ABSTRACT
Conventional chemotherapy against leishmaniasis includes agents exhibiting considerable toxicity. In addition, reports of drug resistance are not uncommon. Thus, safe and effective therapies are urgently needed. Isoselenocyanate compounds have recently been identified with potential antitumor activity. It is well known that some antitumor agents demonstrate effects against Leishmania. In this study, the in vitro leishmanicidal activities of several organo-selenium and organo-sulfur compounds were tested against Leishmania major and Leishmania amazonensis parasites, using promastigotes and intracellular amastigote forms. The cytotoxicity of these agents was measured in murine peritoneal macrophages and their selectivity indexes were calculated. One of the tested compounds, the isoselenocyanate derivative NISC-6, showed selectivity indexes 2- and 10-fold higher than those of the reference drug amphotericin B when evaluated in L. amazonensis and L. major, respectively. The American strain (L. amazonensis) was less sensitive to NISC-6 than L. major, showing a trend similar to that observed previously for amphotericin B. In addition, we also observed that NISC-6 significantly reduced the number of amastigotes per infected macrophage. On the other hand, we showed that NISC-6 decreases expression levels of Leishmania genes involved in the cell cycle, such as topoisomerase-2 (TOP-2), PCNA, and MCM4, therefore contributing to its leishmanicidal activity. The effect of this compound on cell cycle progression was confirmed by flow cytometry. We observed a significant increase of cells in the G1 phase and a dramatic reduction of cells in the S phase compared to untreated cells. Altogether, our data suggest that the isoselenocyanate NISC-6 may be a promising candidate for new drug development against leishmaniasis.
INTRODUCTION
Leishmaniasis is a group of diseases caused by protozoan parasites from the genus Leishmania transmitted by female sand flies. In the 21st century, leishmaniasis remains a major health problem in numerous developing countries. According to World Health Organization (WHO) data, these infections are endemic in 98 countries worldwide (except Australia and Antarctica), with more than 350 million people at risk. Around 2 million cases of leishmaniasis are reported every year and estimated mortality is over 20,000 deaths annually. There are more than 20 pathogenic Leishmania species responsible for the clinical manifestations ranging from localized skin lesions to fatal visceral parasitemia. Cutaneous leishmaniasis (CL) is the most common form of the disease, with up to 1.2 million cases each year in regions where it is endemic (1). Leishmania major is the main causative agent of localized cutaneous leishmaniasis in the Old World. Mucocutaneous leishmaniasis is a clinical form of the disease which usually occurs after resolution of cutaneous lesions, and whose etiologic agent in the New World is, among others, Leishmania amazonensis. Both leishmaniasis forms result in disfiguring scars and lesions with social consequences (2). Currently, effective human vaccines are not available and antileishmanial drugs are unaffordable for most of the affected people. The facts that the treatments are long term and have toxic side effects and that resistant parasites are appearing makes the development of new leishmanicidal compounds an urgent necessity. Therefore, active, safe, and affordable medicines are greatly needed to treat this group of diseases. A strategy currently used to identify new leishmanicidal treatments is the so-called drug repurposing. Consequently, a large battery of antitumor compounds, such as gimatecan and other camptothecin derivatives, indenoisoquinolines, paclitaxel, and methylseleno-imidocarbamates, have demonstrated activity against Leishmania (3–9). However, the only oral drug reported to have an effect against this parasitic disease is miltefosine, an alkylphospholipid with antineoplastic effects and originally developed for breast cancer treatment. Similarly, based on their antitumor activity, several naphthalimide derivatives were postulated as promising leishmanicidal compounds. Although some naphthalimide analogs have been reported as hopeful anticancer drugs (10–12), these compounds exhibited toxicity issues. In 2011, Hossain Sk et al. improved the maximum tolerated dose of some naphthalimide analogs in nude mice by adding isothiocyanate and thiourea functionalities to their structure, leading to the identification of some potent naphthalimide-isothiocyanates (NITC) and -thiourea (NTU) compounds (13). Similarly, in another report, Sharma et al. increased the anticancer properties of the naturally occurring phenylalkyl isothiocyanates by incorporating selenium isosteric into sulfur to generate isoselenocyanate compounds and established phenylbutyl isoselenocyanate (ISC-4) as the most efficacious compound (14, 15). More recently, Karelia et al. analyzed selenium derivatives of these naphthalimide analogs to discover the corresponding naphthalamide-isoselenocyanate (NISC) and -selenourea (NSU) compounds, which led to the identification of dual topoisomerase 2 and the Akt pathway inhibitor activity of NISC-6 (16). In 2015, our group also demonstrated the leishmanicidal activity of selenocompounds (7), previously designed as antitumoral agents (17). In fact, selenium derivatives had been shown to reduce parasitemia and decrease clinical manifestations in infected mice (18). In this study, we assessed the antiproliferative activity of ISC, NITC, NISC, NTU, and NSU compounds against promastigotes and intracellular amastigotes of L. major and L. amazonensis (Fig. 1). First, these drugs fulfilled the criteria for good oral bioavailability. Cytotoxicity assays in murine peritoneal macrophages allowed the selection of ISC-4, NISC-6, and NTU-2 for further experiments. Our results revealed that NISC-6 was more active in the Old World strain L. major than in the American strain L. amazonensis. In fact, NISC-6 exhibited the highest selectivity index (SI) value in L. major, up to 10-fold over the SI for amphotericin B, the reference drug. According to the data obtained from promastigotes, L. major amastigotes also seemed to be more sensitive to NISC-6. We also demonstrated its implication in the inhibition of L. major TOP-2 (topoisomerase-2), PCNA (proliferating cell nuclear antigen; involved in DNA replication and drug resistance), and MCM4 (mini-chromosome maintenance complex) gene expression levels. We confirmed the activity of this mitonafide derivative on the cell cycle of Leishmania, where it induced a significant increase of cells in the G1 phase and a dramatic reduction of cells in the S phase.
Structures of sulfur and selenium compounds.
RESULTS
Druggability and bioavailability.The theoretical values of logP obtained using Molinspiration free online software were within the recommended range of 0 ≤ logP ≤ 5 (Table 1). Hence, all the compounds should have a good hydrophilicity/lipophilicity ratio to be bioavailable compounds. Likewise, the obtained polar surface area values were much lower than 140, predicting adequate intestinal absorption (Table 1). Overall, the sulfur and selenium compounds fulfilled every parameter of the Lipinski’s Rule of Five. Accordingly, these derivatives might present bioavailability, metabolic stability, and transport properties comparable to known drugs.
Parameters for good oral bioavailability of compounds
Cytotoxicity in murine peritoneal macrophages.The cytotoxicity of sulfur and selenium compounds and amphotericin B (Ampho B), a polyene antibiotic with antifungal activity and currently used in the clinic against leishmaniasis, was studied in murine peritoneal macrophages. The 50% inhibitory concentrations (IC50s) were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Mammalian cells extracted from BALB/c mice were seeded in the presence of increased concentrations of the compounds and, 72 h after treatment, dose-response curves were analyzed to calculate the IC50s. All of the compounds exhibited an IC50 higher than that of amphotericin B (Table 2), within a micromolar range. Four (NITC-6, NNITC-2, NNISC-2, and NSU-2) exhibited IC50s at 72 h lower than 10 μM (between 3.7 and 6.0 μM) (Table 2). NISC-6 showed an IC50 around 12 μM and ISC-4 showed an IC50 greater than 10, whereas NTU-2 seemed to be the least cytotoxic compound with an IC50 of >21 μM. Taking into account the cytotoxicity in murine peritoneal macrophages, we also decided to analyze the antileishmanial activity of those compounds exhibiting IC50s greater than 10 μM. Therefore, ISC-4, NISC-6, and NTU-2 were then selected.
IC50 and SI (selectivity index) of isoselenocyanate and amphotericin B on murine peritoneal macrophages and promastigotes at 72 h posttreatmenta
NISC-6 inhibits promastigotes proliferation in vitro.The activity of compounds against promastigote forms is extensively used to estimate the selectivity index (SI) values. L. major and L. amazonensis promastigotes were exposed to increased concentrations of the less toxic compounds, ISC-4, NISC-6, NTU-2 and to Ampho B as the reference drug. For each agent, dose-response curves were plotted to determine the drug’s IC50 at 72 h (Fig. 2). After screening compounds against L. major and L. amazonensis promastigotes using an MTT assay, the results indicated that only NISC-6 exhibited a higher or similar leishmanicidal activity compared to the reference drug (Ampho B) (Table 2). In fact, in vitro assays revealed that the IC50 values of NTU-2 and ISC-4 were 70 times (7.04 and 7.18 μM, respectively), higher than that of Ampho B (0.1 μM) against L. major promastigotes. Against the L. amazonensis strain, the IC50 values for these compounds were around 20-fold (ISC-4, 4.46 μM) and 60-fold (NTU-2, 12.19 μM) above the reference drug’s values (Ampho B, ∼0.2 μM).
Dose-response curves of L. major and L. amazonensis promastigotes treated during 72 h at 26°C with the selected compounds ISC-4 (A), NISC-6 (B), NTU-2 (C) and with the reference drug amphotericin B (Ampho B) (D). Experiments were performed in triplicate. The plots show the means ± the SD of growth inhibition values measured at each concentration.
The most interesting results corresponded to NISC-6. We observed that NISC-6 dramatically inhibited the Leishmania promastigote proliferation in vitro showing an IC50s within a nanomolar range of up to 3-fold higher than that of Ampho B. The IC50 of this drug against L. major promastigotes was 30 nM (0.030 ± 0.0064 μM), while against L. amazonensis parasites, the IC50 was 0.28 μM, a value significantly higher, and similar to that observed for the reference drug (Table 2). These results showed that our compounds were more active in the Old World strain L. major than in the American strain L. amazonensis, which showed less sensitivity compared to most of the studied treatments.
In vitro selectivity index.The SI value is the ratio between the IC50 obtained in macrophages and that in parasites. Our aim here was to find compounds with a great antiparasitic activity and a low toxicity in mammalian macrophages, the typical host cells of Leishmania parasites. For this purpose, a suitable compound will show a high SI value. Each compound tested exhibited an SI above 1.5 (Table 2), indicating higher leishmanicidal activity than cytotoxicity (macrophages). However, ISC-4 and NTU-2 showed SI values lower than those of the reference drug (Ampho B) in both cutaneous strains. For instance, although Ampho B showed leishmanicidal activity values higher than those of ISC-4, NISC-6, and NTU-2, it was more toxic in mammalian cells. The SI values of ISC-4 appeared quite similar (1.4 and 2.2) in both Leishmania strains, whereas NTU-2 seemed to be more suitable against L. major strain (3 versus 1.7 in L. amazonensis) (Table 2). On the other hand, NISC-6 exhibited the highest SI value in L. major (SI = 341.91), up to 10-fold over the SI for the reference drug (SI = 41). Interestingly, since the American strain (L. amazonensis promastigote) was previously described to be less sensitive to Ampho B, a similar trend was observed with NISC-6. In fact, its SI value (SI = 41.7) for L. amazonensis was also lower than the value calculated (SI = 416.7) for L. major (Table 2). However, the value remained 2-fold higher than that obtained for Ampho B (41.7 versus 20.5).
Efficacy of NISC-6 against intracellular forms of the parasite.As described above, NISC-6 showed an SI value higher than that of Ampho B, the reference drug used in our screening studies on the promastigote forms. Although NISC-6 was active against promastigotes, the percentage of infected macrophages remained similar to that of controls (untreated cells) (data not shown). We then assessed its activity on amastigotes. To calculate the activity of the studied compounds against intracellular amastigotes, macrophages from the exudate peritoneal of BALB/c mice were infected with L. major and L. amazonensis parasites. Infected cells were treated during 72 h, and the number of intracellular parasites was microscopically determined as described in Materials and Methods.
Infected macrophages with L. major were treated at different concentrations corresponding to 10 nM (∼IC50 promastigote/3) and 100 nM (∼IC50 promastigote × 3). Similarly, L. amazonensis-infected macrophages were also exposed to concentrations such as 100 nM (∼IC50 promastigote/3) and 1 μM (∼IC50 promastigote × 3). The selected concentrations of the compounds had shown no toxicity for the peritoneal macrophages (Table 2). Due to its high effectiveness on Leishmania infections, Ampho B was selected as the reference treatment. NISC-6 exhibited less activity compared to Ampho B at the same concentration (1 μM) (Fig. 3). Nevertheless, in both Leishmania strains, we observed a significant decrease in the burden of intracellular parasites after treatments with increased concentrations of NISC-6.
Effects of NISC-6 and amphotericin B (Ampho B) on intracellular amastigotes of L. major (A) and L. amazonensis (B). The drug treatments were performed at different concentrations (10 nM, 100 nM, and 1 μM NISC-6 and 1 μM amphotericin B). Bars represent the number of parasites per infected cell with L. major and L. amazonensis after 72 h of treatment. The controls represent the untreated cells. The results show the means from three independent duplicate experiments ± the SD. Significant reductions in the number of amastigotes per infected macrophages were observed (*, P < 0.05; **, P < 0.01; ***, P < 0,001).
According to the results obtained from promastigotes (Table 2), L. major amastigotes also seemed to be more sensitive to NISC-6 (Fig. 3). The percentage of intracellular parasites per macrophage decreased very significantly (**, P < 0.01), i.e., from 19.4 ± 0.2 (untreated infected macrophages) to 13.9 ± 0.7 when infected cells were exposed to the lowest concentration (10 nM = IC50 promastigote/3) for 72 h (Fig. 3). Furthermore, at 100 nM (=IC50 promastigote/3), the percentage of L. amazonensis amastigotes per macrophage was significantly reduced (*, P < 0.05). At a concentration of 1 μM (∼IC50 promastigote × 3), the corresponding reduction was also very significant (*, P < 0.01) (Fig. 3).
NISC-6 induces Leishmania cell cycle arrest.Recently, Karelia et al. (16) demonstrated the effect of NISC-6 on human TOP-2, a molecule involved in cell proliferation. Therefore, we also aimed to analyze the effect of NISC-6 on mRNA expression of Leishmania genes related to cell replication such as TOP-2, PCNA, and MCM4. For this purpose, an L. major promastigote culture was treated with two concentrations of NISC-6 (25 and 50 nM) for 24 h. We observed a significant decrease of TOP-2, PCNA, and MCM4 mRNA expression levels in NISC-6-treated parasites compared to those from untreated cells (Fig. 4A, C, and D). Further, we observed a significant correlation between the mRNA expression of PCNA and TOP-2 (R2 = 0.75, P < 0.0001) and between the mRNA expression of PCNA and MCM4 (R2 = 0.79, P < 0.0001) (data not shown).
Effect of NISC-6 on Leishmania replication. mRNA levels of Leishmania topoisomerase-2 (A), PCNA (C), and MCM4 (D) after 24 h of treatment without or with two concentrations of the compound NISC-6 (0, 25, and 50 nM). (B) Cleavage complex formation, measured as disintegrations per minute (dpm), after 36 h of [methyl-3H]thymidine L. major radiolabeled, followed by 3.5 h of NISC-6 (50 nM), etoposide (20 μM) or no treatment (no drug exposure). The bars represent the means ± the SD from two independent experiments performed in sextuplicate. Significant reduction in the levels of Leishmania TOP-2, PCNA, and MCM4 after NISC-6 treatments was observed (*, P < 0.05).
NISC-6 was examined for its ability to induce cleavable complex in a whole-cell assay. The proportion of covalent DNA-TOP-2 cleavage complexes is increased after the exposure to some drugs, such as etoposide (19). Aforementioned complexes become irreversible upon the addition of a protein denaturant. Afterward, the addition of KCl solution then precipitates these complexes. Our data revealed that NISC-6 also dramatically increased cleavage complex formation in L. major (Fig. 4B), therefore supporting the inhibitor effect of NISC-6 on L. major TOP-2 activity (Fig. 4B).
Subsequently, when we analyzed the percentage of parasites in each cell cycle phase after NISC-6 treatment, we detected a significant increase of cells in the G1 phase and a dramatic reduction of cells in the S phase compared to untreated cells (Fig. 5). These data reinforced the idea that NISC-6 was able to modify cell cycle progression in Leishmania, inducing G1 arrest.
Effect of NISC-6 on Leishmania cell cycle progression. (A) Percent of cells at the different cell cycle phases (G1, S, and G2) after 24 h of treatment without or with two concentrations of the compound NISC-6 (0, 25, and 50 nM). The bars represent the means ± the SD from two independent experiments performed in duplicate. (B) Representative histogram plot of DNA content against cell numbers. A significant increase in the percentage of cells in the G1 phase and significant reduction in the percentage of cells in the S phase after NISC-6 treatments was observed (*, P < 0.05).
DISCUSSION
Leishmaniasis treatment remains a major health priority all over the world. The discovery of novel active molecules is a global challenge. From that perspective, it is crucial to find compounds exhibiting low cytotoxicity and high antileishmanial activity. Host cell functionality is frequently affected by active compounds, since Leishmania is also a eukaryotic cell, although more primitive. In the last decade, anticancer drugs have been explored as candidates against infectious pathologies (9, 20, 21). Recently, several small molecules containing the Se atom have been reported as leishmanicidal agents (7). Here, we evaluated antileishmanial potential of some recently reported sulfur and selenium compounds bearing isothio-/isoselenocyanate and thio-/selenourea moieties attached to a naphthalimide or a phenylalkyl skeleton. All of these compounds have already proven to have very promising antitumor activity and fulfill the Lipinski’s Rule of Five requirements for druglikeness. To our knowledge, this is the first time that isothio- and isoselenocyanate compounds are proposed as novel and potential leishmanicidal agents. The present study focused on the analysis of the cytotoxicity of those compounds on the macrophages as Leishmania’s typical host cells and in their antileishmanial activities.
Our results demonstrated that NISC-6 is relatively potent and the most selective among the compounds tested, including Ampho B. In fact, its SI is about 10-fold higher than that of Ampho B for the L. major strain. In spite of its moderate toxicity shown in mammalian cells, the high leishmanicidal activity of NISC-6 in promastigotes suggested that this drug may be a good candidate against intracellular parasites. This hypothesis was tested through an in vitro infection assay. NISC-6 showed a degree of selectivity against amastigotes of both L. major and L. amazonensis. Interestingly, at low concentrations (10 and 100 nM, around one-third of the IC50 previously calculated in L. major and L. amazonensis promastigotes, respectively), the burden of amastigotes per macrophage was dramatically reduced compared to that observed in controls (untreated infected macrophages). Furthermore, such a reduction was significant in both Leishmania strains. These data indicated that NISC-6 was active against promastigotes and could also reduce Leishmania (L. major and L. amazonensis) burdens in infected macrophages.
Although L. amazonensis promastigotes seemed less sensitive to NISC-6 than L. major when comparing their SI values (41.7 versus 416.7), such a value from L. amazonensis was 2-fold over that obtained with Ampho B (SI = 20.5).
The amastigote is the form of the Leishmania parasite found in the vertebrate host. Due to its relevance, the effectiveness of the tested compounds on this intracellular stage is crucial in screening studies. Our data revealed that NISC-6 may be a useful drug against infections by L. major, as well as L. amazonensis.
NISC-6 was designed using the key functionalities of mitonafide (a naphthalimide analog) and an AKT pathway inhibitor (16). Mitonafide is a well-known TOP-2 inhibitor with systemic toxicity issues (22, 23). In 2011, Karelia et al. used the incorporation of –N=C=S functionality to substantially reduce the cited systemic toxicity (13). Also, the activity of mitonafide on leishmanial nuclear and kinetoplast TOP-2 had been reported (19). Recently, Sk et al. tested NISC-6, a mitonafide derivative and TOP-2A inhibitor, against tumor cells (13). These authors demonstrated that NISC-6 induced cell death in melanoma cells and also inhibited subcutaneous melanoma tumor growth without systemic toxicity. By docking studies, they observed the ability of NISC-6 to fit into the active site of TOP-2, thereby inhibiting its activity. The TOP-2 enzyme is well characterized and related to important cellular processes. Since the TOP-2A protein is conserved in eukaryotic cells and alignment analysis has shown ∼30% homology between human and Leishmania spp. (data not shown), we were prompted to evaluate the modulation of TOP2 in Leishmania at protein level due to the effect of NISC-6. However, currently, specific antibodies against Leishmania TOP-2 are not commercially available. We then performed experiments with nonspecific Leishmania TOP2 antibodies, but the protein was not successfully recognized.
On the other hand, Karelia et al. recently demonstrated that NISC-6 inhibits TOP2 from relaxing the supercoiled DNA (16). It is well known that TOP2α enzyme can convert supercoiled DNA into relaxed DNA strands. Therefore, purified TOP2 enzyme was incubated with supercoiled DNA in the presence of NISC-6 or positive controls (mitonafide or etoposide). After we tested the effect of NISC-6 on TOP2 activity, NISC-6’s ability to inhibit TOP2 activity was concluded (16). Summarizing, supercoiled DNA (pBR322 plasmid DNA) was incubated with TOP2 in the presence of different drug concentration, along with reaction mixture for one hour at 37°C. After incubation, the samples were loaded into wells and separated by electrophoresis on a 1% agarose gel that was subsequently stained with ethidium bromide for visualizing the DNA fragments (16). Consequently, these authors observed that in the presence of just the enzyme and supercoiled DNA, almost all the supercoiled DNA was converted into nicked open DNA. However, in the presence of NISC-6 or increasing dose of a positive control (etoposide or mitonafide), the supercoiled DNA was not converted into nicked open DNA (16).
NISC-6 may also inhibit L. major TOP-2 and dramatically increase DNA-protein complexes formation. We showed that in Leishmania such inhibition also takes place at the gene expression level. Indeed, the gene expression of TOP-2 decreased 24 h after exposure to NISC-6. To our knowledge, these results are complementary to those previously reported (16). Overall, these data demonstrate that NISC-6 is an inhibitor of TOP-2 at both the protein (16) and mRNA levels. In Leishmania, TOP-2 is involved in replication of the kinetoplast DNA (kDNA) network. In Trypanosoma, TOP-2 downregulation by RNAi induces the progressive inhibition of kDNA replication (24). Therefore, topoisomerase-2 is a key enzyme, and its gene and protein are of interest for disease control. The inhibition of TOP-2 is a well-known mechanism of action of several drugs, such as etoposide (25) and some nitroimidazole analogs (26). In addition, Poorrajab et al. postulated that the antiparasitic activity of 1,3,4-thiadiazole derivatives may be by disruption of the DNA-relaxed activities of TOP-1 and TOP-2 (26).
Interestingly, our research confirmed for the first time that some mitonafide derivatives may play a significant role against molecules involved in cell proliferation at the gene expression level. In fact, we also explored some putative mechanisms of action of NISC-6. To achieve this goal, we studied gene expression of some molecules related to TOP-2 activity, such as proliferation markers. In glioma, the relationship between TOP-2 expression and that of proliferating cell nuclear antigen (PCNA) has been noted (27). Furthermore, among the molecules associated with Leishmania replication, some of them related to treatment were considered of interest and studied. For example, PCNA had been described to exhibit a significant role in drug response in Leishmania (7, 28). On the other hand, among the DNA helicases probably involved in DNA replication, MCM4 (minichromosome maintenance complex 4) had been mentioned as one of the most sensitive to drug, such as heliquinomycin (29), and MCM4 had been found in Leishmania, too (30). Moreover, in Leishmania, PCNA colocalizes with MCM4 in the S phase of the cell cycle (30). In 2014, Tandon et al. demonstrated that PCNA plays a significant role in drug resistance in Leishmania clinical specimens (28). Therefore, TOP-2, PCNA, and MCM4 are key molecules for Leishmania biology. In agreement with data previously obtained, we found that the gene expression of these markers of proliferation, TOP-2, PCNA, and MCM4, significantly decreased in parasites treated with NISC-6. Our results revealed a very significant correlation between the mRNA expression of PCNA and TOP-2 and between the mRNA expression of PCNA and MCM4 (data not shown). Finally, through flow cytometry techniques, we confirmed the activity of this mitonafide derivative on the cell cycle of Leishmania, where a significant increase of cells in the G1 phase and a dramatic reduction of cells in the S phase were induced. Therefore, the modification of the cell cycle progression is also a new mechanism of action of NISC-6.
In our case, the molecular basis for the leishmanicidal effect of this agent warrants further analysis. Based on the aforementioned data, we suggest that the isoselenocyanate NISC-6 may be a promising candidate for the development of new drugs against leishmaniasis through its inhibition of the key molecule TOP-2.
MATERIALS AND METHODS
Chemistry.The synthesis, characterization, and purification of the seven sulfur and selenium derivatives (Fig. 1) were carried out according to our previously reported methods (14–16). The purity of the compounds (≥97%) was quantified by analytical high-performance liquid chromatography analysis by comparing the peak areas of the product relative to any impurities.
Amphotericin B (Sigma, St. Louis, MO) was used as reference drug and was dissolved in water at a concentration 250 μg/ml. The compounds studied were dissolved in dimethyl sulfoxide (DMSO) at a concentration of between 0.003 and 0.005 M. Sterile filtrations were achieved using 0.2-μm-pore size filter disks. Serial dilutions with supplemented medium were prepared daily to a final concentration of <2% DMSO in cell culture.
Biological evaluation.(i) Cells and culture conditions. Leishmania major promastigotes (Lv39c5) 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 bovine serum (FBS), and an antibiotic cocktail (50 U/ml penicillin, 50 mg/ml streptomycin). Leishmania amazonensis promastigotes were kindly provided by Basilio Valladares (Instituto de Enfermedades Tropicales y Salud Pública de la Laguna [ULL], Canarias, Spain) and were grown at 26°C in Schneider’s insect medium supplemented with 10% FBS and 40 μg/ml gentamicin. To maintain their infectivity, Leishmania cells were isolated from infected BALB/c mouse spleen and parasites were maintained in culture for not more than five passages.
Murine peritoneal macrophages from 4- to 6-week-old BALB/c mice were used for the study. 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 (31). All the procedures involving animals were approved by the Animal Care Ethics Commission of the University of Navarra.
(ii) Cytotoxicity assays. An MTT test (Sigma, St. Louis, MO) was performed to determine the cytotoxicity of selected compounds in murine macrophages. MTT solutions were prepared at 5 mg/ml in phosphate-buffered saline (PBS), filtered, and maintained at –20°C until use. Briefly, 5 × 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 and, after 72 h of incubation, 100 μg/well of MTT was added, and the plates were incubated 4 h under the same conditions. 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 (32), and the half-maximal inhibitory concentration (IC50) was calculated. The IC50 represents the concentration required for 50% growth inhibition of treated cells with respect to untreated cells (controls). The IC50 was obtained by fitting a sigmoidal Emax model to dose-response curves. The results were expressed as means ± the standard deviations (SD) from three independent experiments.
(iii) Leishmanicidal activity. Activity against promastigotes. 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 the compounds diluted in 100 μl of M199 or Schneider medium and maintained at 26°C. After 72 h of incubation, the IC50 was calculated by the MTT assay as described above.
Activity against intracellular amastigotes. Murine peritoneal macrophages were seeded in 8-well culture chamber slides (Lab-Tek; BD Biosciences) at a density of 5 × 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, stationary L. amazonensis promastigotes and metacyclic L. major promastigotes isolated by the peanut agglutinin method (33) were used to infect macrophages at a macrophage/parasite ratio of 1/20. The plates were incubated for 24 h under the same conditions until promastigotes were phagocytized by macrophages. The wells were then washed with medium to remove the extracellular promastigotes, and plates were incubated with fresh medium supplemented with increasing concentrations of compounds (7). Seventy-two hours later, cells were washed with PBS, fixed with ice-cold methanol for 5 min, and stained with Giemsa stain. To determine parasite burden, the number of amastigotes per 200 infected macrophages was counted under a light microscope. 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.
(iv) Calculation of in vitro therapeutic index (SI). At least three different assays were performed to calculate the SI of each compound, which was determined as the ratio between the IC50 obtained in macrophages and the corresponding IC50 in parasites (34).
(v) Effect of NISC-6 against Leishmania replication. Effect on expression of Leishmania genes involved in the cell cycle. Logarithmic-phase cultures of L. major promastigotes were incubated in 96-well plates at 26°C without or with two concentrations of NISC-6, the IC50 (25 nM) and 2-fold (50 nM) this value. After 24 h, the parasites were recovered by plate centrifugation, and total RNA was extracted using the automated MagMax Express 96 system (Applied Biosystems) total RNA isolation kit (Life Technologies). Reverse transcription was performed as previously reported (35). Real-time PCRs were performed with an iQ SYBR Green supermix (Bio-Rad) in a CFX96 system from Bio-Rad, using specific primers for genes involved in cell cycle: TOP-2, PCNA, and MCM4 (Table 3). The amplification efficiency for each set of primers was also calculated (Table 3). The amount of each transcript was expressed by the formula: 2CT(GAPDH) − CT(gene), with the threshold cycle (CT) being the point at which the fluorescence rises appreciably above the background fluorescence.
Primers used in this study
Effect on Leishmania cell cycle progression. To analyze the effect of NISC-6 on cell cycle progress, L. major parasites in mid-log phase were treated for 14 h with 5 mM hydroxyurea (Sigma) at 26°C (36). Afterward, promastigotes were incubated at 26°C without or with two concentrations of NISC-6, the IC50 (25 nM) and 2-fold (50 nM) this value, for 24 h. The parasites were then collected by centrifugation, washed with cold PBS, fixed in ice-cold 70% ethanol, and incubated at 4°C for 1 h. Subsequently, parasites were collected by centrifugation, treated with RNase A (200 μg/ml; Sigma) at 37°C for 1 h. Propidium iodide (Sigma-Aldrich) was then added to a final concentration of 60 μg/ml. Samples were immediately analyzed using a FACSCanto flow cytometer (BD Biosciences) and the FACSDiva software (BD Biosciences). The percentages of cells in G1, S, and G2 cell cycle phases were obtained using FlowJo software.
Effect on cleavage complex formation. DNA-protein complexes were quantified by the KCl-SDS coprecipitation assay as previously described (19, 37). Briefly, exponentially growing L. major promastigotes (2 × 106 cells/ml) were radiolabeled by adding [methyl-3H]thymidine (Amersham) to the medium to a final concentration of 3 μCi/ml for 36 h at 26°C. The cells were then pelleted by centrifugation, washed twice with PBS, and resuspended in fresh M199 medium supplemented with 10% FBS for 3 h. The cells were exposed to etoposide (20 μM) or NISC-6 (50 nM) or were untreated (no drug exposure) at 26°C for 3.5 h. Finally, the cells were lysed in 250 μl of SDS-containing solution (1.25% [wt/vol] SDS, 5 mM EDTA [pH 8], and 100 μg/ml sheared denatured salmon sperm DNA) during 10 min at 65°C. Then, 200 μl of 325 mM KCl was added to each sample. After vigorous mixing, the samples were cooled on ice for 10 min and centrifuged at 8,000 × g for 30 min at 4°C. The pellets were resuspended in 500 μl of wash solution (10 mM Tris-HCl [pH 8], 100 mM KCl, 1 mM EDTA, and 100 μg/ml sheared denatured salmon sperm DNA) and warmed at 65°C for 10 min with occasional shaking. The suspensions were cooled on ice for 10 min and recentrifuged. The pellets resuspended in 500 μl of wash solution, were mixed with 4 ml of liquid-scintillation, and the radioactivity was determined with a liquid scintillation counter (HIDEX 300 SL).
Statistical analysis.Statistical analysis was performed using PRISM version 5.0 (GraphPad). The data are presented as means ± the SD. Comparisons between two groups were made using Mann Whitney or the two-tailed unpaired t test. The statistical significance was determined (***, P < 0.001; **, P < 0.01; *, P < 0.05).
ACKNOWLEDGMENTS
This study has been funded by Obra Social la Caixa and Fundación Caja Navarra, Gobierno de Navarra Salud (12/2017), Fundación Roviralta, Ubesol, and by Government of Navarre I+D (0011-1383-2018-000005-PI042 NuTeL). J.P.G. was supported by a Ministerio de Educacion Cultura y Deporte fellowship (FPU17/03304).
We acknowledge Paul Miller from the University of Navarra for language editing.
The authors declare that have no competing interests.
FOOTNOTES
- Received 3 May 2018.
- Returned for modification 30 May 2018.
- Accepted 19 November 2018.
- Accepted manuscript posted online 26 November 2018.
- Copyright © 2019 American Society for Microbiology.
