0413

CH2

CH3

81(0.71)

ND

NA

ND

Neq0544

CH2

OC(CH3)3

90(18)

NA

68(28)

35(13)

Neq0551

CH2

Ph

ND

NA

96(13)

27(6.5)

Neq0554

CH2

84(17)

NA

64(30)

19(7.3)

Neq0568

CH2

CH2CH(CH3)2

83(35)

NA

72(32)

28(6.4)

Neq0569

CH2CH(CH3)2

99(58)

NA

88(17)

25(11)

Neq0573

CH2C(CH3)3

91(4.6)

NA

NA

NA

Ph

ND: proteolytic activity or growth inhibition was not determined. NA: not active. Standard deviation is shown in parenthesis. a Cells were treated with 10 μM of the inhibitor for 96 h before the study. After the replacement of the medium, parasites grew without inhibitor for 48 h. The quantification of the growth inhibition (GI) for each compound led to the calculation of the percentage value (GI %) using the negative control (non-treated cells) as 100%. Standard deviations are shown in parenthesis. b Percentage of inhibition of proteolytic activity (IPA %) inside the intact promastigotes calculated at the end of the experiment.

cysteine protease cruzain are pointing to the same direction (Burtoloso et al., 2017). Previous study made with these compounds against mouse fibroblast cells (Balb/C 3T3 clone A31) showed no cytotoxicity (Quilles. et al., 2018). Intriguingly, compounds had high cytotoxicity toward infected macrophage, which impaired the evaluation of amastigote assay, once the amastigotes were released from the host cell, but the parasites nuclei could be kept intact (Fig. 6). This unexpected result was not corroborated by the cytotoxicity assay using non-infected host macrophage cells (Fig. 7), where all compounds were inactive. The cytotoxicity only observed against the infected host cells could arise from the stressing condition of the macrophages promoted by the parasitosis. To study the cell death process, infected and non-infected macrophages could in principle be subjected to the differential analysis of apoptotic markers (for example, caspases 3 and 8) and mitochondrial membrane potential (Zorova et al., 2018). It is now a matter of in-depth studies to get some insights about the putative mechanism of action and the stark contrast in terms of the cytotoxicity between mammalian host cell conditions.

Fig. 4. Proteolytic activity of the enzymatic extract obtained from the lysis of L. infantum (white) and L. amazonensis (gray) promastigotes. Statistical analysis was performed by the Dunnett test comparing with the negative control for P values < 0.05.

for both species showed that these molecules might be acting via an unknown mechanism. Cross-reactivity with other classes of proteases (like the one observed for K777 in Chen et al., 2010; or dipeptidyl nitriles in Bethel et al., 2009; Ndao et al., 2014) or even inhibition of an entirely disparate macromolecule cannot be excluded. For example, HIV aspartate protease inhibitors present cytostatic activity for L. amazonensis promastigotes, but the mechanism of action was unknown (Santos et al., 2009). Only recently, the aspartate protease Ddi1-like from L. major was labeled as a putative target for these HIV aspartate protease inhibitors (White et al., 2011). Interestingly, our ongoing attempts to relate T. cruzi parasiticidal effect and the inhibition of the

5. Conclusion Dipeptidyl nitriles showed cytostatic rather than parasiticidal activity against two Leishmania species. To the best of our knowledge, that is the first report showing this profile for reversible covalent inhibitors of cysteine proteases in Leishmania parasites. L. amazonensis had a higher metabolic ratio in comparison with L. infantum when the cysteine protease selective substrate Z-FR-MCA was used in the cell lysates. It is not yet possible to indicate whether this result is due to the higher expression rates of cysteine proteases in L. amazonensis or even the presence of cysteine proteases with higher catalytic efficiency. 89

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Fig. 5. Cell cycle bar graph (A) and the flow cytometry profile (B) of L. infantum after 72 h of treatment. Phases of the cell cycle: G1 - dark gray; S - light gray; G2/M gray. The Dunnett test was performed to check the pairwise analysis between the negative control (no treatment) and the inhibitors with P values < 0.05. The standard deviation was lower than 5% for all assays performed in duplicate.

opens up a new perspective toward the addressing of the actual macromolecular target(s) for these substances.

Indirectly, the percentage of proteolytic activity using intact cells provided a similar result, where compounds were inactive against L. amazonensis, while some were still active for L. infantum. Moreover, the growth inhibition observed using intact promastigotes is not related to the inhibition of the proteolytic activity, which suggests that these molecules are acting via another mechanism of action. This result is shedding light on an apparent counterintuitive biological profile and

Conflicts of interest There is no competing interest for all authors.

Fig. 6. Cytotoxicity for all compounds was high on the infected host macrophage cell (a) leading to high ratio of amastigotes to host nuclei (b). Assays performed in triplicate. 90

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Fig. 7. Compounds were inactive for the unifected host J774 macrophage cells. Assays performed in duplicate.

Acknowledgment This work had the financial support from CAPES Foundation (grant AUXPE 139/2015), São Paulo Research Foundation (FAPESP – grants 2013/18009-4, 2014/07292-0) and CNPq for the post-doctorate fellowship (CDL). Authors acknowledge Prof. Sergio de Albuquerque for the kind donation of parasites. We acknowledge the anonymous reviewer for the questions that improved the overall scientific discussion of the results. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.exppara.2019.04.001. References Almeida, L., Alves, K.F., Maciel-Rezende, C.M., Jesus, L.O.P., Pires, F.R., Viegas Jr., C., Izidoro, M.A., Júdice, W.A.S., Santos, M.H., Marques, M.J., 2015. Benzophenone derivatives as cysteine protease inhibitors and biological activity against Leishmania (L.) amazonensis amastigotes. Biomed. Pharmacother. 75, 93–99. Avelar, L.A.A., Camilo, C.D., Albuquerque, S., Fernandes, W.B., Gonçalez, C., Kenny, P.W., Leitão, A., McKerrow, J.H., Montanari, C.A., Orozco, E.V.M., Ribeiro, J.F.R., Rocha, J.R., Rosini, F., Saidel, M.E., 2015. Molecular design, synthesis and trypanocidal activity of dipeptidyl nitriles as cruzain inhibitors. PLoS Neglected Trop. Dis. 9, 1–24. Bethel, P.A., Gerhardt, S., Jones, E.V., Kenny, P.W., Karoutchi, G.I., Morley, A.D., Oldham, K., Rankine, N., Augustin, M., Krapp, S., Simader, H., Steinbacher, S., 2009. Design of selective Cathepsin inhibitors. Bioorg. Med. Chem. Lett 19, 4622–4625. Brandt, A.M.L., Batista, P.R., Souza-Silva, F., Alves, C.R., Caffarena, E.R., 2016. Exploring the unbinding of Leishmania (L.) amazonensis CPB derived-epitopes from H2 MHC class I proteins. Proteins 84, 473–487. Bruno, F., Castelli, G., Migliazzo, A., Piazza, M., Galante, A., Verde, V.L., Calderone, S., Nucaloto, G., Vitale, F., 2015. Cytotoxic screening and in vitro evaluation of pentadecane against Leishmania infantum promastigotes and amastigotes. J. Parasitol. 101, 701–715. Burtoloso, A.C.B., de Albuquerque, S., Furber, M., Gomes, J.C., Gonçales, C., Kenny, P.W., Leitão, A., Montanari, C.A., Quilles, J.C.J., Ribeiro, J.F.R., Rocha, J.R., 2017. Antitrypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors. PLoS Neglected Trop. Dis. 11, 1–16. Caroselli, E.E., Assis, D.M., Barbiéri, C.L., Júdice, W.A.S., Juliano, M.A., Gazarini, M.L., Juliano, L., 2012. Leishmania (L.) amazonensis peptidase activities inside the living cells and in their lysates. Mol. Biochem. Parasitol. 184, 82–89. Casgrain, P.A., Martel, C., McMaster, W.R., Mottram, J.C., Oliver, M., Descoteaux, A., 2016. Cysteine peptidase B regulates Leishmania mexicana virulence through the modulation of GP63 expression. PLoS Pathog. 12, 1–17. Chen, Y.T., Brinen, L.S., Kerr, I.D., Hansell, E., Doyle, P.S., McKerrow, J.H., Roush, W.R., 2010. In vitro and in vivo studies of the trypanocidal properties of WRR-483 against trypanosoma cruzi. PLoS Neglected Trop. Dis. 4, e825. Coombs, G.H., 1982. Proteinases of Leishmania mexicana and other flagellate protozoa. Parasitology 84, 149–155. Cruz, E.C.S., Carecho, A.R., Saidel, M.E., Montanari, C.A., Leitão, A., 2017. In silico and cell-based characterization of selective and bioactive compounds for androgen-dependent prostate cancer cell. Bioorg. Med. Chem. Lett 27, 546–550. Denise, H., McNeil, K., Brooks, D.R., Alexander, J., Coombs, G.H., Mottram, J.C., 2003. Expression of multiple CPB genes encoding cysteine proteases is required for

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