Benzyl Farnesyl Amine Mimetics are Potent Inhibitors of the Sterol Biosynthesis Pathway in Leishmania Amazonensis Leading to Oxidative Stress, Growth Arrest, and Ultrastructural Alterations

Leishmaniasis is a neglected disease caused by protozoan parasites of the Leishmania genus spread around the world. Benzyl farnesyl amine mimetics are known class of compounds selectively designed to inhibit the squalene synthase (SQS) enzyme that catalyzes the rst committed reaction on the sterol biosynthesis pathway. Herein, we studied seven new benzyl farnesyl amine mimetics (SBC 37 - 43) against Leishmania amazonensis. After the rst initial screening of cell viability, two inhibitors (SBC 39 and SBC 40) were selected for further studies. Against intracellular amastigotes, SBC 39 and SBC 40 presented selectivity indexes of 117.7 and 180, respectively, indicating that they are highly selective. Analyses of free sterol showed that SBC 39 and SBC 40 inhibit two enzymes, sterol Δ 8 → Δ 7 isomerase and SQS, resulting in depletion of endogenous 24-methyl sterols. Physiological analysis and electron microscopy revealed three main alterations: 1) in the mitochondrion ultrastructure and function; 2) the presence of lipid bodies and autophagosomes; and 3) the appearance of projections in the plasma membrane and extracellular vesicles inside the agellar pocket. In conclusion, our results support the notion that benzyl farnesyl amine mimics have a potent effect against Leishmania amazonensis and should be an interesting novel pharmaceutical lead for the development of new chemotherapeutic alternatives to treat leishmaniasis. inhibitors: isosteric replacements side chain benzyl farnesyl amine.


Introduction
Leishmaniasis is an endemic neglected disease caused by several species of Leishmania genus. The leishmaniasis transmission was reported in a total of 98 countries and 3 territories on 5 continents. The estimated world prevalence for all clinical manifestations of the disease is 12 million, with 58,000 of visceral leishmaniasis (VL) cases and 220,000 cutaneous leishmaniasis (CL) cases per year [1,2]. In the New World, multiple species, including L. amazonensis and L. braziliensis, are the causative agents of the CL. Furthermore, L. amazonensis can also cause mucocutaneous leishmaniasis (MCL), which results in a progressive destruction of the naso-oropharyngeal mucosa, and diffuse cutaneous leishmaniasis (DCL), Trypanosomatids and fungi have an endogenous requirement of ergosterol and other 24-alkylated sterols for growth and survival, which are absent in mammal cells. Thus, the enzymes of sterol biosynthesis pathway are interesting targets for new treatments and several works have shown the effect of different sterol biosynthesis inhibitors (SBIs) in trypanosomatids [11][12][13][14][15][16][17]. Benzyl farnesyl amine mimetics are one class of selective inhibitors of the squalene synthase (SQS), an important enzyme that catalyzes the condensation of two molecules of farnesyl pyrophosphate to produce squalene. This is the rst committed step in the sterol biosynthesis, and its inhibition does not affect the synthesis of isoprenoids that are also important molecules for eukaryotic cells. For years, the research for speci c squalene synthase inhibitors (SQSi) has involved intensive efforts of industrial and academic investigators, because of the potential use of these inhibitors in the treatment of coronary heart disease and hypercholesterolemia [18][19][20].
In this work, the effect of another class of selective SQS inhibitors, novel mimetics of benzyl farnesyl amine, were evaluated against Leishmania amazonensis. Several aspects of the anti-Leishmania activity of these compounds were investigated in different times of treatment, such as antiproliferative, ultrastructural and biochemical effects. Moreover, we found one derivative that is among the most selective compounds for the parasite with low toxicity for the mammalian cells.

Ethics statements.
The experiments using BALB/c mice to isolate macrophages and maintained Leishmania parasite were approved by the Ethics Committee for Animal Experimentation of the Health Sciences Centre, Federal University of Rio de Janeiro (Protocols n. IBCCF 096/097/106), according to the Brazilian federal law (11.794/2008, Decreto no 6.899/2009). All animals received humane care in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research, and the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences, USA.

Parasites
The MHOM/BR/75/Josefa strain of L. amazonensis used in this study was gently provided by the Leishmania Collection of the Instituto Oswaldo Cruz (code IOCL 0071-FIOCRUZ). It has been maintained via inoculation into the base of BALB/c mouse tails. Amastigotes were obtained from the lesions of infected mice and transformed into promastigotes that were maintained in Warren's medium [brain heart infusion plus hemin and folic acid] [24] supplemented with 10% fetal bovine serum (FBS) at 25°C. Infective metacyclic promastigotes were used to obtain intracellular amastigotes in macrophage culture.
Firstly, peritoneal macrophages from BALB/c mice were harvested by washing them with Hanks' solution and plated in 24-well tissue culture chamber slides, allowing them to adhere to the slides for 24 h in RPMI medium (Gibco) supplemented with 10% FBS at 37°C in 5% CO 2 . After this, adherent macrophages were infected with metacyclic promastigotes at a macrophage-to-parasite ratio of 1:10 at 35°C in 5% CO 2 for 2 h. These cultures were maintained for 24 h in RPMI medium supplemented with 10% FBS for the assays with intracellular amastigotes.

Drugs.
The benzyl farnesyl amine mimetics, SBCs 37 -43, were prepared by chemical synthesis at IQ-UNICAMP according to an experimental procedure previously described by Cämmerer and Souza [25]. Structures of those N- [4-[benzyloxy] benzyl]-benzene-methaneamine derivatives, SBCs 37 -43, are displayed in scheme 1. These compounds have been recently reported to exhibit signi cant biological activity against intracellular amastigotes of Trypanosoma cruzi [25]. Compounds were used as hydrochloride salts, which were puri ed by one recrystallization from analytical grade ethanol and dried in high vacuum at room temperature.

Cell viability and cytotoxicity assays
For primary screening of the antileishmanial effects of the SBCs 37-43, we evaluated the cell viability and cytotoxicity effects in L. amazonensis promastigotes and peritoneal macrophages by CellTiter 96® Aqueous MTS Assay (Promega, United States) [16,29]. For analysis in promastigotes, we started the culture at cell density of 1 x 10 6 cells/ml in Warren´s medium supplemented with 10% FBS. After 24 h, different concentrations of SBC37-43 were added to the cultures. Cell viability and the cytotoxic effects were measured at 24, 48, and 72 h of treatment, when all groups, including untreated, were transferred to clear 96-well plate in triplicate. MTS/PMS assay reaction was quanti ed by optical density measurement at 490 nm in a microplate reader and SpectraMax M 2 /M 2 e spectro uorometer (Molecular Devices, United States). As a negative control, parasites were xed with 0.4% nascent formaldehyde for 10 min at room temperature before the incubation. Cytotoxicity effects of SBCs 37 -43 in murine macrophages were also evaluated using the same MTS/PMS assay reaction described above. Murine macrophages were obtained from BALB/c mouse after washing with Hanks's solution and cultivated in a clear 96-well plate with RPMI medium supplemented with 10% FBS and maintained at 37°C in 5% CO 2 . After 24 h of cultivation, SBCs 37 -43 were added at different concentrations. Macrophages viability was measured at 24, 48, and 72 h of treatment. MTS/PMS assay reaction was also quanti ed by optical density measurement at 490 nm in a microplate reader and SpectraMax M 2 /M 2 e spectro uorometer (Molecular Devices, United States). The cytotoxicity concentration to reduce 50% of viable macrophages (CC 50 ) was determined.

Growth inhibition of promastigotes and amastigotes of L. amazonensis
After the evaluation of the cell viability and cytotoxic effects by MTS assay in promastigote forms, we also analyzed the effects of the SBCs 37 -43 in the growth of promastigotes. For this, promastigote cultures were initiated at a cell density of 1.0 x 10 6 cells/ml. After 24 h of growth, SBCs 37 -43 were added at different concentrations from concentrated stock solutions, and cell densities were evaluated daily over 96 h of growth using a Neubauer chamber. Based on the analysis of CC 50 and IC 50 in promastigotes, and the cytotoxic effects in murine macrophages, three of the benzyl farnesyl amine mimetics (SBCs 37, 39 and 40) were chose for evaluation against amastigotes infected macrophages. To evaluate the effects of compounds on L. amazonensis intracellular amastigotes, macrophages were infected as described previously and incubated with different concentrations of compounds after 24 h of infection. Fresh medium was added daily until 3 days (24, 48, and 72 h of treatment). After this time, cultures were xed in Bouin's solution [17], and washed with 70% ethanol, distilled water and then stained with Giemsa solution for 1 h. The number of intracellular amastigotes was obtained after count in light microscopy. Association indexes ((mean number of parasites internalized X percentage of infected macrophages) / total number of macrophages) were determined and used as a parameter to calculate the percentage of infection for each condition used in this study. The concentration that inhibited 50% of growth (IC 50 ) and selective index (SI) were calculated.
2.6. Estimation of the mitochondrial transmembrane electric potential Mitochondrial transmembrane electric potential (Δψ m ) of the untreated and treated promastigotes was analyzed using the JC-1 uorochrome (Molecular Probes, United States), a lipophilic and cationic mitochondrial vital dye that accumulates in the mitochondria in response to Δψ m , since its uorescence is considered an indicator of an energized mitochondrial state [15]. JC-1 exits as a J-monomer that in absence of Δψ m accumulate in low concentration with emission wavelength at 530 nm (green uorescence), however, in presence of Δψ m JC-1 accumulated as J-aggregates with emission at 590 nm (red uorescence). Parasites were prepared as previously described [15,16]. For each sample, 1 x 10 7 parasites were incubated with 10 µg/mL JC-1 for 25 min, with readings made every minute using a microplate reader and spectro uorometer SpectraMax M2/M2 e (Molecular Devices, United States). Cells were incubated in the presence of 2 µM FCCP (a mitochondrial protonophore) during the initial 20 min of experiment as positive control for the mitochondrial membrane depolarization. After 20 min of readings, 2 µM FCCP was added at all samples to abolish the Δψ m . The relative Δψ m values were obtained calculating the ratio between the reading at 590 nm and 530 nm (590:530 nm). Experiments were independently repeated at least three times in triplicate, and graphic shows the mean and standard deviation of one representative experiment.

Evaluation of ROS production
Intracellular ROS levels were evaluated in control and compound-treated promastigotes as described previously [17]. For this, 3 x 10 7 promastigotes were harvested, washed twice in PBS (pH 7.2) and incubated with 10 µg/ml H 2 DCFDA [a cell-permeable green probe; Molecular Probes, United States] in PBS for 1h at 25°C. After 1 h, cells were washed and resuspended in PBS, added in a black 96-well plate, and then analyzed in a microplate reader and spectro uorometer SpectraMax M2/M2 e (Molecular Devices, United States), using the pair of 507 nm and 530 nm wavelengths as emission and excitation wavelengths, respectively.

Electron tomography
For electron tomography, ribbons of 200 nm thick serial sections were produced from transmission electron microscopy blocks described above. These ribbons were collected in a formvar-coated copper slot grids. After that, colloidal gold particles (10 nm) were deposited on both surfaces of the sections, being used as ducial markers for alignment of the tilted views. Single-axis tilt series (±60° with 1°i ncrements) were produced from samples using Xplore3D software and a Tecnai-G2 (FEI Company, Eindhoven, Netherlands) electron microscope operating at 200 kV. 3D reconstruction was performed using the IMOD software package [27]. Furthermore, tomogram generation by R-weighted back-projection was performed using ETOMO, and virtual slices were manually segmented using 3DMOD that was also used to produce 3D models.
2.11. Extraction, separation of neutral lipids and free sterol analysis.
For the analysis of the effects of SBC 39 and SBC 40 on the free sterol composition of the promastigotes, total lipids were extracted from control and drug-treated L. amazonensis promastigotes, as described previously [17,28]. Neutral lipids were analyzed by MS and mass spectra were obtained by electron ionization (EI) at 70 eV according to the protocol published previously [17,28]. The assignment of structures was based on relative chromatographic behaviors, as well as the characteristic fragmentation patterns in MS and by comparison of the mass spectra with those available in the National Institute of Standards and Technology (NIST) Research Library located at the NIST Mass Spectrometry Data Center.

Calibration for cholesterol and ergosterol determination.
A set of ve calibration standards was prepared from the pure standard of cholesterol and ergosterol purchased from Sigma-Aldrich Co. Different calibration solutions were prepared using ethyl acetate as solvent. For the quanti cation of cholesterol and ergosterol, standards were used at different concentrations of 0.08, 0.10, 0.25, 0.50 and 1.0 mM to plot the standard curve. From each calibration solution, 1 µL was injected (run in triplicate) into the GC-MS system to achieve the regression plot of various concentrations versus their peak area.

Statistical analysis
All the graphics in the gures were created using the means of three independent experiments, and the bars represent the standard deviations of the means. The statistical signi cance of differences among the groups was assessed using the one-way or two-way analysis of variance (ANOVA) test, followed by Bonferroni's multiple-comparison test in the GraphPad Prisma 5 software. Results were considered statistically signi cant when P was < 0.05( * ), <0.01( ** ), and <0.001( *** ).  We also evaluated the cytotoxic effects of the SBCs 37 -43 against murine macrophages using MTS/PMS assay after 72 h of treatment. Figure 3 shows that SBC 37, SBC 38, SBC 39, and SBC 40 presented low cytotoxicity to mammalian cells, with CC 50 of 33.94 µM, 40.53 µM, 40.65 µM, and 39.15 µM, respectively. Based on the results obtained for macrophage and promastigotes, we decided to evaluate the effects of three of them (SBCs 37, 39 and 40) against intracellular amastigotes. Figure 4 shows the antiproliferative effects of SBC 37, SBC 39, and SBC 40 after 72h of treatment, presenting IC 50 values of 740.48 nM, 345.35 nM, and 217.5 nM, respectively (Fig. 4A-C). Thus, the selectivity index obtained was 45.83, 117.7, and 180, respectively, after 72 h of treatment.

SBC 39 and SBC 40 alter the morphology of promastigotes
Scanning electron microscopy revealed important changes in the morphology of promastigotes treated with SBC 39 and SBC 40 for 48 h (Fig. 5-F). Figure 5A shows a control L. amazonensis promastigote without any alteration in the morphology of cell body, surface and agellum (Fig. 5A). Treatment with lower concentrations of SBCs induced several alterations such as the presence of parasites rounded and swollen (Fig. 5B, C, F), also presenting vesicles budding from the region near the agellar pocket (Fig. 5E) and sometimes more than two agella (Fig. 5C, D). After 48 h of treatment with 300 nM SBC 40, all parasites were completely rounded (Fig. 5F). These results indicate the potent effect of these inhibitors to alter the morphology of promastigotes.

SBC 39 and SBC 40 alter mitochondrion function and induce lipid bodies accumulation
Nanomolar concentrations of SBC 39 and SBC 40 were able to reduce signi cantly the mitochondrial membrane potential (ΔΨ m ) after 48 h of treatment (Fig. 6A). This effect was similar to those observed for the positive control group treated with FCCP, a mitochondrial protonophore. The readings were done 25 min after addition of JC-1 in all groups, when both forms of the uorochrome (monomer and Jaggregate) are stabilized in inner portion of the mitochondrion. Although both inhibitors were able to reduce potential, only SBC 40 was able to increase signi cantly the ROS production at low concentrations (Fig. 6B). Furthermore, both inhibitors were able to induce lipid bodies accumulation after 48 h of treatment (Fig. 7). However, for SBC 39 the concentration to increase the presence of lipid bodies was 3-times higher than those for SBC 40.

SBC 39 and SBC 40 alters the ultrastructure of promastigotes
Transmission electron microscopy was used to analyze the ultrastructural alterations induced by SBC 39 and SBC 40. Figure 8A shows a control promastigote presenting a structural organization without any alteration for the nucleus (N), mitochondrion (M), kinetoplast (k), agellum (f) and cell surface. After 48 h of treatment with 500 nM or 1 µM SBC 39, several alterations were observed, such as: 1) loss of the mitochondrial matrix content and vesiculation of its inner membrane (Fig. 8C, D, F); 2) presence of several large vacuoles similar to autophagosomes engul ng parts of the cytosol (Fig. 8B, E); 3), increased number of lipid bodies (Fig. 8E); and 4) disorganization of the kinetoplast (Fig. 8C-E). For the treatment with SBC 40, in concentrations much lower, the ultrastructural alterations were similar for those with SBC 39. A signi cant accumulation of lipid bodies randomly distributed throughout the cytosol were observed (Fig. 9C, D). The presence of glycosomes were easily observed in ultrathin sections of treatedpromatigotes, probably indicating an increase number of them, since they are di cult to observe in control parasites (Fig. 9C). Furthermore, several extracellular vesicles inside the agellar pocket (Fig. 9D), and the presence of autophagosome-like vacuoles in close association with nucleus and mitochondrion ( Fig. 9A, E, F) were observed. Alterations in the trans-Golgi network (Fig. 9E), disorganizing of the kinetoplast (Fig. 9B), and intense mitochondrial swelling (Fig. 9B, F) were also induced by the treatments.
Trying to understand better the ultrastructural effects induced by SBs, we decided to carry out treatments with high concentrations and short time of incubation. For this, we used the concentration of 5 µM for just 6 h. The results obtained indicated signi cant alterations showing that these new compounds present a potent activity against Leishmania. Plasma membrane projections were observed after treatment with 5 µM SBC 39 (Fig. 10B-D, arrowhead). Interesting, these projections appeared frequently in regions of the membrane close to endoplasmic reticulum (Fig. 10D, arrowhead). Therefore, we decided to observe these projections by electron tomography. Figure 11A-D shows a serial section tomography of L. amazonensis promastigotes treated with 5 µM SBC 39 for 6 h. From these sections, a speci c area of the parasite surface was reconstructed (Fig. 11E, F) revealing the ultrastructure of this plasma membrane projection. Images con rmed the presence of the endoplasmic reticulum pro le near the projection, and the absence of microtubules associated to this projection. The alignment of the electron tomograms allows us to observe the relation between projection and endoplasmic reticulum (Suppl. Figs. 1 and 2movies).
Furthermore, the treatment with SBC 39 and SBC 40 for short time caused several other alterations in the ultrastructure of the promastigotes such as: mitochondrial swelling with an increased numbers of mitochondrial cristae (Fig. 10B, C, F); presence of large vacuoles and myelin-like gures (Fig. 10B-F); and disorganization of kinetoplast ( Figure 11E). After alignment of several electron tomograms, it is possible to observe some of these changes in a movie containing a large volume of one treated promastigote with 5 µM SBC 39 (Suppl. Fig. 3 -movie).

Effects of SBC 40 on the ne structure of intracellular amastigotes
Transmission electron microscopy was also used to study the ne structure of intracellular amastigotes and the effects induced by the treatments. Figure 12 shows the ultrathin sections of L. amazonensis intracellular amastigotes treated with 1 µM SBC 40 for 48 h. Several alterations in the ultrastructure were observed, which are absent in the control parasites (Fig. 12A) such as: mitochondrial swelling (Fig. 12C); presence of autophagosome-like vacuoles containing membrane pro les (Fig. 12C-D); alterations in the plasma membrane ultrastructure ( Figure 12B-D, arrowhead); and disorganization of the kinetoplast ( Figure 12D). Together, these images suggest the potent effect of benzyl farnesyl amine mimetics in L. amazonensis intracellular amastigotes.
These changes in the relative percentages of endogenous sterols are compensated with an increase in the percentage of cholesterol, which account for ca. 19% and 26% for SBC 39 and SBC 40 respectively.
When performing the quantitative analysis of cholesterol and endogenous sterol (Table 2), it can be seen that both drugs induce a concomitant 10 and 14 -fold (from 5.12 µg/ X 10 8 control cells to 0.4 or 0.5 µg/ X 10 8 treated cells; 90%) reduction of the content of endogenous sterols when compared with untreated cells. While cholesterol mass reduction is related to the decrease in cell density and not by the drugs actions.

Discussion
Although leishmaniasis has a number of treatment options, its therapy has a lot of problems as extensive toxicity, lack of e cacy, parenteral route of administration affecting compliance, high costs, and emerging drug resistance [29]. Visceral, cutaneous and mucocutaneous leishmaniasis remain some of the most devastating neglected tropical diseases. Thus, there is an urgent need for development of the new anti-leishmanial compounds that have more e cacy, low toxicity and cost, and preferentially administrated by oral or topic routes.
Sterol biosynthesis (SB) is an important metabolic pathway in Leishmania sp. For many years, several SB inhibitors have been studied against both developmental stages of the parasite in vitro [15-17, 23, 28,  [30][31][32]. Some of them have also been tested in vivo against Trypanosoma cruzi, inducing a potent suppressive effect in models of acute Chagas' disease [30,[33][34][35]. An important step of the SB is catalyzed by the enzyme squalene synthase (SQS). SQS is responsible for the reaction that catalyze the rst committed step in the SB pathway; thus, not interfering with isoprenoid production and its metabolites [11,36]. Several classes of squalene synthase inhibitors, such as quinuclidine derivatives, have been studied as potent SB inhibitors. The rst quinuclidine derivatives tested against Leishmania sp. was BPQ-OH, ER-119884 and E5700 inducing cell death in association with the depletion of the parasite's endogenous sterols [23,31]. Furthermore, other quinuclidine derivative, WSP 1267, showed potent effect against Candida albicans, C. parapsilosis, and C. tropicalis, with a MIC 50 of 2 µg/ml [37]. Some quinuclidine derivatives were also able to inhibit the recombinant L. major SQS at submicromolar concentrations, exhibiting selectivity action for the parasite enzyme [20][21]. Benzyl farnesyl amine mimics were also reported as to be selective inhibitors of human SQS. They were explored for their potential use for development of cholesterol lowering treatment options for hypercholesterolemia in man [38].
In this study, we report the activity of several benzyl farnesyl amine mimetics against Leishmania amazonensis. The compounds SBC 39 and SBC 40 showed the most pronounced effects on the growth of L. amazonensis intracellular amastigotes, associated with a low cytotoxicity in mammal cells and a higher selectivity index of 117.7 and 180, respectively. These selectivity indexes are higher than those found after treatment with posaconazole and itraconazole, two potent inhibitors of the growth of L. amazonensis [16]. Herein, the antiproliferative activities of SBC 39 and SBC 40 were in the nanomolar range against both extracellular promastigotes and intracellular amastigotes. The biological activity against Leishmania amazonensis was lower than those found for E5700 and ER-119884, two SQS inhibitors from Eisai Pharmaceutical Company, previously studied against Leishmania [17,26]. Nevertheless, it is noteworthy to mention, that further development of E5700 and ER 119884 has been stopped, because it caused testicle atrophy in a small animal experiment. Beyond this, quinuclidine derivatives often bear a certain risk of neurological side effects (neurotoxicity of antimalarial drug quinine and other quinuclidine containing drugs). Therefore, medicinal chemistry has today an increasing interest, to avoid the quinuclidine moiety in early phase drug development. Last but not least, benzyl farnesyl amine mimetics have a much lower production cost, what is especially highly attractive for antiinfectious drug design in tropical emerging countries, where cost restrictions play sometimes a limiting factor in pharmaceutical development. Thus, these results should regarded as a highly valuable contribution to SB inhibitors research in tropical parasites with a high potential for further drug development.
To analyze morphological and ultrastructural alterations, we used scanning and transmission electron microscopy, respectively. Some morphological alterations such as the swollen of the cell body and the presence of several agella were observed. Although the number of agella in treated promastigotes was altered, we did not observe arrest of the cell cycle or presence of multiple numbers of nucleus and kinetoplast, similar to those observed by our group with other SB inhibitors [16,17,28]. Transmission electron microscopy images indicated that mitochondrion ultrastructure was dramatically altered after treatment with SBC 39 and SBC 40 (Figures 8 and 9). Using uorescence markers for mitochondrial membrane potential and ROS production, as JC-1 and H 2 DCFA respectively, it was possible to con rm the mitochondrial damage provoked by the treatments (Fig. 6). Alterations in the mitochondrion structure and function could be related to signi cant changes in the lipidic composition of the mitochondrial membranes, since previous studies showed that the unique and rami ed mitochondrion of the trypanosomatid have a special composition of 24-methyl sterols [14]. Interestingly, in some images, several glycosomes were observed after treatment with SBC 40 (Fig. 9C), which could indicate an effort of the treated parasite to supply the mitochondrial damages and a possible decrease in the oxidative phosphorylation. An increase number of glycosomes could help the parasite to compensate the ATP production using the glycolytic pathway.
Another important alteration was observed in the plasma membrane (Fig. 5), which could be consequence of 24-methyl sterols depletion that was replaced by toxic intermediates of the sterol biosynthesis. This phenotype was also observed after treatment with several other SB inhibitors in Leishmania [15-17, 26-27, 38]. Moreover, several projections on plasma membrane were observed after only 6 h of treatment, which were better observed by electron tomography (Figure 10, 11). Projections could also be related with the secretory pathway, since in some images, as shown in gure 9D, we observed several extracellular vesicles secreted by treated promastigotes. The presence of several giant vacuoles containing portions of the cytosol, damaged organelles and membranes could be related with an intense autophagic process, which could increase the secretory activity by the treated promastigotes.
After analyses of sterol composition, our results suggest that the compounds SBC 39 and SBC 40 have at least two mechanisms of action, both affecting the integrity of the plasma membrane. The rst one, both act as inhibitors of the enzyme sterol Δ 8 → Δ 7 isomerase, being the SBC 39 better inhibitor. The second mechanism of action would be the blockade of endogenous sterol biosynthesis at the level of SQS, where the SBC 40 has the greatest effect.
In conclusion, our results support the notion that SBC 39 and SBC 40 are promising new chemotherapeutic agents against Leishmania sp, since they presented a very high speci city for the parasite. Furthermore, our ndings justify future studies to better understand the mode of action and also using in combination therapy with other SB inhibitors as a new therapeutic strategy that could reduce toxicity, but increase e cacy of treatment.  Figure 1 Evaluation of cytotoxicity effects of SBCs on Leishmania amazonensis promastigotes. Cell viability and cytotoxicity were evaluated against promastigotes using the MTS/PMS assay after 72 h of treatment.

Declarations
The cytotoxicity concentration to reduce 50 % of viable promastigotes (CC 50 ) was determined. SBC37, SBC39, SBC40, SBC41, SBC42, and SBC43 presented CC 50 lower than 5 µM. Bars represent standard deviation; *p<0.05, **p<0.01, and ***p<0.001.    with vesicles next to agellar pocket (E). Sometimes more than two agella could be observed (C, D).  Analysis of Nile Red accumulation. Fluorometric analyses indicate that there is a signi cant increase in Nile Red accumulation after treatment with 300 nM SBC39 and 100 nM SBC40. Bars represent standard deviation. *p<0.05, **p<0.01, and ***p<0.001.    Electron microscopy tomography and 3D reconstruction showing a projection of the plasma membrane of L. amazonensis promastigotes treated with 5 µM SBC39 for 6h. The images con rmed the presence of the endoplasmic reticulum pro le near the projection, and the absence of microtubules associated to it. Figure 12