Withanolides from Withania aristata as Antikinetoplastid Agents through Induction of Programmed Cell Death

Leishmaniasis and American trypanosomiasis are parasitic diseases that cause significant clinical, social and economic impact on the population of tropical and subtropical countries. Their current treatment is limited and presents multiple drawbacks, including high toxicity, high cost, lengthy treatment plans, as well as the emergence of resistant species. Therefore, there is a need to find new lead compounds with high potency against parasites and low toxicity in patients. In the present work, the bioguided fractionation of an endemic plant from the Canary Islands, Withania aristata, led to the identification of withanolide-type metabolites (1–3) with leishmanicidal and trypanocidal activities. Compounds 1 and 3 showed a significant dose-dependent inhibition effect on the proliferation of L. amazonensis promastigotes and T. cruzi epimastigotes, higher than the reference drugs, miltefosine and benznidazole, respectively. Moreover, compounds 1–3 were more potent (IC50 0.055–0.663 µM) than the reference drug against the intracellular amastigote stage of L. amazonensis, with a high selectivity index on murine macrophage cells (SI 58.66–216.73). Studies on the mechanism of death showed that the compounds induced programmed cell death or that which was apoptosis-like. The present findings underline the potential of withanolides as novel therapeutic antikinetoplastid agents.


Introduction
Leishmaniasis and American trypanosomiasis (Chagas disease), protozoal diseases, are neglected tropical diseases causing considerable morbidity, affecting millions of people every year worldwide [1]. Leishmaniasis is an infectious disease caused by protozoal parasites of the genus Leishmania and is transmitted by the bite of a sand fly belonging to the genera Lutzomyia and Phlebotomus. Leishmaniasis

Bioassay-Guided Fractionation
The hexanes and acetone extracts of the leaves of Withania aristata were evaluated against promastigote forms of L. amazonensis and L. donovani, and on the epimastigote stage of T. cruzi. Cytotoxicity on murine macrophages was also assessed searching for selectivity (Table 1). In addition, miltefosine and benznidazole were evaluated for comparative purposes as reference drugs against leishmaniasis and Chagas disease respectively. Miltefosine shows IC 50 s of 2.64 µg/mL and 1.35 µg/mL against L. amazonensis and L. donovani, respectively, and CC 50 of 29.42 µg/mL, whereas benznidazole showed an IC 50 of 1.81 µg/mL against T. cruzi, with a CC 50 of 104.1 µg/mL. The acetone extract showed activity against the three tested species, with IC 50 s between 2.87 and 20.25 µg/mL. The most sensitive species was L. amazonensis and the less sensitive one was L. donovani. The selectivity index (SI), ranging from 2.1 to 14.9, was a promising fact to continue with the bioassay-guided fractionation. Therefore, the acetone extract was submitted to vacuum liquid chromatography on silica gel affording three fractions, F1-F3 (Scheme 1). The most active fractions, F2 (IC 50 values ranging from 1.02 to 12.73 µg/mL) and F3 (IC 50 values ranging from 3.63 to 22.28 µg/mL), exhibited potent activity on L. amazonesis and T. cruzi, similar to the reference drugs. Furthermore, both fractions showed a good selectivity index (SI around 11.0) on murine macrophages (Table 1), highlighting these two fractions as the most promising ones.

Mechanisms of Cell Death
The programmed cell death or apoptosis-like death, is crucial for parasite development and pathogenesis, and the ability of a drug to modulate the life or death of a parasite is recognized for its immense therapeutic potential [16]. In this work, we carried out several experimental approaches to research the apoptotic potential of the withanolides 1-3 on L. amazonesis and T. cruzi.

Withanolides induced Mitochondrial Damage in L. amazonensis
The effect of the tested compounds on the mitochondrial membrane potential was determined by JC-1 fluorescence measure. Moreover, the tested compounds induced a noticeable decrease in the depolarization of L. amazonensis mitochondrial membrane (∆Ψm), since the JC-1 dye remained in the cytoplasm in its monomeric form (Figure 2), thereby showing no clear effect on T. cruzi. The mitochondrial damage was confirmed by quantifying the ATP level after 24 h. Compound 1 at IC 90 produced a strong decrease in the total ATP level for L. amazonensis ( Figure 3). Furthermore, this effect was not seen in T. cruzi, with an ATP level similar to the untreated cells and with a slight decrease when incubated with compound 1.

Mechanisms of Cell Death
The programmed cell death or apoptosis-like death, is crucial for parasite development and pathogenesis, and the ability of a drug to modulate the life or death of a parasite is recognized for its immense therapeutic potential [16]. In this work, we carried out several experimental approaches to research the apoptotic potential of the withanolides 1-3 on L. amazonesis and T. cruzi.

Withanolides induced Mitochondrial Damage in L. amazonensis
The effect of the tested compounds on the mitochondrial membrane potential was determined by JC-1 fluorescence measure. Moreover, the tested compounds induced a noticeable decrease in the depolarization of L. amazonensis mitochondrial membrane (ΔΨm), since the JC-1 dye remained in the cytoplasm in its monomeric form (Figure 2), thereby showing no clear effect on T. cruzi. The mitochondrial damage was confirmed by quantifying the ATP level after 24 h. Compound 1 at IC90 produced a strong decrease in the total ATP level for L. amazonensis ( Figure 3). Furthermore, this effect was not seen in T. cruzi, with an ATP level similar to the untreated cells and with a slight decrease when incubated with compound 1. Due to T. cruzi epimastigotes being seemingly unaffected at the mitochondrial level by the assayed compounds, analysis of the cell death mechanism was continued only in the L. amazonensis species.

Withanolides Caused Chromatin Condensation in Treated Cells
In view of the obtained results, we decided to analyze the chromatin condensation event, a hallmark of apoptosis-like death, in which small and compact chromatin nuclei appear. To this end, parasites of L. amazonensis were incubated for 24 h with the IC90 of the withanolides, and Due to T. cruzi epimastigotes being seemingly unaffected at the mitochondrial level by the assayed compounds, analysis of the cell death mechanism was continued only in the L. amazonensis species.

Withanolides Caused Chromatin Condensation in Treated Cells
In view of the obtained results, we decided to analyze the chromatin condensation event, a hallmark of apoptosis-like death, in which small and compact chromatin nuclei appear. To this end, parasites of L. amazonensis were incubated for 24 h with the IC 90 of the withanolides, and subsequently, stained with Hoechst and propidium iodide. Condensation of chromatin in the parasites was clearly observed by fluorescence microscopy, since the treated cells showed a higher amount of bright blue in the nucleus (Figure 4). In addition, propidium iodide staining in red was observed in parasites of L. amazonensis treated with compound 1, indicating an advanced process of death, as confirmed by the transmitted light image, in which parasites appeared to be visibly damaged.

Withanolides Caused Chromatin Condensation in Treated Cells
In view of the obtained results, we decided to analyze the chromatin condensation event, a hallmark of apoptosis-like death, in which small and compact chromatin nuclei appear. To this end, parasites of L. amazonensis were incubated for 24 h with the IC90 of the withanolides, and subsequently, stained with Hoechst and propidium iodide. Condensation of chromatin in the parasites was clearly observed by fluorescence microscopy, since the treated cells showed a higher amount of bright blue in the nucleus (Figure 4). In addition, propidium iodide staining in red was observed in parasites of L. amazonensis treated with compound 1, indicating an advanced process of death, as confirmed by the transmitted light image, in which parasites appeared to be visibly damaged. The cytoplasmic membrane permeability assays on promastigotes of L. amazonensis with withanolides 1-3 ( Figure 5) showed green fluorescence inside the cells, indicating that the plasmatic membrane permeability was slightly damaged, since the cells preserved their integrity and shape.

Withanolides Induce Oxidative Stress in L. amazonensis
As could be observed in Figure 6, after 24 h of incubation of L. amazonensis promastigotes with the IC 90 of the withanolides 1-3, the treated cells showed a higher amount of red staining in the cytoplasm. This corresponds with the reactive oxygen species (ROS) accumulation inside the cell, a well-established event on cells before undergoing a programmed cell death. ROS induced by chemotherapeutic agents is closely associated with mitochondrial function, cytochrome c release, and the apoptosis-like mechanism.
Some species belonging to the Withania genera have been shown to present antileishmanial activity. One of the most studied is Withania somnifera, a well-known plant in the Ayurvedic medicine system, which extracts exhibited activity against L. donovani parasites on in vivo models as well as against L. major promastigotes and intracellular amastigotes [17,18]. Withania coagulans aerial parts demonstrated activity against L. major [19]. Furthermore, to the best of our knowledge, only a study on the evaluation of Withania somnifera against T. cruzi has been reported [20].  The cytoplasmic membrane permeability assays on promastigotes of L. amazonensis with withanolides 1-3 ( Figure 5) showed green fluorescence inside the cells, indicating that the plasmatic membrane permeability was slightly damaged, since the cells preserved their integrity and shape.

Withanolides Induce Oxidative Stress in L. amazonensis
As could be observed in Figure 6, after 24 h of incubation of L. amazonensis promastigotes with the IC90 of the withanolides 1-3, the treated cells showed a higher amount of red staining in the cytoplasm. This corresponds with the reactive oxygen species (ROS) accumulation inside the cell, a well-established event on cells before undergoing a programmed cell death. ROS induced by chemotherapeutic agents is closely associated with mitochondrial function, cytochrome c release, and the apoptosis-like mechanism. Some species belonging to the Withania genera have been shown to present antileishmanial activity. One of the most studied is Withania somnifera, a well-known plant in the Ayurvedic medicine system, which extracts exhibited activity against L. donovani parasites on in vivo models as well as against L. major promastigotes and intracellular amastigotes [17,18]. Withania coagulans aerial parts demonstrated activity against L. major [19]. Furthermore, to the best of our knowledge, only a study on the evaluation of Withania somnifera against T. cruzi has been reported [20].
Withanolides have attracted considerable attention due to their potential in drug research and development [7]. In particular, withaferin A, a well-known anticancer drug candidate, exerts its anticancer effect via induction of apoptosis in several human cancer cells [21]. Recent studies have revealed that withaferin A-analogues are potent apoptotic inducers in different tumor cell lines, evidenced by DNA fragmentation, chromatin condensation and phosphatidylserine exposure, indicating an apoptosis mechanism of action in cancer cells [22,23]. Moreover, the leishmanicidal activity of withaferin A has been reported against L. donovani, inducing an apoptosis-like cell death mechanism by inhibiting protein-kinase C, leading to the depolarization of mitochondrial membrane potential and releasing of cytochrome c into the cytosol, which induces formation of ROS inside cells, causing oxidative DNA lesions [24]. Our data agree with these previous studies, because Withanolides have attracted considerable attention due to their potential in drug research and development [7]. In particular, withaferin A, a well-known anticancer drug candidate, exerts its anticancer effect via induction of apoptosis in several human cancer cells [21]. Recent studies have revealed that withaferin A-analogues are potent apoptotic inducers in different tumor cell lines, evidenced by DNA fragmentation, chromatin condensation and phosphatidylserine exposure, indicating an apoptosis mechanism of action in cancer cells [22,23]. Moreover, the leishmanicidal activity of withaferin A has been reported against L. donovani, inducing an apoptosis-like cell death mechanism by inhibiting protein-kinase C, leading to the depolarization of mitochondrial membrane potential and releasing of cytochrome c into the cytosol, which induces formation of ROS inside cells, causing oxidative DNA lesions [24]. Our data agree with these previous studies, because depolarization of mitochondrial membrane potential was detected, as well as accumulation of ROS, DNA condensation and a lack of ATP, that proves the mechanism of apoptosis-like in L. amazonensis induced by withaferin A (1), as well as by compounds 2 and 3. These compounds could induce apoptosis-like through the mitochondrial pathway initiated by ROS production. Therefore, our proposal is that withanolides-type compounds could induce the formation of ROS, leading this excessive ROS to trigger apoptosis-like death by altering the mitochondrial membrane potential and damaging the respiratory chain, as has been proven in a human leukemia cell line [25].

General Procedures
Optical rotations were recorded at 25 • C on a Perkin Elmer 241 automatic polarimeter in CHCl 3 and the [α] D values are given in 10 −1 deg cm 2 /g. IR (film) spectra were recorded on a Bruker IFS 55 spectrophotometer. NMR spectra were performed on Bruker Avance 500 and 600 spectrometers at 300 0 K. The EIMS and HREIMS data were obtained on a Micromass Autospec spectrometer. HRESIMS (positive mode) were performed on an LCT Premier XE Micromass Electrospray spectrometer. Silica gel 60 (particle size 15-40 and 63-200 µm, Macherey-Nagel) was used for column chromatography (CC), while silica gel 60 F254 was used for analytical thin layer chromatography (TLC). Centrifugal

Leishmanicidal Activity Assay
Promastigote in logarithmic phase were used for plate preparation, and the in vitro antiprotozoal assay was performed in 96-well plates. The different fractions or compounds were serially diluted on the wells and 10 6 parasites/mL were finally added. The activity of the fraction was calculated by the alamarBlue ® method [26]. In addition, the active pure molecules were tested against the amastigote stage of L. amazonensis.
The amastigote activity was performed according to Jain et al. [27]. After allowing the transformation of rescued amastigotes to promastigotes, the activity was evaluated by AlamarBlue method. After incubation, the fluorescence was measured in a Perkin Elmer EnSpire spectrofluorometer and fluorescence was measured at emission peak 585 nm.

Trypanocidal Capacity Assay
For the evaluation against the epimastigote stage of T. cruzi, assays were performed following the same procedure mentioned in Section 3.5.1., modifying the density to 2 × 10 5 parasite/well and using LIT medium.

Cytotoxicity Assay
The cytotoxicity was evaluated using a macrophage cell line J774A.1 in RPMI medium. Macrophages were plated and at 10 5 macrophages/well and, subsequently, serial dilution of samples were added to the plates. After 24 h of incubation cell viability was determined by the alamarBlue ® [28].

Measurement of ATP
To determine the ATP level of the parasites, the Cell Titer-Glo ® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used following the manufacturer's instructions after incubation of the parasites with the calculated IC 90 of the tested withanolides for 24 h.

Chromatin Condensation Determination
The detection of condensed chromatin, was performed using the Vybrant™ Apoptosis Assay Kit #5, Hoechst 33342/Propidium Iodide (Invitrogen, Carlsbad, CA, USA). Parasites were incubated with the IC 90 of the pure compounds for 24 h, then collected and centrifuged. The cell pellet was resuspended in RPMI, added to a black plate and incubated with the Hoechst 33342 and the propidium iodide. Then the plate was protected from light for 20 min at 4 • C. The EVOS FL Cell Imaging System (Invitrogen, ThermoFisher, Carlsbad, CA, USA) was used to observe the cells, using the DAPI (Hoechst) and RFP (PI) Light Cubes.

Plasma Membrane Permeability
In order to detect membrane permeability alterations SYTOX ® Green dye was used. Briefly, 10 6 parasites/mL previously incubated with the IC 90 of the compounds for 24 h, were incubated with SYTOX ® Green (Molecular Probes, Eugene, OR, USA) at 1 µM. The fluorescence was monitored in an EVOS FL Cell Imaging System AMF4300, Life Technologies, USA. This dye is impermeable to intact cells, but when their membrane permeability is changed, SYTOX Green has the capacity to get inside the cell and attach to DNA, increasing its fluorescence >500 times.

Oxidative Stress
CellRox Deep Red Oxidative Stress Reagent (Thermo Fisher Scientific, Waltham, MA, USA) is a probe designed to measure reactive oxygen species (ROS) in live cells, exhibiting strong fluorogenic signal under oxidative state. Following the manufacturer's protocol, parasites were incubated with the compounds at the IC 90 concentration for 24 h, then washed, and incubated with 5 µM of CellRox Reagent for 30 min at 26 • C. Then, parasites were centrifuged and resuspended in buffer. H 2 O 2 at 600 µM for 30 min was used as positive control [29]. A fluorescence microscope (EVOS FL) was used to observe the cells. The signal for Deep Red is localized in the cytoplasm.

Statistical Analysis
Data are presented as mean ± SE. Non-linear regression analysis was used for the IC 50 (inhibitory concentration 50) and CC 50 (cytotoxic concentration 50) calculations. All determinations were performed in triplicate and the data shown are representative results from at least three independent experiments. Statistical differences between means were tested using one-way analysis of variance (ANOVA; three or more samples), using the SigmaPlot 12.0 software. A significance level of p < 0.05 was used.

Conclusions
In summary, the bioassay-guided fractionation of W. aristata against Leishmania spp. and T. cruzi was employed to identify drug candidates for the treatment of leishmaniasis and/or Chagas disease. We have successfully identified three potent and selective withanolides with significant leishmanicidal profiles, and high selectivity indexes for L. amazonensis. In addition, withanolides induced several ultrastructural and morphological changes in promastigotes of L. amazonensis. Mitochondrial membrane potential changes, ROS production, phosphatidylserine externalization, cell shrinkage, a rounded shape of the parasites, nuclear condensation and no change in membrane permeability were observed in parasites that were treated with the mentioned molecules. The major finding in the present study was that these withanolides induced programmed cell death in L. amazonensis, sharing several phenotypic characteristics with other cases of programmed cell death in metazoans, and exhibiting them as promising candidates against leishmaniasis. These findings support future investigations for further biochemical studies to unveil the mechanism of action of these withanolides, which can lead to the optimization of treatment for leishmaniasis.