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The phenotypic activity of two 5-nitroindazolinones, i.e. 2-benzyl-1-propyl (22) and 2-benzyl-1-butyl (24) derivatives, previously proposed as anti-Trypanosoma cruzi prototypes, was presently assayed on bloodstream trypomastigotes (BT) of the moderately drug-resistant Y strain. Further exploration of putative targets and cellular mechanisms involved in their activity was also carried out. Therefore, transmission electron microscopy, high-resolution respirometry and flow cytometry procedures were performed on BT treated for up to 24 h with the respective EC50 value of each derivative. Results demonstrated that although 22 and 24 were not as active as benznidazole in this in vitro assay on BT, both compounds triggered important damages in T. cruzi that lead to the parasite death. Ultrastructural alterations included shedding events, detachment of plasma membrane and nuclear envelope, loss of mitochondrial integrity, besides the occurrence of a large number of intracellular vesicles and profiles of endoplasmic reticulum surrounding cytoplasmic organelles such as mitochondrion. Moreover, both derivatives affected mitochondrion leading to this organelle dysfunction, as reflected by the inhibition in oxygen consumption and the loss of mitochondrial membrane potential. Altogether, the findings exposed in the present study propose autophagic processes and mitochondrial machinery as part of the mode of action of both 5-nitroindazolinones 22 and 24 on T. cruzi trypomastigotes.

Cascade reactions of amidines with nitroallylic acetates and α-bromonitroalkenes provide potentially bioactive imidazoles in good to excellent yields in most cases. While 2,4-disubstituted imidazol-5-yl acetates are formed in the first case, 2,4-disubstituted imidazoles, bearing no substituent at position 5, are the products in the second case. These two series of imidazoles, viz. 2,4,5-trisubstituted and 2,4-disubstituted, were screened for their activity against the protozoan parasite Trypanosoma cruzi which is responsible for Chagas disease. As many as three compounds were as active as the standard benznidazole and two others were 2-3-fold more active highlighting the potential of substituted imidazoles, easily accessible from nitroalkenes, as possible anti-parasitic agents.

A one-pot, two step synthesis of highly substituted imidazoles has been carried out in good to excellent yields for the first time via a cascade intermolecular aza-SN2'-intramolecular aza-Michael addition involving a variety of Morita-Baylis-Hillman acetates of nitroalkenes and amidines in the presence of DABCO at room temperature. The synthetic and biological utility of the products has been demonstrated. In particular, some of the imidazoles exhibited potent activity against T. cruzi, the etiological agent of Chagas disease.

Background: Aspartic peptidase inhibitors have shown antimicrobial action against distinct microorganisms. Due to an increase in the occurrence of Chagas' disease/AIDS co-infection, we decided to explore the effects of HIV aspartic peptidase inhibitors (HIV-PIs) on Trypanosoma cruzi, the etiologic agent of Chagas' disease., Methodology and Principal Findings: HIV-PIs presented an anti-proliferative action on epimastigotes of T. cruzi clone Dm28c, with IC50 values ranging from 0.6 to 14 µM. The most effective inhibitors, ritonavir, lopinavir and nelfinavir, also had an anti-proliferative effect against different phylogenetic T. cruzi strains. The HIV-PIs induced some morphological alterations in clone Dm28c epimastigotes, as reduced cell size and swollen of the cellular body. Transmission electron microscopy revealed that the flagellar membrane, mitochondrion and reservosomes are the main targets of HIV-PIs in T. cruzi epimastigotes. Curiously, an increase in the epimastigote-into-trypomastigote differentiation process of clone Dm28c was observed, with many of these parasites presenting morphological alterations including the detachment of flagellum from the cell body. The pre-treatment with the most effective HIV-PIs drastically reduced the interaction process between epimastigotes and the invertebrate vector Rhodnius prolixus. It was also noted that HIV-PIs induced an increase in the expression of gp63-like and calpain-related molecules, and decreased the cruzipain expression in epimastigotes as judged by flow cytometry and immunoblotting assays. The hydrolysis of a cathepsin D fluorogenic substrate was inhibited by all HIV-PIs in a dose-dependent manner, showing that the aspartic peptidase could be a possible target to these drugs. Additionally, we verified that ritonavir, lopinavir and nelfinavir reduced drastically the viability of clone Dm28c trypomastigotes, causing many morphological damages., Conclusions and Significance: The results contribute to understand the possible role of aspartic peptidases in T. cruzi physiology, adding new in vitro insights into the possibility of exploiting the use of HIV-PIs in the clinically relevant forms of the parasite.

Chagas disease is a neglected tropical illness endemic to Latin America, and its treatment remains unsatisfactory. This disease is caused by the hemoflagellate protozoan Trypanosoma cruzi, which has a complex life cycle involving three evolutive forms in both vertebrate and invertebrate hosts. Targeting metabolic pathways in the parasite for rational drug design represents a promising research field. This research area requires high performance techniques and proteomics become a powerful tool in this context. Here, we review advances in the construction of proteomic maps of the different forms of T. cruzi, emphasizing their biological applications towards the identification of alternative candidates for drug intervention.

The available treatment for the prevention and cure of Chagas disease, caused by the protozoan Trypanosoma cruzi, is still unsatisfactory. Thus, there is an urgent need to develop new drugs. In the last few years, our research group has focused on finding a new chemical entity able to target the infectious bloodstream trypomastigotes. In this study, we assayed 16 β-lapachone analogous with modifications in the pyran and aromatic ring to find a new prototype with high trypanocidal activity. Interestingly, two ortho-naphthoquinones presented the best trypanocidal profile (8c and 8d with an IC50/24 h of 26.9 ± 1.3 and 23.5 ± 2.5 μM, respectively), which were 4 to 17 times more effective than β-lapachone (391.5 ± 16.5 μM) and the standard drug benznidazole (103.6 ± 0.6 μM). The introduction of a hydroxyl group on the compounds' aromatic ring modulated their biological profile by increasing their activity not only for cancer cells (MDAMB435), as previously described in literature, but also against T. cruzi. The Structure-Activity Relationship (SAR) study indicated that this introduction modulated HOMO and MEP parameters, improving the trypanocidal activity.

Background: Naphthoquinones (NQs) are privileged structures in medicinal chemistry due to the biological effects associated with the induction of oxidative stress. The present study evaluated the activities of sixteen NQs derivatives on Trypanosoma cruzi., Results: Fourteen NQs displayed higher activity against bloodstream trypomastigotes of T. cruzi than benznidazole. Further assays with NQ1, NQ8, NQ9 and NQ12 showed inhibition of the proliferation of axenic epimastigotes and intracelulluar amastigotes interiorized in macrophages and in heart muscle cells. NQ8 was the most active NQ against both proliferative forms of T. cruzi. In epimastigotes the four NQs induced mitochondrial swelling, vacuolization, and flagellar blebbing. The treatment with NQs also induced the appearance of large endoplasmic reticulum profiles surrounding different cellular structures and of myelin-like membranous contours, morphological characteristics of an autophagic process. At IC50 concentration, NQ8 totally disrupted the ΔΨm of about 20% of the parasites, suggesting the induction of a sub-population with metabolically inactive mitochondria. On the other hand, NQ1, NQ9 or NQ12 led only to a discrete decrease of TMRE + labeling at IC50 values. NQ8 led also to an increase in the percentage of parasites labeled with DHE, indicative of ROS production, possibly the cause of the observed mitochondrial swelling. The other three NQs behaved similarly to untreated controls., Conclusions: NQ1, NQ8, NQ9 and NQ12 induce an autophagic phenotype in T. cruzi epimastigoted, as already observed with others NQs. The absence of oxidative stress in NQ1-, NQ9- and NQ12-treated parasites could be due to the existence of more than one mechanism of action involved in their trypanocidal activity, leaving ROS generation suppressed by the detoxification system of the parasite. The strong redox effect of NQ8 could be associated to the presence of the acetyl group in its structure facilitating quinone reduction, as previously demonstrated by electrochemical analysis. Further experiments using biochemical and molecular approaches are needed to better characterize ROS participation in the mechanism of action of these NQs.

Five 2-hydroxy-3-substituted-aminomethyl naphthoquinones, nine 1,2,3-triazolic para-naphthoquinones, five nor-β-lapachone-based 1,2,3-triazoles, and several other naphthoquinonoid compounds were synthesized and evaluated against the infective bloodstream form of Trypanosoma cruzi, the etiological agent of Chagas disease, continuing our screening program for new trypanocidal compounds. Among all the substances, 16-18, 23, 25-29 and 30-33 were herein described for the first time and fifteen substances were identified as more potent than the standard drug benznidazole, with IC(50)/24h values in the range of 10.9-101.5 μM. Compounds 14 and 19 with Selectivity Index of 18.9 and 6.1 are important structures for further studies., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Chagas' disease, caused by the protozoan Trypanosoma cruzi, represents a serious health problem in Latin America, and the available chemotherapy, which is based on 2 nitro-derivatives, is not satisfactory. In folk medicine, natural products including naphthoquinones have been employed for the treatment of different parasitic diseases. In the pursuit of alternative drugs for Chagas' disease, we investigated the mechanism of action of the triazolic naphthoquinone (TN; 2,2-dimethyl-3-(4-phenyl-1H-1,2,3-triazol-1-yl)-2,3-dihydronaphtho[1,2-b]furan-4,5-dione), which is the most active compound against T. cruzi trypomastigotes among a series of naphthofuranquinones. TN was active against the 3 parasite forms producing a dose-dependent inhibitory effect. In epimastigotes, TN induced reservosome disruption, flagellar blebbing, Golgi disorganization, the presence of cytosolic concentric membrane structures and abnormal multiflagellar parasites. The treatment also led to the appearance of well-developed endoplasmic reticulum profiles surrounding organelles that associated with an increase in monodansylcadaverine labelling, suggesting autophagy as part of the TN mechanism of action. Interestingly, no ultrastructural damage was detected in the mitochondria of naphthoquinone-treated epimastigotes. Flow cytometric analysis demonstrated an impairment of mitosis, an increase in ROS production and the maintenance of mitochondrial membrane potential. TN could be a good starting point in the investigation of a chemotherapeutic approach for the treatment of Chagas' disease.

Background: Trypanosoma cruzi is the etiological agent of Chagas' disease. During the parasite life cycle, many molecules are involved in the differentiation process and infectivity. Peptidases are relevant for crucial steps of T. cruzi life cycle; as such, it is conceivable that they may participate in the metacyclogenesis and interaction with the invertebrate host., Methodology/principal Findings: In this paper, we have investigated the effect of the calpain inhibitor MDL28170 on the attachment of T. cruzi epimastigotes to the luminal midgut surface of Rhodnius prolixus, as well as on the metacyclogenesis process and ultrastructure. MDL28170 treatment was capable of significantly reducing the number of bound epimastigotes to the luminal surface midgut of the insect. Once the cross-reactivity of the anti-Dm-calpain was assessed, it was possible to block calpain molecules by the antibody, leading to a significant reduction in the capacity of adhesion to the insect guts by T. cruzi. However, the antibodies were unable to interfere in metacyclogenesis, which was impaired by the calpain inhibitor presenting a significant reduction in the number of metacyclic trypomastigotes. The calpain inhibitor also promoted a direct effect against bloodstream trypomastigotes. Ultrastructural analysis of epimastigotes treated with the calpain inhibitor revealed disorganization in the reservosomes, Golgi and plasma membrane disruption., Conclusions/significance: The presence of calpain and calpain-like molecules in a wide range of organisms suggests that these proteins could be necessary for basic cellular functions. Herein, we demonstrated the effects of MDL28170 in crucial steps of the T. cruzi life cycle, such as attachment to the insect midgut and metacyclogenesis, as well as in parasite viability and morphology. Together with our previous findings, these results help to shed some light on the functions of T. cruzi calpains. Considering the potential roles of these molecules on the interaction with both invertebrate and vertebrate hosts, it is interesting to improve knowledge on these molecules in T. cruzi.

Chagas' disease, caused by the protozoan Trypanosoma cruzi, is endemic in Latin America, which current treatment presents variable efficacy and serious side effects. A previous screening of naphthoquinone derivatives pointed to the naphthoimidazoles N1, N2 and N3 as the most active compounds against T. cruzi. In this study, a proteomic approach was employed to identify proteins involved in the N1, N2 and N3 trypanocidal activity. In epimastigotes, the naphthoimidazoles are involved in multiple mechanisms: (a) redox metabolism; (b) energy production; (c) ergosterol biosynthesis; (d) cytoskeleton assembly; (e) protein metabolism and biosynthesis; and (f) chaperones modulation. They induce an imbalance in crucial pathways of the parasite, leading to the loss of metabolic homeostasis and T. cruzi death., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Objectives: There is a general lack of effective and non-toxic chemotherapeutic agents for treating Chagas' disease. In the present work, we evaluated the in vitro activity of the calpain inhibitor MDL28170 against Trypanosoma cruzi relevant clinical forms., Methods: The effect of MDL28170 on bloodstream trypomastigotes at different concentrations was assessed by counting the parasites in a Neubauer chamber, which allowed the determination of IC(50) values. Subsequently, parasite-macrophage interaction was assessed by two approaches: (i) peritoneal mouse macrophages were pre-infected with trypomastigotes for 3 h and then treated daily for 72 h with MDL28170; or (ii) bloodstream trypomastigotes were pre-treated with the calpain inhibitor for 1 h and then subjected to the infection assay., Results: MDL28170 was capable of significantly reducing the viability of bloodstream trypomastigotes, presenting an IC(50)/24 h value of 20.4 microM. Also, parasites pre-treated with the inhibitor, at subinhibitory drug concentrations, prior to macrophage infection presented a clear dose-dependent inhibition profile, where the inhibition increased from 20% to 50% (in relation to control) as MDL28170 concentration rose from 6.25 to 50 microM. In addition, macrophages experimentally infected with T. cruzi that were treated with the calpain inhibitor presented a significant reduction in the percentage of infection even at the lowest concentrations (6.25 microM)., Conclusions: These data may contribute to the study of the calpains in T. cruzi infection and add new in vitro insights into the possibility of exploiting calpains as promising targets to treat Chagas' disease.

In continuing our screening program of naphthoquinone activity against Trypanosoma cruzi, the aetiological agent of Chagas' disease, new beta-lapachone-based 1,2,3-triazoles, 3-arylamino-nor-beta-lapachones, 3-alkoxy-nor-beta-lapachones and imidazole anthraquinones were synthesised and evaluated against bloodstream trypomastigote forms of the parasite. Compounds 2,2-dimethyl-3-(2,4-dibromophenylamino)-2,3-dihydro-naphtho[1,2-b]furan-4,5-dione, IC(50)/24h 24.9+/-7.4 and 4-azido-3-bromo-2,2-dimethyl-3,4-dihydro-2H-benzo[h]chromene-5,6-dione with 23.4+/-3.8 microM showed a trypanosomicidal activity higher than benznidazole. These results demonstrate the potential of naphthoquinone derivatives as novel structures for the development of alternative drugs for Chagas' disease., ((c) 2010 Elsevier Ltd. All rights reserved.)

Despite ongoing efforts, the current treatment for Chagas disease is still unsatisfactory, mainly because of the severe side effects and variable efficacy of the available nitroheterocycles. Our group has been assaying natural quinones isolated from Brazilian flora, and their derivatives, as alternative chemotherapeutic agents against Trypanosoma cruzi. From C-allyl lawsone three naphthofuranquinones were synthesized, which were active against trypomastigotes and epimastigotes. Here, we further investigated the activity and the mechanisms of action of these quinones. They exhibited powerful effects on intracellular amastigotes, presenting low toxicity to the host cells. Ultrastructural analyses of treated epimastigotes and trypomastigotes indicated a potent effect of the three naphthofuranquinones on the parasite mitochondrion, which appeared drastically swollen and with a washed-out matrix profile. Fluorescence-activated cell sorting analysis of rhodamine 123-stained T. cruzi showed that the three naphthofuranquinones caused a potent dose-dependent collapse of the mitochondrial membrane potential, especially in the epimastigote form. Naphthofuranquinones also decreased specifically mitochondrial complex I-III activity in both epimastigotes and trypomastigotes, parallel to a reduction in succinate-induced oxygen consumption. Mitochondrial hydrogen peroxide formation was also increased in epimastigotes after treatment with the naphthofuranquinones. Our results indicate that the trypanocidal action of the naphthofuranquinones is associated with mitochondrial dysfunction, leading to increased reactive oxygen species generation and parasite death.

Summary: In a screening of 65 derivatives of natural quinones using bloodstream trypomastigotes of Trypanosoma cruzi, the 3 naphthoimidazoles derived from beta-lapachone - N1, N2 and N3--were selected as the most active. Investigation of their mode of action led to the characterization of mitochondrion, reservosomes and DNA as their main targets, and stimulated further studies on death pathways. Ultrastructural analysis revealed both autophagic (autophagosomes) and apoptotic-like (membrane blebbing) phenotypes. Flow cytometry analysis showed, in N2-treated trypomastigotes, a small increase of phosphatidylserine exposure, and a large increase in the percentage of necrosis, caused by N1 or N2. These death phenotypes were not detected in treated epimastigotes. The strong increase in labelling of monodansyl cadaverine, the inhibition of the death process by wortmannin or 3-methyladenine, the overexpression of ATG genes in treated epimastigotes, together with ultrastructural evidence point to autophagy as the predominant phenotype induced by the naphthoimidazoles. However, there are other pathways occurring concomitantly with variable intensities, justifying the need to detail the molecular features involved.

Electron microscopy has proven to be a reliable and essential tool to determine morphological alterations and target organelles in the investigation of new drugs for Chagas disease. In this review, we focused on evaluating different agents that induce death of Trypanosoma cruzi, i.e. lysophospholipids analogues, naphthoquinones and derivatives, cytoskeletal inhibitors and natural products. Apoptosis-like presents as morphological characteristics DNA fragmentation, membrane blebbing and apoptotic body formation. Autophagy involves autophagosome formation, with the appearance of membranes surrounding organelles and cytosolic structures. Necrosis causes the loss of osmotic balance, an increase of cytoplasmic vacuolization and plasma membrane disruption. Mitochondrion appears as a central checkpoint in both apoptosis and necrosis. Our evidences of ultrastructural changes to T. cruzi treated with the different classes of compounds point to dramatic mitochondrial alterations and similar autophagic phenotypes. Lysophospholipid analogues interfere in the lipid biosynthesis in epimastigotes, altering the amount of both phospholipids and sterols, and consequently the physical properties of the membrane. Naphthoquinone derivatives led to a strong DNA fragmentation in trypomastigotes and to the release of cysteine proteases from reservosomes to cytosol in epimastigotes, starting a proteolytic process which results in parasite death. The susceptibility of reservosomes was also observed in parasites treated with propolis, suggesting impairment of lipid metabolism, compromising membrane fluidity and leading to lysis. The cytoskeletal agents blocked mitosis of epimastigotes, arresting cell cycle and impairing the parasite proliferation. The variety of drug stimuli converge to the same pathway of death suggests an intense cross-talking between the three types of PCD in the protozoa.

P2X7 is a member of the purinergic receptors family, with extracellular adenosine triphosphate (ATP) as the main agonist, promoting cations influx and membrane permeabilization that can lead to cell death. We previously proposed that extracellular ATP is involved in thymus atrophy induced by Trypanosoma cruzi infection through the induction of CD4+/CD8+ double-positive cell death and that P2X7 could be involved in this process. To further elucidate this possibility raised by in vitro assays, in this study, we used P2X7-/- mice and observed no difference in thymus atrophy or parasitemia when compared to C57Bl/6. We then decided to investigate other aspects of purinergic receptor interplay that could be better evidenced by the infection and observed that (1) thymocytes from infected and noninfected C57Bl/6 mice express P2X4 and P2X7 receptors (Western blotting), but ATP-induced membrane permeabilization only occurs in thymocytes from infected mice; (2) peritoneal macrophages from noninfected C57Bl/6 mice (P2X4+ and P2X7+) are permeabilized by ATP. Although macrophages from infected C57Bl/6 mice are P2X7- but P2X4+, they are resistant to ATP, either through permeabilization or Ca++ influx (fluorimetry); (3) using noninfected P2X7-/- mice, C57Bl/6 infected mice, and different agonistic stimuli, we observed interesting cross-talks among P2X and P2Y receptors (flow cytometry).

Proteasomes are intracellular complexes that control protein degradation in organisms ranging from Archaebacteria to mammals. In some parasitic protozoa, the proteasome is involved in cell differentiation and replication. In this study, we have used proteasome inhibitors to determine the biological role of proteasomes during the replication and in vitro metacyclogenesis of Trypanosoma cruzi. We used light and transmission electron microscopy to analyze morphological data and flow cytometry to analyze changes in the cell cycle. The growth of T. cruzi epimastigote culture forms in liver infusion tryptose medium was inhibited by the presence of up to 10 microM lactacystin. Inhibition was dose-dependent, with IC50 (50% inhibitory concentration) of 4.35 microM after 24 or 72 h. The metacyclogenesis process in vitro was strongly (95%) inhibited by 5 microM lactacystin treatment. The adhesion phase was not affected, but the epimastigotes did not differentiate into metacyclic trypomastigotes. Most treated epimastigotes had replicated DNA, with swelling of the mitochondrion and an altered distribution of nuclear and kinetoplast DNA. Our findings suggest that inhibition of the ubiquitin-proteasome pathway in T. cruzi epimastigotes does not block adhesion, but disrupts cell division and affects factors triggering differentiation.

[1,2,3]-Triazole derivatives of nor-beta-lapachone were synthesized and assayed against the infective bloodstream trypomastigote form of Trypanosoma cruzi, the etiological agent of Chagas disease. All the derivatives were more active than the original quinones, with IC(50)/1 day values in the range of 17 to 359 microM, the apolar phenyl substituted triazole 6 being the most active compound. These triazole derivatives of nor-beta-lapachone emerge as interesting new lead compounds in drug development for Chagas disease.

In the search for new therapeutic agents for Chagas' disease, we screened extracts obtained from the Brazilian plant Pterodon pubescens found commercially in the medicinal flora market. We investigated the potential trypanocidal effect of the oleaginous ethanolic extract of P. pubescens seeds and its fractions (PF1, PF1.1, PF1.2, and PF1.3) and of geranylgeraniol (GG-OH), the sole component of the hexane fraction (PF1.2). In experiments with bloodstream trypomastigotes of Trypanosoma cruzi, performed at 37 degrees C in culture medium, PF1.2 and GG-OH showed similar potency, while the oleaginous extract from P. pubescens seeds and the other fractions were about three times less active. GG-OH inhibited the proliferation of intracellular amastigotes, at concentrations which do not affect the mammalian host cell. Transmission electron microscopy and flow cytometry analysis indicate the mitochondrion, an organelle that plays a central role in apoptosis, of both epimastigotes and of trypomastigotes as the major target of GG-OH. On the other hand, the ultrastructural images of the endoplasmic reticulum profiles, myelin-like figures, and concentric membranous arrangements inside damaged mitochondrion are suggestive of an autophagic pathway leading to parasite death. Because the different forms of cell death share some morphological features such as mitochondrial collapse, further studies are needed to disclose the trypanocidal action of GG-OH.

New naphthoquinone derivatives were synthesized and assayed against bloodstream trypomastigote forms of Trypanosoma cruzi, the etiological agent of Chagas' disease. The compounds were rationalized based on hybrid drugs and appear as important compounds against this parasite. From nor-lapachol were prepared five substituted ortho-naphthofuranquinones, a non-substituted para-naphthofuranquinone, a new oxyrane and an azide and from alpha-lapachone a new non-substituted para-naphthofuranquinone. Other five substituted ortho-naphthofuranquinones recently designed as cytotoxic, were also evaluated. The most active compounds were the ortho naphthofuranquinones 3-(4-methoxyphenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione and 3-(3-nitrophenylamino)-2,3-dihydro-2,2-dimethylnaphtho[1,2-b]furan-4,5-dione with trypanocidal activity higher than that of benznidazole, the standard drug. The compounds were rationalized based on hybrid drugs and appear as important compounds against T. cruzi. The trypanocidal activity of these substances endowed with redox properties representing a good starting point for a medicinal chemistry program aiming the chemotherapy of Chagas' disease.

Three naphthoimidazoles presenting aromatic groups attached to the imidazole ring were the most active against trypomastigotes of Trypanosoma cruzi between 45 derivatives from beta-lapachone. N1 is active against the three forms of the parasite. In this work, we investigated N2 and N3 and analyzed the effect of the three derivatives on metacyclogenesis, endocytosis, and cell cycle. In epimastigotes, N2 and N3 blocked the cell cycle, inhibited succinate cytochrome c reductase, metacyclogenesis, and induced damage to mitochondrion, Golgi, and reservosomes. In treated trypomastigotes, there were alterations in the mitochondrion, nucleus and kinetoplast, and DNA fragmentation. Preincubation with cysteine protease inhibitors reversed the effect of N1, N2, and N3. Such reversion and ultrastructural alterations suggest the involvement of autophagy in parasite death. Ultrastructural, flow cytometry, and biochemical studies suggest that naphthoimidazoles interferes with the energetic metabolism and induces DNA fragmentation.

This study describes the design, synthesis and trypanocidal evaluation of new azaheterocyclic derivatives (4-8). These compounds were designed as megazol (1) analogs based on bioisosterism tools and were synthesized to investigate the possible pharmacophoric contribution of the 1,2,4-triazole nucleus, the position of the heterocyclic nucleus and presence of the nitro group, to the activity against the bloodstream trypomastigote forms of Trypanosoma cruzi. The most potent compound was 6, a nitro derivative obtained by substitution of a thiadiazole by a triazole ring and by moving the nitro group from C-5 position, as in 1, to the C-4 position. Finally, we have used semi-empirical theoretical calculations to discuss the correlation of some stereo electronic properties with biological activity in an attempt to understand the possible mechanism of action of the designed series of compounds.

In the present work, we have investigated the effect of essential oils obtained from Origanum vulgare L. (oregano) and Thymus vulgaris L. (thyme) on growth and ultrastructure of diverse evolutive forms of Trypanosoma cruzi. Culture epimastigotes and bloodstream trypomastigotes were incubated for 24 h with different concentrations of oregano or thyme essential oils and with thymol (the main constituent of thyme), and the inhibitory concentration (IC)(50) was determined by cell counting. Crude extract of oregano essential oil inhibited epimastigote growth (IC(50)/24 h = 175 microg/ml) and also induced trypomastigote lysis (IC(50)/24 h = 115 microg/ml). Thyme essential oil presented IC(50)/24 h values of 77 microg/ml for epimastigotes and 38 mug/ml for trypomastigotes, while treatment with thymol resulted in an IC(50)/24 h of 62 microg/ml for epimastigotes and 53 microg/ml for trypomastigotes. Scanning electron microscopy of treated cells showed few morphological alterations at the plasma membrane. Observation by transmission electron microscopy showed cytoplasmic swelling with occasional morphological alterations in plasma and flagellar membrane. Our data indicate that oregano and thyme essential oils are effective against T. cruzi, with higher activity of thyme, and that thymol may be the main component responsible for the trypanocidal activity.

Objectives: Investigation of the mode of action of the naphthoimidazole N1, obtained from the reaction of beta-lapachone with benzaldehyde, which among 45 semi-synthetic derivatives of naphthoquinones isolated from Tabebuia sp. was one of the most active compounds against Trypanosoma cruzi trypomastigotes., Methods: Quantification of the effect of N1 against the proliferative forms of T. cruzi, and investigation of potential targets in the parasite using electron microscopy and flow cytometry techniques., Results: N1 presented the following order of activity: amastigotes > trypomastigotes > epimastigotes. The effect on intracellular forms was approximately 25 times higher than on macrophages and heart muscle cells. N1-treated parasites presented an abnormal chromatin condensation and mitochondrial damage. In epimastigotes, alterations of reservosomes were observed, and in trypomastigotes, a decrease in the electron density of acidocalcisomes was observed. In epimastigotes, the naphthoimidazole inhibited the activity of succinate cytochrome c reductase. Labelling with rhodamine 123 or Acridine Orange was decreased in both forms treated with N1., Conclusions: The results suggest that epimastigotes, reservosomes, mitochondrion, and nucleus contain N1 targets. In trypomastigotes, in which reservosomes are absent, the organelles affected by the compound were also the mitochondrion and nucleus, as well as acidocalcisomes, in which the decrease in electron density could be due to the use of polyphosphate as an alternative energy supply.

We synthesized new naphthoimidazoles from beta-lapachone with an aromatic moiety linked to the imidazole ring, using phenylic and heterocyclic aldehydes. The most active compound against Trypanosoma cruzi had a p-methyl group linked to the phenyl ring, presenting an EC(50) value of 15.5 +/- 2.9 microM. No reliable correlation could be established with the biological activity and the structure of in the phenylic series. For the heterocyclic series, activity was associated with a three bond-distance from nitrogen to the imidazole ring, in accordance with our previous work.