Leishmanicidal Activities of Novel Methylseleno-Imidocarbamates

Abstract

The generation of new antileishmanial drugs has become a priority. Selenium and its derivatives stand out as having promising leishmanicidal activity. In fact, some parasites express selenoproteins and metabolize selenium. Recently, selenium derivatives have shown the potential to reduce parasitemia, clinical manifestations, and mortality in parasite-infected mice. In this paper, after selecting four candidates according to drug similarity parameters, we observed that two of them, called compounds 2b [methyl-N,N′-di(thien-2-ylcarbonyl)-imidoselenocarbamate] and 4b [methyl-N,N′-di(5-nitrothien-3-ylcarbonyl)-imidoselenocarbamate], exhibit low 50% inhibitory concentrations (IC₅₀s) (<3 μM) and good selectivity indexes (SIs) (>5) in Leishmania major promastigotes and lack toxicity on macrophages. In addition, in analysis of their therapeutic potential against L. major in vitro infection, both compounds display a dramatic reduction of amastigote burden (∼80%) with sublethal concentrations. Furthermore, in macrophages, these selenocompounds induce nitric oxide production, which has been described to be critical for defense against intracellular pathogens. Compounds 2b and 4b were demonstrated to cause cell cycle arrest in G₁. Interestingly, evaluation of expression of genes related to proliferation (PCNA), treatment resistance (ABC transporter and alpha-tubulin), and virulence (quinonoid dihydropteridine reductase [QDPR]) showed several alterations in gene expression profiling. All these results prompt us to propose both compounds as candidates to treat leishmanial infections.

INTRODUCTION

Leishmaniasis, one of the 17 neglected tropical diseases recognized by the World Health Organization (WHO), is caused by the protozoan parasites of the genus Leishmania. It exhibits three main clinical forms: visceral, cutaneous, and mucocutaneous leishmaniasis. Considering the impact and disease burdens of tropical infections, leishmaniasis ranks second in mortality and fourth in morbidity (1). Leishmania major is one of the causative agents of cutaneous leishmaniasis, afflicting around 1 million people each year in the world.

Due to the lack of an effective vaccine, existing means for disease control are limited to the management of vector and reservoir hosts in order to reduce transmission and treatment with chemotherapeutic agents. However, the efficiency of current treatment strategies is reduced by a growing incidence of drug resistance. The generation of new antileishmanial drugs has become a priority.

Based on the significant antitumor activity of some selenocompounds developed by our group (2), these agents were postulated as promising drugs. In addition, there is growing evidence suggesting a connection between selenium (Se) and parasites, particularly trypanosomatids. In fact, some parasites express selenoproteins (3, 4) and metabolize Se (5).

Se derivatives have been shown to reduce parasitemia (3, 6). Furthermore, they also decrease clinical manifestations (7, 8) and mortality in infected mice. The increased concentration of Se in plasma has demonstrated leishmanicidal properties, and therefore, it may be a novel strategy against these protozoa (9). More recently, Se has been recognized as an antioxidant, antiviral, antitumoral, and antiparasitic agent (2, 10, 11). Methylseleno-imidocarbamates have been described to be antiproliferative molecules against several tumor cell lines, to induce cell cycle arrest, and to trigger apoptosis (12).

In this study, we observed that the selenocompounds called 2b [methyl-N,N′-di(thien-2-ylcarbonyl)-imidoselenocarbamate] and 4b [methyl-N,N′-di(5-nitrothien-3-ylcarbonyl)-imidoselenocarbamate] show antiproliferative activities against promastigotes and the intracellular forms of Leishmania major likely through PCNA downregulation and apparently without affecting ABC transporter levels. Interestingly, they alter alpha-tubulin and quinonoid dihydropteridine reductase (QDPR) gene expression in promastigotes. In addition, these drugs dramatically reduce parasite burden within macrophages. A hypothetical mechanism of induction of parasite death and inhibition of proliferation may be mediated by nitric oxide (NO) production, as both compounds notably increase NO levels, a key factor inducing leishmania cell death (13).

MATERIALS AND METHODS

Cells and culture conditions.

Leishmania major promastigotes (Lv39c5) were kindly provided by Manuel Soto (Centro de Biología Molecular Severo Ochoa [CSIC-UAM], Madrid, Spain) and were grown at 26°C in M199 medium supplemented with 25 mM HEPES (pH 7.2), 0.1 mM adenine, 0.0005% (wt/vol) hemin, 2 mg/ml biopterin, 0.0001% (wt/vol) biotin, 10% (vol/vol) heat-inactivated fetal calf serum (FCS), and an antibiotic cocktail (50 U/ml penicillin, 50 mg/ml streptomycin).

Murine RAW 264.7 macrophages were cultured in complete RPMI medium supplemented with 2 mM l-glutamine, 0.5% HEPES, 5 μg/ml penicillin, 100 U/ml streptomycin, and 10% fetal bovine serum (BioWhittaker, Walkersville, MD).

Murine peritoneal macrophages of 4- to 6-week-old BALB/c mice were used for the study. All the procedures involving animals were approved by the Animal Care Ethics Commission of the University of Navarra. Animals were inoculated with 2 ml sterile thioglycolate (3%) broth (BD Difco) prior to peritoneal cavity lavage with 5 ml of cold RPMI medium, and macrophages were removed by a syringe as previously described (14).

Compounds.

Paromomycin (Sigma, St. Louis, MO, USA) was used as a reference drug and dissolved at a concentration of 50 mg/ml in water. The studied compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration between 0.003 and 0.005 M. Sterile filtration was achieved using 0.2-mm filter disks. Serial dilutions with supplemented medium were prepared daily to a final concentration of less than 2% DMSO in cell culture.

Structure.

Compounds 2b [methyl-N,N′-di(thien-2-ylcarbonyl)-imidoselenocarbamate], 4b [methyl-N,N′-di(5-nitrothien-3-ylcarbonyl)-imidoselenocarbamate], 5b [methyl-N,N′-di(thienaphthen-2-ylcarbonyl)-imidoselenocarbamate], and 8b [methyl-N,N′-di(quinolin-3-ylcarbonyl)-imidoselenocarbamate] were synthesized in good yield and purity as previously described by our group (12). Briefly, synthesis started from Se-methyl-imidoselenocarbamate and the corresponding heteroaryl acyl chloride in a 1:2 molar ratio, respectively, in chloroform in the presence of pyridine as a catalyst at room temperature for 48 h. All compounds were characterized by routinely used methods, such as nuclear magnetic resonance (NMR) and infrared (IR) and mass spectrometry (MS).

Leishmanicidal activity.

To determine the antileishmanial activities of the compounds analyzed in this study, exponentially growing cells (2 × 10⁶ promastigotes/ml) were seeded in 96-well plates (100 μl per well) with increasing concentrations of selenocompounds (2b, 4b, 5b, and 8b [Fig. 1]) diluted in 100 μl of M199 medium and maintained at 26°C. After 48 and 72 h of incubation, the half-maximal inhibitory concentration (IC₅₀) was calculated. IC₅₀ represents the concentration required for 50% growth inhibition of treated cells with respect to untreated controls. The decrease in the number of viable cells was determined using the colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO, USA). MTT solutions were prepared at 5 mg/ml in phosphate-buffered saline (PBS), filtered, and maintained at −20°C until use. After adding 20 μl of MTT solution to each well, the plates were incubated for 4 h at 26°C. Therefore, 100 μl of a solution of 50% isopropanol and 10% sodium dodecyl sulfate (SDS; pH 5.4) was added to each well to dissolve formazan crystals. The optical density (OD) was measured in a Multiskan EX microplate photometer plate reader at 540 nm (15). The IC₅₀ was obtained by fitting a sigmoidal E_max model to dose-response curves. The results were expressed as means (± standard deviations [SDs]) from three independent experiments.

FIG 1.

Structures of selenium compounds: 2b, methyl-N,N′-di(thien-2-ylcarbonyl)-imidoselenocarbamate; 4b, methyl-N,N′-di(5-nitrothien-3-ylcarbonyl)-imidoselenocarbamate; 5b, methyl-N,N′-di(thienaphthen-2-ylcarbonyl)-imidoselenocarbamate; 8b, methyl-N,N′-di(quinolin-3-ylcarbonyl)-imidoselenocarbamate.

Cytotoxicity assays.

The alamarBlue assay (Invitrogen Life Technologies), a colorimetric test involving the cellular reduction of resazurin to resorufin, was performed to determine the cytotoxicity of selected compounds in RAW 264.7 cells. Briefly, 2 × 10⁴ cells were seeded per well in 96-well plates and allowed to adhere for 24 h at 37°C in a 5% CO₂ humidified atmosphere. The culture medium was replaced by fresh medium with increasing concentrations of compounds with the lowest IC₅₀ in promastigotes. After 48 h of incubation, alamarBlue reagent was added in an amount equal to 10% of the culture volume and plates were incubated under the same conditions for 4 h. Fluorescence was monitored at a 545-nm excitation wavelength and a 595-nm emission wavelength using a BMG FLUORstar Galaxy microplate reader. The assay was performed in triplicate. The macrophage viability was evaluated based on a comparison with untreated control cells. The IC₅₀ was calculated by sigmoid regression analysis.

Calculation of in vitro therapeutic index (SI).

At least three different assays were performed to calculate the selectivity index (SI) of each compound, which was determined as the ratio between the IC₅₀ obtained in macrophages and the corresponding IC₅₀ in parasites (16).

Intracellular amastigote assay.

Murine peritoneal macrophages were seeded in 8-well culture chamber slides (LabTek; BD Bioscience) at a density of 2 × 10⁴ cells per well in RPMI medium and allowed to adhere overnight at 37°C in a 5% CO₂ incubator. In order to perform the infection assay, metacyclic promastigotes were isolated by the peanut agglutinin (PNA) method (17) and used to infect macrophages at a macrophage/parasite ratio of 1/10. The plates were incubated for 24 h under the same conditions until promastigotes were phagocytosed by macrophages. Afterwards, wells were washed with preheated medium to remove the extracellular promastigotes, and plates were incubated with fresh medium supplemented with increasing concentrations of compounds. Seventy-two hours later, cells were washed with PBS, fixed with ice-cold methanol for 5 min, and stained with Giemsa stain. To determinate the parasite burden, the number of amastigotes per 200 macrophages was counted under a light microscope. The percentage of macrophage infection was calculated by dividing the number of infected macrophages by the number of counted macrophages. The mean number of amastigotes per infected macrophage was determined by dividing the total number of amastigotes counted by the number of infected macrophages. Three independent experiments were performed with duplicates.

Effects on cell cycle progression.

To analyze the effect of selected compounds on cell cycle progress, the IC₅₀ of compounds was added to logarithmic-phase cultures and cultures were incubated for 24 h. Parasites were collected by the centrifugation method, washed twice with cold PBS, fixed with ice-cold fixative solution (30% PBS-70% methanol), and incubated at 4°C for 1 h. Afterwards, parasites were collected by centrifugation, resuspended in PBS containing 20 μg/ml of RNase A (Roche, Mannheim, Germany), and incubated for 20 min at 37°C. Then, 7-aminoactinomycin D (7AAD) (Sigma-Aldrich) was added to a final concentration of 10 μg/ml. Samples were immediately analyzed with an Attune acoustic focusing cytometer.

RNA extraction and quantitative reverse transcription-PCR (RT-PCR) gene expression analysis.

Total RNA from L. major promastigotes was purified using the TRIzol reagent (Sigma-Aldrich) as described by the manufacturer. Any possible genomic DNA contamination was eliminated by treatment with DNase (Ambion Life Technologies). RNA concentrations were measured with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA). Two micrograms of RNA was reverse transcribed using Transcriptor reverse transcriptase (GoScript reverse transcription system; Promega) to cDNAs which were then used as the PCR template. The PCR was performed in a 96-well plate using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA) with SYBR Green PCR master mix (Applied Biosystems) according to the manufacturer's instructions. The Leishmania major glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene was used to normalize gene expression. The sequences of the primers used for real-time PCR were as follows: PCNA, 5′-AGATGGACTACCGCAGCA-3′ and 5′-CTCTGATTTCACCTCCGACTTG-3′; ABC transporter, 5′-CGGGTTTGTCTTTCAGTCGT-3′ and 5′-CACCAGAGAGCATTGATGGA-3′; alpha-tubulin, 5′-ATGCGTGAGGCTATCTGCATCCACAT-3′ and 5′-TAGTGGCCACGAGCGTAGTTGTTCG-3′; quinonoid dihydropteridine reductase (QDPR), 5′-ATGAAAAATGTACTCCTCATCG-3′ and 5′-TTCACCCTGCGTACTGAACACAT-3′ (18); GAPDH, 5′-ACCACCATCCACTCCTACA-3′ and 5′-CGTGCTCGGGATGATGTTTA-3′.

Drug similarity parameters.

The properties for drug similarity were analyzed according to Lipinski's rule of five (molecular mass of ≤500 Da, logP [logarithm of compound partition coefficient between n-octanol and water] of ≤5, H-bond donors [HBD] of ≤5, and H-bond acceptors [HBA] of ≤10) and using topological polar surface area (TPSA) values from the Molinspiration online property calculator tool kit, using the Molinspiration property calculation program (http://www.molinspiration.com/services/properties.html). TPSA was used to calculate the percentage of absorption (% Abs) according to the equation % Abs = 109 − (0.345 × TPSA) (19).

Assay of drug-induced nitric oxide production in murine macrophages.

The assay to measure nitric oxide production by murine peritoneal macrophages was previously described (20). In brief, murine macrophages were seeded in 24-well plastic plates at a density of 2 × 10⁵ cells per well and allowed to adhere for 24 h at 37°C in a humidified atmosphere containing 5% CO₂. Afterwards, the medium was replaced by fresh RPMI medium containing two different concentrations of the selected compounds and cells were incubated for 72 h. Then, 100 μl of culture supernatants was incubated with an equal volume of Griess reagent (Panreac) (0.5% sulfanilamide and 0.05% naphthylene-diamide dihydrochloride in 2.5% H₃PO₄) for 30 min at room temperature. Absorbance was measured using a Multiskan EX microplate photometer plate reader at 540 nm. The nitrite concentration in the medium was determined from the calibration curve obtained by using different concentrations of sodium nitrite (Sigma, MO, USA).

Statistical analysis.

The statistical analyses were carried out with GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Comparisons between two groups were performed by the Student t test. Statistical differences between more than two groups were examined with the analysis of variance (ANOVA). P values of <0.05 were considered statistically significant, and P values of <0.01 or P values of <0.001 were considered very significant. Data were represented as means ± SDs.

RESULTS

Ability to be administered as a drug and bioavailability.

The physicochemical properties of the molecules are important aspects to be considered in a drug discovery process. As a filter for drug-like properties, Lipinski descriptors for bioavailability estimation were calculated for the compounds using the freely accessible program MIPC (Molinspiration Property Calculator) (http://www.molinspiration.com/services/properties.html). Results are shown in Table 1. Lipinski parameters revealed some important pharmacokinetic molecular properties of the drugs in the human body, especially their oral absorption. According to those criteria, a good oral drug must not violate more than one of the following criteria: <5 hydrogen donors (nOHNH), <10 hydrogen acceptors (nON), molecular weight (MW) of <500, logP [logarithm of compound partition coefficient between n-octanol and water] value of <5. Moreover, the topological polar surface area (TPSA), a predictive indicator of membrane penetration value, was also calculated. The compounds 2b, 4b, and 8b largely complied with Lipinski's rule of five, although 5b violated only one parameter (Table 1). Our results strongly suggest that these methylseleno-imidocarbamates might be orally administered, especially those with the thiophene templates. These molecules could then be considered promising candidate drugs for further development of leishmanicidal agents.

TABLE 1.

Pharmacokinetic parameters important for good oral bioavailability of compounds^a

Compound	LogP (preferred value, ≤5)	TPSA	% Abs	MW (preferred value, ≤500)	nON (preferred value, ≤10)	nOHNH (preferred value, ≤5)	Vol (Å3)	No. of Lipinski violations (preferred value, ≤1)
2b	2.52	58.53	88.81	357.32	4	1	250.56	0
4b	2.06	150.18	57.19	447.31	10	1	297.23	0
5b	5.13	58.53	88.81	457.44	4	1	338.54	1
8b	2.69	84.32	79.91	447.36	6	1	348.80	0

^aAbbreviations: LogP, logarithm of compound partition coefficient between n-octanol and water; TPSA, topological polar surface area; % Abs, percentage of absorption; MW, molecular weight; nON, number of hydrogen bond acceptors; nOHNH, number of hydrogen bond donors.

Compounds 2b and 4b inhibit L. major proliferation in vitro.

Four methylseleno-imidocarbamates and paromomycin were screened against the Leishmania major promastigote stage using the MTT method, and results were expressed as IC₅₀s at 48 and 72 h. As the reference drug for the assays, cells were grown in the presence of different concentrations of paromomycin, an aminoglycoside antibiotic which has been used in cutaneous leishmaniasis treatment.

For each agent, dose-response curves were drawn to determine the drug's IC₅₀ at 48 h (Fig. 2). From the resultant dose-response curve, the IC₅₀ of this control drug was less than 20 μM after 48 h of incubation (Table 2). After screening selenocompounds against L. major promastigotes, our results indicated that all the drugs showed more effectiveness in vitro (IC₅₀ of <10 μM) against parasites than did paromomycin (IC₅₀ of ≈20 μM) at 48 h (Table 2). Of the four compounds tested, two exhibited an IC₅₀ lower than 3 μM (2b and 4b), one showed an IC₅₀ of approximately 5 μM (8b), and the last one showed an IC₅₀ near 10 μM (5b). For all compounds, a time-dependent decrease of effectiveness was observed (Fig. 2; Table 2). Therefore, these selenocompounds dramatically inhibit the proliferation of L. major promastigote forms.

FIG 2.

Dose-response curves of Leishmania major promastigotes treated for 48 and 72 h at 26°C with the selected selenocompounds 2b (A), 4b (B), 5b (C), and 8b (D) and with a reference drug, paromomycin (PM) (E). Experiments were conducted in triplicate. The plots indicated represent the means (± standard deviations) of growth inhibition values measured at each concentration.

TABLE 2.

IC₅₀s of bisacylimidocarbamate selenocompounds and paromomycin on Leishmania major promastigotes (24- and 72-h treatment) and RAW 264.7 macrophages (48-h treatment)

Drug	IC50 (μM) for cell type and duration of treatment			SI
	L. major promastigotes		RAW macrophages (48 h)
	48 h	72 h
Compounds
2b	2.25 ± 0.43	4.96 ± 0.17	13.42 ± 1.56	5.96
4b	2.78 ± 0.19	3.19 ± 0.10	22.47 ± 2.92	8.08
5b	8.67 ± 1.20	12.14 ± 2.32	NTa
8b	4.77 ± 0.72	8.79 ± 1.26	NT
Paromomycin	18.51 ± 0.92	29.72 ± 4.35	>1 mM	>54

^aNT, not tested.

Cytotoxicity in macrophages and in vitro therapeutic index (SI).

We decided to continue the screening with those compounds that had IC₅₀s below 5 μM for promastigotes. Of the four compounds tested, 2b and 4b had IC₅₀s of <5 μM, with IC₅₀s of 2.25 ± 0.43 and 2.78 ± 0.19 μM, respectively. Therefore, compounds 2b and 4b, displaying antiparasitic activity, were assayed for cytotoxicity against the macrophage cell line RAW 264.7. Both molecules showed IC₅₀s of >10 μM (13.42 ± 1.56 for 2b and 22.47 ± 2.92 μM for 4b). Their corresponding selective toxicity values (SI), defined as the ratio between the IC₅₀ obtained in macrophages and that in parasites, were determined. In both cases, SI was higher than 5 (Table 2). All these data demonstrated that the leishmanicidal activity of these two compounds is selective, inhibiting L. major growth notably without inducing significant host cell toxicity.

Burden of amastigotes after treatment is highly reduced.

The effectiveness of compounds against intracellular parasites is crucial and does not necessarily correlate with the activity on cultured parasites. When exposed to different concentrations (∼0, 1.25, or 2.5 μM IC₅₀ of compound 2b or ∼3 μM IC₅₀ of compound 4b) of the drugs, peritoneal murine macrophages were viable and their survival was more than 80% (data not shown). We then treated for 72 h the infected peritoneal macrophages at the same concentrations of each drug and checked whether these treatments could efficiently trigger amastigote death. The number of infected macrophages was microscopically determined, counting 200 cells in three independent duplicate experiments. As positive controls, cells were treated with 50 μM paromomycin, a leishmanicidal drug. Compared to untreated cells (considered 100%), there was a significant decrease in the number of infected macrophages after treatment at those doses (Fig. 3A). Only 28.72% ± 10.67% and 23.90% ± 4.15% of macrophages remained infected after 72 h of exposure to 2b (∼2.5 μM IC₅₀) and 4b (∼3 μM IC₅₀), respectively (Fig. 3A). However, with 50 μM paromomycin for the same period of time, 54.26% ± 4.48% of macrophages remained infected.

FIG 3.

Effects of selenocompounds and paromomycin (PM) on Leishmania major-infected peritoneal murine macrophages. The drug treatments were performed at different concentrations (1.25 and 2.5 μM compound 2b, 1.25 and 3 μM compound 4b, and 50 μM paromomycin). (A) Percentages of macrophages that remained infected after 72 h of treatment. Both compounds, as well as paromomycin, induced significant reductions (*, P < 0.05; **, P < 0.01) in numbers of infected macrophages. (B) The number of parasites per infected cell after 72 h of treatment also decreased. The controls represent the untreated cells. The results indicate the means from three independent duplicate experiments.

Additionally, the analysis of infected macrophages allowed us to determine the number of amastigotes per macrophage at the abovementioned drug concentrations (Fig. 3B). In both cases, a reduction from 3.89 ± 1.50 in control to 2.26 ± 0.60 when treated with 2.5 μM (∼IC₅₀) 2b and to 2.50 ± 0.34 with 3 μM (∼IC₅₀) 4b was observed. These levels were similar to those observed with our reference drug paromomycin (2.11 ± 0.19) (Fig. 3B).

Compounds 2b and 4b dramatically alter the cell cycle.

Because both compounds inhibited cell growth, we also studied their role in the cell cycle. Exponentially growing cells were treated with 2.5 μM 2b or 3 μM 4b for 24 h. Significant effects of 2b and 4b on the cell cycle were observed in L. major promastigotes compared to untreated cells (Table 3), consisting in an accumulation of cells in the G₁ phase and a reduction of the percentage of cells in the G₂/M phase. However, the proportion of cells in the S phase remained unchanged. Compound 2b induced a cell cycle arrest in G₁ (from 42.94% ± 2.35% to 49.49% ± 3.85%, P = 0.0657) and a significant cell decrease in the amount of cells in G₂/M (from 42.66% ± 3.78% to 34.04% ± 1.54%, P < 0.05). Similarly, 4b dramatically increased the parasite population in the G₁ phase (from 42.94% ± 2.35% to 50.16% ± 0.85%, P < 0.01) and produced a reduction in the number of cells in the G₂/M phase (35.93% ± 2.77%, P = 0.0677) (Table 3).

TABLE 3.

Effects of compounds 2b and 4b on cell cycle distribution in promastigotes of Leishmania major

Compound	% of cells in phase of cell cyclea:
	Sub-G0	G1	S	G2/M
Control	1.81 ± 0.60	42.94 ± 2.35	11.85 ± 3.89	42.66 ± 3.78
2b	2.47 ± 0.21	49.49 ± 3.85	13.59 ± 2.26	34.04 ± 1.54*
4b	2.65 ± 1.40	50.16 ± 0.85**	10.75 ± 2.80	35.93 ± 2.77

^aSignificance: *, P < 0.05; **, P < 0.01.

Both compounds reduce PCNA gene expression.

Since both drugs (2b and 4b) significantly affected proliferation, we asked whether expression of genes related to proliferation would be altered. For this purpose, we selected PCNA, the gene for which is highly expressed in actively proliferating promastigotes (21). PCNA expression was then measured by quantitative RT-PCR in L. major promastigotes treated with 2.5 μM 2b or 3 μM 4b for 24 h. mRNA levels of PCNA were significantly downregulated in parasites exposed to 2.5 μM 2b (P < 0.01; Fig. 4), but no significant changes were observed for 4b. Therefore, the PCNA gene, a member of the proliferating gene family, is downregulated by our methylseleno-imidocarbamate compound 2b.

FIG 4.

PCNA gene expression analysis by quantitative real-time PCR in Leishmania major promastigotes exposed to 2.5 μM compound 2b and 3 μM compound 4b for 24 h. Data are presented as mean fold changes (± standard errors) of gene expression compared to control (untreated) (**, P < 0.01).

These novel drugs affect alpha-tubulin and QDPR gene expression.

Different genes (ABC transporter, alpha-tubulin, and QDPR genes) related to treatment, resistance, and virulence were also analyzed by quantitative PCR to assess whether their gene expression may be affected by our compounds at 2.5 μM 2b and 3 μM 4b for 24 h. Recent data have reported the role of ABC transporters in conferring drug resistance in Leishmania (22). In our experimental settings, both drugs slightly increased gene expression (∼1.3- to 1.5-fold compared to the levels in untreated parasites) of ABC transporter, but without statistical significance (P > 0.05) (Fig. 5A). Next, we studied the quinonoid dihydropteridine reductase (QDPR) gene, a gene whose high levels throughout the parasite infectious cycle may contribute to virulence (22). It has been suggested that parasite QDPR may be a useful target for chemotherapy (23). These data prompted us to measure QDPR gene expression after treatment with 2b and 4b. A significant downregulation was observed in both cases, particularly when cells were exposed to 2b (P < 0.05) (Fig. 5B).

FIG 5.

Gene expression analysis by quantitative real-time PCR in Leishmania major promastigotes exposed to 2.5 μM compound 2b and 3 μM compound 4b for 24 h. (A) ABC transporter. (B) Quinonoid dihydropteridine reductase (QDPR). (C) Alpha-tubulin. Data are presented as mean fold changes (± standard errors) of gene expression compared to control (untreated) (*, P < 0.05; **, P < 0.01).

Finally, we analyzed alpha-tubulin, a Leishmania structural protein whose gene expression changes depending on the morphological variation during the parasite life cycle. It is a key component of the cytoskeleton, responsible for cell shape and involved in cell division, ciliary and flagellar motility, and intracellular transport (24). Recently, alpha-tubulin has been related to drug resistance (25, 26). In fact, proteomic analyses demonstrated that this protein is commonly more abundant in Sb(III)-resistant Leishmania lines (27). The two compounds in our study significantly reduced alpha-tubulin gene expression (100% in control versus 71.16% ± 12.88% in parasites exposed to 2b and 30.23% ± 10.94% in those treated with 4b) (P < 0.01) (Fig. 5C).

NO production in murine macrophages.

Nitric oxide (NO) production is critical in macrophages for defense against intracellular pathogens such as Leishmania major (13, 28). Nitric oxide was measured indirectly by the Griess method in the supernatant of macrophages treated over a period of 72 h with 2b (1.25 and 2.5 μM) and 4b (1.25 and 3 μM) compounds and paromomycin (50 μM) as a positive control, since it has been demonstrated to be an inducer of nitric oxide in vitro (29). We observed that NO was increased by both compounds by more than 1.6-fold compared to the control compound paromomycin (P < 0.05) (Fig. 6). Although statistical significance was obtained only for compound 2b (P < 0.05), these data demonstrated that these compounds induced nitric oxide production in macrophages.

FIG 6.

Induction (mean ± standard deviation) of nitric oxide (NO) production in murine macrophages after 72 h of treatment with compound 2b (1.25 and 2.5 μM) or 4b (1.25 and 3 μM) compared to that after paromomycin (PM) (50 μM) exposure. Both drugs dramatically augmented nitric oxide levels. Such an increase was significant with 2b (*, P < 0.05).

DISCUSSION

The generation of new leishmanicidal drugs has become a worldwide priority. Selenium derivatives have shown promise for the treatment of several diseases, including leishmaniasis (10, 30). It is well known that oral administration of a drug remains one of the most convenient routes and generally carries the lowest cost. However, oral drugs against cutaneous, mucocutaneous, and visceral leishmaniasis are almost nonexistent. Recently, miltefosine was approved as a new oral drug in the United States to treat disease caused by specific Leishmania species. The discovery of novel oral agents effective against leishmaniasis is therefore an outstanding challenge. These criteria were used to select the compounds used in our study. Based on ability to be administered as a drug and on bioavailability criteria (such as Lipinski's rule of five), four compounds (2b, 4b, 5b, and 8b) were selected from our database representing those with good pharmacokinetic parameters, useful for suitable oral bioavailability (19; also see http://www.molinspiration.com/services/properties.html). Afterwards, the analysis of their activity against Leishmania major was achieved. It is notable that 2b and 4b previously exhibited the best pharmacokinetic profile and were the most active in vitro against L. major (<3 μM). More experiments were performed to assess their action on Leishmania proliferation. Those two drugs showed a low IC₅₀ and, subsequently, a dramatic parasite proliferation inhibition and were therefore chosen for our studies. Their cytotoxicity in macrophages was low (IC₅₀s of >10 μM), and they were more than 5 times more active for promastigotes of L. major than for macrophages.

A relationship between the bulk of the heteroaryl group and antiproliferative activity has been previously described. In fact, when the size of the ring was increased, a loss of antiproliferative activity was observed (12). This coincides with the IC₅₀s obtained for promastigotes.

In addition, the effectiveness of both compounds against intracellular parasites was assessed. There was a dramatic reduction in numbers of infected macrophages after 72 h of treatment. Such a decrease was higher than that observed with paromomycin, our reference leishmanicidal drug. Moreover, the burden of amastigotes in infected macrophages exposed to the drugs was similar to that in infected cells treated with paromomycin. Taken together, these results show that both compounds are potential drugs against Leishmania infection.

These compounds also presented a potent effect on the cell cycle, inducing arrest in G₁. The induction of G₁ cell cycle arrest and cell death by selenium compounds has been postulated to contribute to their biological activities in several cell lines, including tumors (31–33). Interestingly, a protein associated with some vital cellular processes such as the abovementioned cell cycle is the well-known PCNA (proliferating cell nuclear antigen). PCNA is a eukaryotic evolutionarily well-conserved protein. Besides cell cycle control, it is involved in DNA replication, chromatin remodeling, and DNA repair (34). In the G₁ phase, PCNA was shown to be highly expressed in actively proliferating promastigotes throughout the cell cycle (21). During this phase, cells grow in size and prepare for division if the metabolic conditions are optimum. Proteins participating in this phenomenon are cyclins, cyclin-dependent kinases, and PCNA. Inhibition of the cell's DNA repair systems is a promising strategy to improve therapies. In cancer and other pathologies, including leishmaniasis, PCNA may be a novel, promising therapeutic target. Here, we demonstrated that exposure to treatments effectively downregulated PCNA mRNA levels. Therefore, this process may explain the inhibition of proliferation induced by both drugs. More papers have also been published explaining some similar mechanistic exploratory assays that were performed and showing the downregulation of PCNA by different drugs. Recently, PCNA downmodulation was described to be induced by the antitumor drug trastuzumab at mRNA levels in cancer cells (35).

Resistance to the current leishmanicidal drugs is one of the major therapeutic challenges in the treatment of this disease. Several genes have been described to be involved in virulence and resistance to therapy (22, 25–27). Therefore, some of those genes related to treatment resistance and parasite virulence were also assessed. On the one hand, alpha-tubulin, which had been demonstrated to be more abundant in Sb(III)-resistant Leishmania strains (27), was analyzed. Interestingly, its gene expression was significantly reduced after treatment. In fact, a downregulation of alpha-tubulin mRNA levels had been described in response to stress (24). These data reinforced the abovementioned action of compounds 2b and 4b against Leishmania spp. In addition, quinonoid dihydropteridine reductase (QDPR), a novel target for chemotherapy and a virulence gene product required for regeneration and maintenance of tetrahydrobiopterin (BH4) pools (23), was also altered. This reduction of its mRNA levels supports the finding that the evaluated drugs may decrease the virulence of L. major parasites.

Since, in addition to catalyzing tetrahydrobiopterin (BH4) regeneration, QDPR is a cofactor for monoamine synthesis, phenylalanine hydroxylation, and nitric oxide (NO) production (23), we were finally prompted to assess NO generation. Nitric oxide production had been shown previously to provide tissue-wide immunity during Leishmania infection (36). Furthermore, some studies had mentioned that the killing of L. major is mainly dependent on the production of nitric oxide (13). We then studied nitric oxide (NO) induction after exposure of macrophages to the drugs. Interestingly, upon paromomycin treatment, an increase in NO levels was observed in macrophages (37). Both drugs studied (2b and 4b) notably enhanced NO levels above those with the reference compound paromomycin. Therefore, the ability of both drugs to decrease parasite burden may depend on the stimulation of NO production.

The small increase of ABC transporter gene expression in parasites treated with the analyzed molecules would indicate a startup mechanism of resistance in Leishmania major. However, Leprohon et al. demonstrated in Leishmania infantum that the overexpression of some ABC transporter genes may not result in drug resistance (38). Therefore, the potential role of ABC transporter in resistance to selenium derivatives requires further experimental work.

Finally, as our methylseleno-imidocarbamates seem to exhibit good oral administration properties, especially those with the thiophene templates, they might be promising scaffolds for future structural optimization and as potential leishmanicidal drugs. Taken together, these data show an initially encouraging potential for the use of both compounds (2b and 4b) to treat cutaneous leishmaniasis.