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Approximately 6-7 million people around the world are estimated to be infected with Trypanosoma cruzi, the causative agent of Chagas disease. The current treatments are inadequate and therefore new medical interventions are urgently needed. In this paper we describe the identification of a series of disubstituted piperazines which shows good potency against the target parasite but is hampered by poor metabolic stability. We outline the strategies used to mitigate this issue such as lowering logD, bioisosteric replacements of the metabolically labile piperazine ring and use of plate-based arrays for quick diversity scoping. We discuss the success of these strategies within the context of this series and highlight the challenges faced in phenotypic programs when attempting to improve the pharmacokinetic profile of compounds whilst maintaining potency against the desired target.

Leishmaniasis is a parasitic neglected tropical disease and result in a broad spectrum of clinical manifestations, ranging from a single ulceration to a progressive and fatal visceral disease. Comprising a limited and highly toxic therapeutic arsenal, new treatments are urgently needed. Targeting delivery of drugs has been a promising approach for visceral leishmaniasis (VL). Phosphatidylserine-liposomes have demonstrated superior efficacy in VL, targeting intracellular parasites in host cells through macrophage scavenger receptors. In this work, we investigated the in vitro and in vivo efficacy of the antihelminthic drug nitazoxanide in a nanoliposomal formulation against Leishmania (L.) infantum. Physicochemical parameters of liposomes containing nitazoxanide (NTZ-LP) were determined by dynamic light scattering and small angle X-ray scattering. The efficacy of the formulation was verified in an intracellular amastigote model and in an experimental hamster model. Our findings showed that NTZ-LP was able to eliminate the amastigotes inside the host cell with an IC50 value of 16 μM. NTZ-LP was labelled a fluorescent probe and by spectrofluorimetry, we observed that the infected macrophages internalized similar levels of the drug to the uninfected cells. The confocal microscopy images confirmed the uptake and demonstrated a diffuse distribution of the NTZ-LP in the cytoplasm of Leishmania-infected macrophages, with the vesicles in a closer proximity to the parasites. For the in vivo efficacy, the liposomal NTZ-LP was administrated intraperitoneally to Leishmania-infected hamsters for 10 consecutive days at 2 mg/kg/day. By qPCR we demonstrated a reduction of the parasite burden by 82% and 50% in the liver (p, Graphical abstract Image 1, Highlights • Nanoliposomal nitazoxanide (NTZ-LP) eliminates amastigotes of Leishmania. • The uptake of NTZ-LP by infected macrophages is similar to uninfected cells. • NTZ-LP localizes in a closer proximity to the amastigotes inside the macrophages. • NTZ-LP reduces the parasite burden by 82% (liver) and 50% (spleen) of hamsters.

Liposomes containing phosphatidylserine (PS) has been used for the delivery of drugs into the intramacrophage milieu. Leishmania (L.) infantum parasites live inside macrophages and cause a fatal and neglected viscerotropic disease, with a toxic treatment. Sertraline was studied as a free formulation (SERT) and also entrapped into phosphatidylserine liposomes (LP-SERT) against intracellular amastigotes and in a murine model of visceral leishmaniasis. LP-SERT showed a potent activity against intracellular amastigotes with an EC50 value of 2.5 μM. The in vivo efficacy of SERT demonstrated a therapeutic failure. However, when entrapped into negatively charged liposomes (−58 mV) of 125 nm, it significantly reduced the parasite burden in the mice liver by 89% at 1 mg/kg, reducing the serum levels of the cytokine IL-6 and upregulating the levels of the chemokine MCP-1. Histopathological studies demonstrated the presence of an inflammatory infiltrate with the development of granulomas in the liver, suggesting the resolution of the infection in the treated group. Delivery studies showed fluorescent-labeled LP-SERT in the liver and spleen of mice even after 48 h of administration. This study demonstrates the efficacy of PS liposomes containing sertraline in experimental VL. Considering the urgent need for VL treatments, the repurposing approach of SERT could be a promising alternative.

EtOH extracts from the leaves and twigs of Nectandra oppositifolia Nees & Mart. shown activity against amastigote forms of Trypanosoma cruzi. These extracts were subjected to successive liquid-liquid partitioning to afford bioactive CH2Cl2 fractions. UHPLC-TOF-HRMS/MS and molecular networking were used to obtain an overview of the phytochemical composition of these active fractions. Aiming to isolate the active compounds, both CH2Cl2 fractions were subjected to fractionation using medium pressure chromatography combined with semi-preparative HPLC-UV. Using this approach, twelve compounds (1–12) were isolated and identified by NMR and HRMS analysis. Several isolated compounds displayed activity against the amastigote forms of T. cruzi, especially ethyl protocatechuate (7) with EC50 value of 18.1 μM, similar to positive control benznidazole (18.7 μM). Considering the potential of compound 7, protocatechuic acid and its respective methyl (7a), n-propyl (7b), n-butyl (7c), n-pentyl (7d), and n-hexyl (7e) esters were tested. Regarding antitrypanosomal activity, protocatechuic acid and compound 7a were inactive, while 7b-7e exhibited EC50 values from 20.4 to 11.7 μM, without cytotoxicity to mammalian cells. These results suggest that lipophilicity and molecular complexity play an important role in the activity while efficiency analysis indicates that the natural compound 7 is a promising prototype for further modifications to obtain compounds effective against the intracellular forms of T. cruzi.

Background: Considering the high toxicity and limited therapies available for treating visceral leishmaniasis (VL), the drug repositioning approach represents a faster way to deliver new therapies to the market. Methods: In this study, we described for the first time the activity of a potent antiarrhythmic, amiodarone (AMD), against L. (L.)infantum and its in vitro and in vivo activity. Results: The evaluation against promastigotes has shown that amiodarone presents leishmanicidal effect against the extracellular form, with an IC50 value of 10 μM. The activity was even greater against amastigotes in comparison with promastigotes with an IC50 value of 0.5 μM. The selectivity index in relation to the intracellular form demonstrated that the antiparasitic activity was approximately 56 times higher than its toxicity to mammalian cells. Investigation of the in vivo AMD activity in the L. infantum-infected hamster model showed that 51 days after the initial infection, amiodarone was unable to reduce the parasite burden in the spleen and liver when treated for 10 consecutive days, intraperitoneally, at 50 mg/kg/day, as determined by qPCR. Although not statistically significant, AMD was able to reduce the parasite burden by 20% in the liver when treated for 10 consecutive days, orally, at 100 mg/kg/day; no reduction in the spleen was found by qPCR. Conclusions: Our findings may help further drug design studies seeking new AMD derivatives that may provide new candidates with an in vitro selectivity close to or even greater than that observed in the prototype delivering effectiveness in the experimental model of VL.

Bioactivity-guided fractionation of the MeOH extract from the leaves of Alchornea glandulosa afforded a new guanidine alkaloid named alchornedine, as well as two other inactive derivatives (pteroginine and pteroginidine). The structure of alchornedine, which shows a very rare ring system, was elucidated based on NMR, IR, and MS spectral analyses. This compound displayed antiprotozoal activity against Trypanosoma cruzi (Y strain). By using the MTT assay, the trypomastigotes showed an IC50 value of 93 µg/mL (443 µM), a similar effectiveness to the standard drug benznidazole. Alchornedine also showed activity against the intracellular amastigotes, with an IC50 value of 27 µg/mL (129 µM). Using benznidazole as a standard drug, this guanidine alkaloid was approximately 3-fold more effective against the intracellular form of T. cruzi. The mammalian cytotoxicity of alchornedine was verified against NCTC cells and demonstrated an IC50 of 50 µg/mL (237 µM), but this compound demonstrated a selective elimination of parasites inside macrophages without affecting the morphology of the host cells. Alchornedine was effective against both clinical forms of T. cruzi and could be used as a scaffold for future drug design studies against American trypanosomiasis.

Leishmaniasis and Chagas disease are infectious diseases caused by parasite Leishmania sp. and Trypanosoma cruzi, respectively, and are included among the most neglected diseases in several underdeveloped and developing countries, with an urgent demand for new drugs. Considering the antiparasitic potential of MeOH extract from leaves of Casearia sylvestris Sw. (Salicaceae), a bioguided fractionation was conducted and afforded four active clerodane diterpenes (casearins A, B, G, and J). The obtained results indicated a superior efficacy of tested casearins against trypomastigotes of T. cruzi, with IC50 values ranging from 0.53 to 2.77 μg/ml. Leishmania infantum promastigotes were also susceptible to casearins, with IC50 values in a range between 4.45 and 9.48 μg/ml. These substances were also evaluated for mammalian cytotoxicity against NCTC cells resulting in 50% cytotoxic concentrations (CC50) ranging from 1.46 to 13.76 μg/ml. Additionally, the action of casearins on parasite membranes was investigated using the fluorescent probe SYTOX Green. The obtained results demonstrated a strong interaction of casearins A and B to the plasma membrane of T. cruzi parasites, corroborating their higher efficacy against these parasites. In contrast, the tested casearins induced no alteration in the permeability of plasma membrane of Leishmania parasites, suggesting that biochemical differences between Leishmania and T. cruzi plasma membrane might have contributed to the target effect of casearins on trypomastigotes. Thus, considering the importance of studying novel and selective drug candidates against protozoans, casearins A, B, G, and J could be used as tools to future drug design studies.

a b s t r a c t Studying the cellular death pathways in Leishmania is an important aspect of discovering new antileish- manials. While using a drug repositioning approach, the lethal action of the nitrothiazolyl-salicylamide derivative nitazoxanide (NTZ) was investigated against Leishmania (L.) infantum. The in vitro antileish- manial activity and cytotoxicity were assessed using both parasite stages and mammalian NCTC cells, respectively. The lethal action of NTZ was investigated by detecting the phosphatidylserine (PS) exposure, reactive oxygen species (ROS) regulation, plasma membrane permeability, mitochondrial membrane potential and ultrastructural modifications by transmission electron microscopy. NTZ's activity against L. infantum was confirmed, producing IC50 values of 42.71 g/mL against promastigotes and 6.78 g/mL against intracellular amastigotes. NTZ rapidly altered the cellular metabolism of promastigotes by depo- larising the mitochondrial membrane and up-regulating the reactive oxygen species (ROS). In addition, the flow cytometry data revealed an intense and time-dependent exposure of PS in promastigotes. When using SYTOX® Green as a fluorescent probe, NTZ demonstrated no interference in plasma membrane permeability. The ultrastructural alterations in promastigotes were time-dependent and caused chro- matin condensation, plasma membrane blebbing and mitochondrial swelling. These data suggest that NTZ induced oxidative stress in L. (L.) infantum and might be a useful compound for investigating new therapeutic targets. © 2013 Elsevier B.V. All rights reserved.

Iron is an essential growth component in all living organisms and plays a central role in numerous biochemical processes due to its redox potential and high affinity for oxygen. The use of iron chelators has been suggested as a novel therapeutic approach towards parasitic infections, such as malaria, sleeping sickness and leishmaniasis. Known iron chelating agents such as Deferoxamine and the 3-hydroxypyridin-4-one (HPO) Deferiprone possess anti-parasitic activity but suffer from mammalian toxicity, relatively modest potency, and/or poor oral availability. In this study, we have developed novel derivatives of Deferiprone with increased anti-parasitic activity and reduced cytotoxicity against human cell lines. Of particular interest are several new derivatives in which the HPO scaffold has been conjugated, via a linker, to the 4-aminoquinoline ring system present in the known anti-malaria drug Chloroquine. We report the inhibitory activity of these novel analogues against four parasitic protozoa, Trypanosoma brucei, Trypanosoma cruzi, Leishmania infantum and Plasmodium falciparum, and, for direct comparison, against human cells lines. We also present data, which support the hypothesis that iron starvation is the major cause of growth inhibition of these new Deferiprone-Chloroquine conjugates in T. brucei.

Synthetic analogues of marine sponge guanidine alkaloids showed in vitro antiparasitic activity against Leishmania (L.) infantum and Trypanosoma cruzi. Guanidines 10 and 11 presented the highest selectivity index when tested against Leishmania. The antiparasitic activity of 10 and 11 was investigated in host cells and in parasites. Both compounds induced depolarization of mitochondrial membrane potential, upregulation of reactive oxygen species levels, and increased plasma membrane permeability in Leishmania parasites. Immunomodulatory assays suggested an NO-independent effect of guanidines 10 and 11 on macrophages. The same compounds also promoted anti-inflammatory activity in L. (L.) infantum-infected macrophages cocultived with splenocytes, reducing the production of cytokines MCP-1 and IFN-γ. Guanidines 10 and 11 affect the bioenergetic metabolism of Leishmania, with selective elimination of parasites via a host-independent mechanism.

Made available in DSpace on 2018-12-11T17:00:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-01-08. Added 1 bitstream(s) on 2021-07-15T15:21:35Z : No. of bitstreams: 1 S1678-91992016000100301.pdf: 817296 bytes, checksum: 878ad3ccede8d1ab9c78f50c1e845e71 (MD5) Background: Apis mellifera venom, which has already been recommended as an alternative anti-inflammatory treatment, may be also considered an important source of candidate molecules for biotechnological and biomedical uses, such as the treatment of parasitic diseases. Methods: Africanized honeybee venom from Apis mellifera was fractionated by RP-C18-HPLC and the obtained melittin was incubated with promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Cytotoxicity to mice peritoneal macrophages was evaluated through mitochondrial oxidative activity. The production of anti- and pro-inflammatory cytokines, NO and H2O2 by macrophages was determined. Results: Promastigotes and intracellular amastigotes were susceptible to melittin (IC50 28.3 μg.mL-1 and 1.4 μg.mL-1, respectively), but also showed mammalian cell cytotoxicity with an IC50 value of 5.7 μg.mL-1. Uninfected macrophages treated with melittin increased the production of IL-10, TNF-aα, NO and H2O2. Infected melittin-treated macrophages increased IL-12 production, but decreased the levels of IL-10, TNF-aα, NO and H2O2. Conclusions: The results showed that melittin acts in vitro against promastigotes and intracellular amastigotes of Leishmania (L.) infantum. Furthermore, they can act indirectly on intracellular amastigotes through a macrophage immunomodulatory effect. São Paulo State University (UNESP - Univ Estadual Paulista) Graduate Program in Tropical Diseases Botucatu Medical School Adolfo Lutz Institute Department of Parasitology University of São Paulo (USP) Laboratory of Protozoology Institute for Tropical Medicine São Paulo State University (UNESP - Univ Estadual Paulista) Department of Animal Production School of Veterinary Medicine and Animal Husbandry São Paulo State University (UNESP - Univ Estadual Paulista) Center for the Study of Venoms and Venomous Animals (CEVAP), Rua José Barbosa de Barros, 1780 Butantan Institute Laboratory of Biochemistry and Biophysics São Paulo State University (UNESP - Univ Estadual Paulista) Graduate Program in Tropical Diseases Botucatu Medical School São Paulo State University (UNESP - Univ Estadual Paulista) Department of Animal Production School of Veterinary Medicine and Animal Husbandry São Paulo State University (UNESP - Univ Estadual Paulista) Center for the Study of Venoms and Venomous Animals (CEVAP), Rua José Barbosa de Barros, 1780

MeOH extract from the leaves of Plectranthus barbatus Andrews (Lamiaceae), showed in vitro anti-trypanosomal activity. The bioassay-guided fractionation resulted in the isolation of a gallic acid derivative, identified as 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG), after thorough NMR and MS spectral analysis. Finally, this compound was tested against trypomastigote forms of T. cruzi and displayed an EC50 value of 67 µM, at least 6.6-fold more effective than the standard drug benznidazole. This is the first occurrence of PGG in the Plectranthus genus and the first anti-parasitic activity described for PGG in the literature.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Essential oils of ripe fruits from Schinus terebinthifolius (Anacardiaceae), obtained using a pilot extractor and a Clevenger apparatus were chemically characterized. Due the high amount of (-)- α-pinene in both oils, this monoterpene was tested against the protozoan parasite Trypanosoma cruzi, showing a moderate potential (IC50 63.56 µg/mL) when compared to benznidazole (IC50 43.14 µg/mL). Otherwise, (-)- α-pinene oxide did not showed anti-trypanosomal activity (IC50 > 400 µg/mL) while (-)-pinane showed an IC50 of 56.50 µg/mL. The obtained results indicated that the epoxydation of α-pinene results to the loss of the anti-parasitic activity while its hydrogenation product, contributed slightly to the increased activity. Universidade Federal de São Paulo (UNIFESP) Instituto de Ciências Ambientais, Químicas e Farmacêuticas Universidade de São Paulo Instituto de Medicina Tropical de São Paulo Instituto Adolfo Lutz Departamento de Parasitologia Universidade de São Paulo Faculdade de Ciências Farmacêuticas Departamento de Farmácia Universidade Federal de Alfenas Instituto de Química Universidade Federal de Viçosa Departamento de Química UNIFESP, Instituto de Ciências Ambientais, Químicas e Farmacêuticas SciELO

HPLC-UV-ELSD-MS-guided fractionation of the anti-parasitic extract obtained from the marine sponge Monanchora arbuscula, collected off the southeastern coast of Brazil, led to the isolation of a series of guanidine and pyrimidine alkaloids. The pyrimidines monalidine A (1) and arbusculidine A (7), as well as the guanidine alkaloids batzellamide A (8) and hemibatzelladines 9-11, represent new minor constituents that were identified by analysis of spectroscopic data. The total synthesis of monalidine A confirmed its structure. Arbusculidine A (7), related to the ptilocaulin/mirabilin/netamine family of tricyclic guanidine alkaloids, is the first in this family to possess a benzene ring. Batzellamide A (8) and hemibatzelladines 9-11 represent new carbon skeletons that are related to the batzelladines. Evaluation of the anti-parasitic activity of the major known metabolites, batzelladines D (12), F (13), L (14), and nor-L (15), as well as of synthetic monalidine A (1), against Trypanosoma cruzi and Leishmania infantum is also reported, along with a detailed investigation of parasite cell-death pathways promoted by batzelladine L (14) and norbatzelladine L (15).

Neglected parasitic diseases as Leishmaniasis and Chagas disease affect 12 and 6 million people in tropical and subtropical countries, respectively. Co-infections with HIV and other parasitic diseases represent a major threat to Public Health. With a limited number of drugs in clinical use, the treatment is still deficient, including severe adverse effects [1]. Guanidine compounds has been widely found in natural products and especially in marine invertebrates. Marine sponges, in particular, are by far the most abundant source of all natural guanidine derivatives; the structural complexity of these compounds and their potent biological activities make these compounds a target of choice for studies in organic synthesis [2]. In this work, we studied the in vitro antileishmanial activity of a synthetic batzelladine alkaloid derivative and a ptilomycalin A derivative and their capability to alter the permeability of the plasma membrane of Leishmania. The in vitro assays demonstrated that Leishmania (L.) infantum was susceptible to the guanidine derivatives, EB-3 and GB-118, with IC50 values of 57 µg/mL and 33 µg/mL, respectively. The compounds also eliminated the intracellular forms of Leishmania with an IC50 value of 5 µg/mL and 2 µg/mL, respectively. The cytotoxicity of both compounds against NCTC cells resulted in CC50 values of 137 µg/mL and 45 µg/mL, demonstrating selectivity indexes of 27 and 22, respectively. Using the fluorescent probe SYTOX Green, it was possible to demonstrate that both compounds altered the plasma membrane permeability of Leishmania at the early time of incubation (< 60 min), but EB-3 affected the membrane to a higher extent, when compared to the positive control with Triton-X 100. These results indicate the potential of synthetic guanidine derivatives as novel drug prototypes for Leishmaniasis.

Considering the limited and toxic therapeutic arsenal available for visceral leishmaniasis (VL), the drug repositioning approach could represent a promising tool to the introduction of alternative therapies. Histamine H1-receptor antagonists are drugs belonging to different therapeutic classes, including antiallergics and anxyolitics. In this work, we described for the first time the activity of H1-antagonists against L . (L.) infantum and their potential effectiveness in an experimental hamster model. The evaluation against promastigotes demonstrated that chlorpheniramine, cinnarizine, hydroxyzine, ketotifen, loratadine, quetiapine and risperidone exerted a leishmanicidal effect against promastigotes, with IC 50 values in the range of 13–84 μM. The antihistaminic drug cinnarizine demonstrated effectiveness against the intracellular amastigotes, with an IC 50 value of 21 μM. The mammalian cytotoxicity was investigated in NCTC cells, resulting in IC 50 values in the range of 57–229 μM. Cinnarizine was in vivo studied as a free formulation and entrapped into phosphatidylserine-liposomes. The free drug was administered for eight consecutive days at 50 mg/kg by intraperitoneal route (i.p.) and at 100 mg/kg by oral route to L. infantum -infected hamsters, but showed lack of effectiveness in both regimens, as detected by real time PCR. The liposomal formulation was administered by i.p. route at 3 mg/kg for eight days and reduced the parasite burden to 54% in liver when compared to untreated group; no improvement was observed in the spleen of infected hamsters. Cinnarizine is the first antihistaminic drug with antileishmanial activity and could be used as scaffold for drug design studies for VL.

Bioassay-guided fractionation of the EtOH extract of the aerial parts of Baccharis uncinella C. DC. (Asteraceae) led to identification of two cinnamic acid derivatives (caffeic and ferulic acids), two flavones (hispidulin and pectolinaringenin) and a mixture of three chlorogenic acids (3,4-, 3,5- and 4,5-O-dicaffeoylquinic acids), which displayed in vitro anti-trypanosomal activity. Pectolinaringenin, hispidulin and caffeic acid showed activity against trypomastigotes of Trypanosoma cruzi, exhibiting 50% inhibitory concentration (IC50) values of 52, 81 and 56 microg/mL, respectively, while the chlorogenic acid mixture showed an IC50 value of 61 microg/mL. The flavonoids and cinnamic acid derivatives were evaluated for cytotoxicity against NCTC cells resulting in a 50% cytotoxic concentration (CC50) ranging from 33.82 to 129.1 microg/mL while the chlorogenic acids did not display cytotoxicity (CC50150 microg/mL). This is the first report of anti-trypanosomal activity of compounds from B. uncinella.

Naphtoquinones have been used as promising scaffolds for drug design studies against protozoan parasites. Considering the highly toxic and limited therapeutic arsenal, the global negligence with tropical diseases and the elevated prevalence of co-morbidities especially in developing countries, the parasitic diseases caused by various Leishmania species (leishmaniasis) became a significant public health threat in 98 countries. The aim of this work was the evaluation of antileishmanial in vitro potential of thirty-six 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones obtained by a three component reaction of lawsone, the appropriate aldehyde and thiols adequately substituted, exploiting the in situ generation of o-quinonemethides (o-QM) via the Knoevenagel condensation. The antileishmanial activity of the naphthoquinone derivatives was evaluated against promastigotes and intracellular amastigotes of Leishmania (Leishmania) infantum and their cytotoxicity was verified in mammalian cells. Among the thirty-six compounds, twenty-seven were effective against promastigotes, with IC50 values ranging from 8 to 189 µM; fourteen compounds eliminated the intracellular amastigotes, with IC50 values ranging from 12 to 65 µM. The compounds containing the phenyl groups at R1 and R2 and with the fluorine substituent at the phenyl ring at R2, rendered the most promising activity, demonstrating a selectivity index higher than 15 against amastigotes. A QSAR (quantitative structure activity relationship) analysis yielded insights into general structural requirements for activity of most compounds in the series. Considering the in vitro antileishmanial potential of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones and their structure-activity relationships, novel lead candidates could be exploited in future drug design studies for leishmaniasis.

Nature has provided inspiration for Drug Discovery studies and amphibian secretions have been used as a promising source of effective peptides which could be explored as novel drug prototypes for neglected parasitic diseases as Leishmaniasis and Chagas disease. In this study, we isolated four antimicrobial peptides (AMPs) from Phyllomedusa nordestina secretion, and studied their effectiveness against Leishmania (L.) infantum and Trypanosoma cruzi. The antiparasitic fractions were characterized by mass spectrometry and Edman degradation, leading to the identification of dermaseptins 1 and 4 and phylloseptins 7 and 8. T. cruzi trypomastigotes were susceptible to peptides, showing IC50 values in the range concentration of 0.25-0.68 μM. Leishmania (L.) infantum showed susceptibility to phylloseptin 7, presenting an IC50 value of 10 μM. Except for phylloseptin 7 which moderate showed cytotoxicity (IC50=34 μM), the peptides induced no cellular damage to mammalian cells. The lack of mitochondrial oxidative activity of parasites detected by the MTT assay, suggested that peptides were leishmanicidal and trypanocidal. By using the fluorescent probe SYTOX(®) Green, dermaseptins 1 and 4 and phylloseptins 7 and 8 showed time-dependent plasma membrane permeabilization of T. cruzi; phylloseptin 7 also showed a similar effect in Leishmania parasites. The present study demonstrates for the first time that AMPs target the plasma membrane of Leishmania and T. cruzi, leading to cellular death. Considering the potential of amphibian peptides against protozoan parasites and the reduced mammalian toxicity, they may contribute as scaffolds for drug design studies.

Chagas disease is caused by the parasitic protozoan Trypanosoma cruzi. It has high mortality as well as morbidity rates and usually affects the poorer sections of the population. The development of new, less harmful and more effective drugs is a promising research target, since current standard treatments are highly toxic and administered for long periods. Fractioning of methanol (MeOH) extract of the stem bark of Calophyllum brasiliense (Clusiaceae) resulted in the isolation of the coumarin soulamarin, which was characterized by one- and two-dimensional 1H- and 13C NMR spectroscopy as well as ESI mass spectrometry. All data obtained were consistent with a structure of 6-hydroxy-4-propyl-5-(3-hydroxy-2-methyl-1-oxobutyl)-6″,6″-dimethylpyrane-[2″,3″:8,7]-benzopyran-2-one for soulamarin. Colorimetric MTT assays showed that soulamarin induces trypanocidal effects, and is also active against trypomastigotes. Hemolytic activity tests showed that soulamarin is unable to induce any observable damage to erythrocytes (cmax. = 1,300 µM). The lethal action of soulamarin against T. cruzi was investigated by using amino(4-(6-(amino(iminio)methyl)-1H-indol-2-yl)phenyl)methaniminium chloride (SYTOX Green and 1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[4-(chloromethyl)phenyl]-2,3,6,7,12,13,16,17-octahydro-chloride (MitoTracker Red) as fluorimetric probes. With the former, soulamarin showed dose-dependent permeability of the plasma membrane, relative to fully permeable Triton X-100-treated parasites. Spectrofluorimetric and fluorescence microscopy with the latter revealed that soulamarin also induced a strong depolarization (ca. 97%) of the mitochondrial membrane potential. These data demonstrate that the lethal action of soulamarin towards T. cruzi involves damages to the plasma membrane of the parasite and mitochondrial dysfunction without the additional generation of reactive oxygen species, which may have also contributed to the death of the parasites. Considering the unique mitochondrion of T. cruzi, secondary metabolites of plants affecting the bioenergetic system as soulamarin may contribute as scaffolds for the design of novel and selective drug candidates for neglected diseases, mainly Chagas disease., Author Summary Chagas disease is a parasitic protozoan that affects the poorest population in the world, causing a high mortality and morbidity. As a result of highly toxic and long-term treatments, the discovery of novel, safe and more efficacious drugs is essential. Natural products isolated from plants are commonly used as drug prototypes or precursors to treat parasitic diseases. As part of our investigation of bioactive compounds from Brazilian flora, the present study was undertaken in order to determine the antitrypanosomal effects of the soulamarin, a coumarin isolated from the stem bark of Callophyllum brasiliense (Clusiaceae), against Trypanossoma cruzi. This study moreover investigated the lethal action of soulamarin towards the parasite. Considering the obtained results, secondary metabolites of plants affecting the bioenergetic system as soulamarin may contribute as scaffolds for the design of novel and selective drug candidates for neglected diseases, mainly Chagas disease.

Background: Among the tropical parasitic diseases, those caused by protozoans are considered a challenge to public health, being represented by leishmaniasis and Chagas disease. In view of the low effectiveness and toxicity of the current therapy, animal venoms such as amphibian secretions have been used as a promising source of new drug prototypes. The present work aimed to achieve bioguided fractionation of metabolites present in a cutaneous secretion of the caecilian Siphonops annulatus (Amphibia: Gymnophiona: Siphonopidae) with antileishmanial and antitrypanosomal activity. Methods: Through liquid-liquid partition and chromatographic techniques, the secretion was fractionated using bioguided assays. The 50% inhibitory concentration (IC50) of the main fraction (SaFr1) was studied against Leishmania (L.) infantum promastigotes and intracellular amastigotes, trypomastigotes of Trypanosoma cruzi and mammalian cells; viability was detected by the colorimetric MTT assay. By using a spectrofluorimetric assay with the probe SYTOX® Green and transmission electron microscopy (TEM), we also investigated the potential damage caused by SaFr1 in the plasma membrane and mitochondria of Leishmania. Results: The bioguided assay enabled isolation of a highly purified fraction (SaFr1) with an IC50 of 0.065 μg/mL against promastigotes and 2.75 μg/mL against trypomastigotes. Due to its high toxicity to peritoneal macrophages, SaFr1 showed no selectivity towards the intracellular forms of Leishmania. Ultrastructural studies with Leishmania demonstrated severe mitochondrial damage and the formation of large cytoplasmic vacuoles, leading to the parasite’s death within a few hours. Nevertheless, it caused no alteration in the plasma membrane permeability as detected by the fluorescent probe and TEM. Conclusions: The present study demonstrated for the first time the antiparasitic activity of the skin secretion of the caecilian S. annulatus against Leishmania and T. cruzi, confirming that skin secretions of these amphibians, similarly to those of anurans and salamanders, are also potential tools for the development of new drug candidates against neglected diseases.