Furan derivatives impair proliferation and affect ultrastructural organization of Trypanosoma cruzi and Leishmania amazonensis.

Chagas disease and leishmaniasis are neglected diseases caused by parasites of the Trypanosomatidae family and together they affect millions of people in the five continents. The treatment of Chagas disease is based on benznidazole, whereas for leishmaniasis few drugs are available, such as amphotericin B and miltefosine. In both cases, the current treatment is not entirely efficient due to toxicity or side effects. Encouraged by the need to discover valid targets and new treatment options, we evaluated 8 furan compounds against Trypanosoma cruzi and Leishmania amazonensis, considering their effects against proliferation, infection, and ultrastructure. Many of them were able to impair T. cruzi and L. amazonensis proliferation, as well as cause ultrastructural alterations, such as Golgi apparatus disorganization, autophagosome formation, and mitochondrial swelling. Taken together, the results obtained so far make these compounds eligible for further steps of chemotherapy study.

Crystal structure of Q4D6Q6, a conserved kinetoplastid-specific protein from Trypanosoma cruzi.

Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ßßαßßαßß topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction.

In vitro activities of adamantylidene-substituted alkylphosphocholine TCAN26 against Trypanosoma cruzi: Antiproliferative and ultrastructural effects.

Phospholipids are the main component of membranes and are responsible for cell integrity. Alkylphospholipid analogues (APs) were first designed as antitumoral agents and were later tested against different cell types. Trypanosoma cruzi, the Chagas disease etiological agent, is sensitive to APs (edelfosine, miltefosine and ilmofosine) in vitro. We investigated the effect of synthetic ring substituted AP against epimastigotes, amastigotes and trypomastigotes. TCAN26, could inhibit the in vitro growth of epimastigotes and amastigotes with the 50% inhibitory concentrations (IC50) in the nanomolar range. Trypomastigotes lysis was also induced with 24-h treatment and a LC50 of 2.3 µM. Ultrastructural analysis by electron microscopy demonstrated that TCAN26 mainly affected the parasite's membranes leading to mitochondrial and Golgi cisternae swelling, membrane blebs, and autophagic figures in the different parasite developmental stages. While the Golgi of the parasites was significantly affected, the Golgi complex of the host cells remained normal suggesting a specific mechanism of action. In summary, our results suggest that TCAN 26 is a potent and selective inhibitor of T. cruzi growth probably due to disturbances of phospholipid biosynthesis.

Morphobiological, morphometric and ultrastructural characterization of sylvatic Trypanosoma cruzi isolates from Rio de Janeiro state, Brazil / Caracterização morfobiológica, morfométrica e ultraestrutural de isolados silvestres de Trypanosoma cruzi do estado do Rio de Janeiro, Brasil

Abstract Triatoma vitticeps is a triatomine with geographic distribution restrict to Brazil, which exhibits high prevalence of Trypanosoma cruzi natural infection. Of special epidemiologic concern, this species often invades households in the states of Rio de Janeiro, Minas Gerais and Espírito Santo. The objective of this study was to evaluate morphological and ultrastructural parameters on three T. cruzi isolates obtained from wild T. vitticeps specimens. The growth and cell differentiation of the parasite was evaluated through epimastigote and trypomastigote forms obtained in the growth curves for three distinct isolates. The maximum growth showed differences at the 20th day of the curve. Our in vitro results show a heterogeneity, regarding these features for samples cultivated under the same conditions. Morphometric analyzes based on the shape of epimastigotes and trypomastigotes corroborated such differentiation. These results highlight the need of better understanding the meaning of this diversity under an eco-epidemiological perspective.

Resumo Triatoma vitticeps é um triatomíneo com distribuição geográfica restrita ao território brasileiro, apresentando alta prevalência de infecção natural pelo Trypanosoma cruzi. Esta espécie é relevante sob o ponto de vista epidemiológico por invadir domicílios com frequência nos estados do Rio de Janeiro, Minas Gerais e Espírito Santo. O objetivo deste estudo foi avaliar parâmetros morfológicos e ultraestruturais, em três isolados de T. cruzi obtidos a partir de triatomíneos silvestres. O crescimento e a diferenciação celular do parasita foi avaliado através das formas epimastigotas e tripomastigotas obtidas nas curvas de crescimento para os três isolados. O crescimento máximo mostrou diferenças no 20º dia da curva. Nossos resultados in vitro mostram uma heterogeneidade, em relação a essas características para amostras cultivadas nas mesmas condições. As análises morfométricas baseadas na conformação de epimastigotas e trypomastigotes corroboraram essa diferenciação. Estes resultados ressaltam a necessidade de uma melhor compreensão do significado desta diversidade sob uma perspectiva eco-epidemiológica.

Primary targets of the sesquiterpene lactone deoxymikanolide on Trypanosoma cruzi.

BACKGROUND: Deoxymikanolide is a sesquiterpene lactone isolated from Mikania micrantha and M. variifolia which, has previously demonstrated in vitro activity on Trypanosoma cruzi and in vivo activity on an infected mouse model. PURPOSE: Based on these promising findings, the aim of this study was to investigate the mechanism of action of this compound on different parasite targets. METHODS: The interaction of deoxymikanolide with hemin was examined under reducing and non- reducing conditions by measuring modifications in the Soret absorption band of hemin; the thiol interaction was determined spectrophotometrically through its reaction with 5,5'-dithiobis-2-nitrobenzoate in the presence of glutathione; activity on the parasite antioxidant system was evaluated by measuring the activity of the superoxide dismutase and trypanothione reductase enzymes, together with the intracellular oxidative state by flow cytometry. Superoxide dismutase and trypanothione reductase activities were spectrophotometrically tested. Cell viability, phosphatidylserine exposure and mitochondrial membrane potential were assessed by means of propidium iodide, annexin-V and rhodamine 123 staining, respectively; sterols were qualitatively and quantitatively tested by TLC; ultrastructural changes were analyzed by transmission electron microscopy. Autophagic cells were detected by staining with monodansylcadaverine. RESULTS: Deoxymikanolide decreased the number of reduced thiol groups within the parasites, which led to their subsequent vulnerability to oxidative stress. Treatment of the parasites with the compound produced a depolarization of the mitochondrial membrane even though the plasma membrane permeabilization was not affected. Deoxymikanolide did not affect the intracellular redox state and so the mitochondrial dysfunction produced by this compound could not be attributed to ROS generation. The antioxidant defense system was affected by deoxymikanolide at twenty four hours of treatment, when both an increased oxidative stress and decreased activity of superoxide dismutase and trypanothione reductase (40 and 60% respectively) were observed. Both the oxidative stress and mitochondrial dysfunction induce parasite death by apoptosis and autophagy. CONCLUSION: Based on our results, deoxymikanolide would exert its anti-T cruzi activity as a strong thiol blocking agent and by producing mitochondrial dysfunction.

Treatment of Trypanosoma cruzi with 2-bromopalmitate alters morphology, endocytosis, differentiation and infectivity.

BACKGROUND: The palmitate analogue 2-bromopalmitate (2-BP) is a non-selective membrane tethered cysteine alkylator of many membrane-associated enzymes that in the last years emerged as a general inhibitor of protein S-palmitoylation. Palmitoylation is a post-translational protein modification that adds palmitic acid to a cysteine residue through a thioester linkage, promoting membrane localization, protein stability, regulation of enzymatic activity, and the epigenetic regulation of gene expression. Little is known on such important process in the pathogenic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. RESULTS: The effect of 2-BP was analyzed on different developmental forms of Trypanosoma cruzi. The IC50/48 h value for culture epimastigotes was estimated as 130 µM. The IC50/24 h value for metacyclic trypomastigotes was 216 nM, while for intracellular amastigotes it was 242 µM and for cell derived trypomasigotes was 262 µM (IC50/24 h). Our data showed that 2-BP altered T. cruzi: 1) morphology, as assessed by bright field, scanning and transmission electron microscopy; 2) mitochondrial membrane potential, as shown by flow cytometry after incubation with rhodamine-123; 3) endocytosis, as seen after incubation with transferrin or albumin and analysis by flow cytometry/fluorescence microscopy; 4) in vitro metacyclogenesis; and 5) infectivity, as shown by host cell infection assays. On the other hand, lipid stress by incubation with palmitate did not alter epimastigote growth, metacyclic trypomastigotes viability or trypomastigote infectivity. CONCLUSION: Our results indicate that 2-BP inhibits key cellular processes of T. cruzi that may be regulated by palmitoylation of vital proteins and suggest a metacyclic trypomastigote unique target dependency during the parasite development.

New 1,3-thiazole derivatives and their biological and ultrastructural effects on Trypanosoma cruzi.

In previous studies, the compound 3-(bromopropiophenone) thiosemicarbazone was described as a potent anti-Trypanosoma cruzi and cruzain inhibitor. In view to optimize this activity, 1,3-thiazole core was used as building-block strategy to access new lead generation of anti T. cruzi agents. In this way a series of thiazole derivatives were synthesized and most of these derivatives exhibited antiparasitic activity similar to benznidazole (Bzd). Among them, compounds (1c) and (1g) presented better selective index (SI) than Bzd. In addition, compounds showed inhibitory activity against the cruzain protease. As observed by electron microscopy, compound (1c) treatment caused irreversible and specific morphological changes on ultrastructure organization of T. cruzi, demonstrating that this class of compounds is killing parasites.

The Uptake of GABA in Trypanosoma cruzi.

Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.

Trypanosoma cruzi Calreticulin Topographical Variations in Parasites Infecting Murine Macrophages.

Trypanosoma cruzi calreticulin (TcCRT), a 47-kDa chaperone, translocates from the endoplasmic reticulum to the area of flagellum emergence. There, it binds to complement components C1 and mannan-binding lectin (MBL), thus acting as a main virulence factor, and inhibits the classical and lectin pathways. The localization and functions of TcCRT, once the parasite is inside the host cell, are unknown. In parasites infecting murine macrophages, polyclonal anti-TcCRT antibodies detected TcCRT mainly in the parasite nucleus and kinetoplast. However, with a monoclonal antibody (E2G7), the resolution and specificity of the label markedly improved, and TcCRT was detected mainly in the parasite kinetoplast. Gold particles, bound to the respective antibodies, were used as probes in electron microscopy. This organelle may represent a stopover and accumulation site for TcCRT, previous its translocation to the area of flagellum emergence. Finally, early during T. cruzi infection and by unknown mechanisms, an important decrease in the number of MHC-I positive host cells was observed.

4-Nitrobenzaldehyde thiosemicarbazone: a new compound derived from S-(-)-limonene that induces mitochondrial alterations in epimastigotes and trypomastigotes of Trypanosoma cruzi.

Trypanosoma cruzi is the causative agent of Chagas' disease, a parasitic disease that remains a serious health concern with unsatisfactory treatment. Drugs that are currently used to treat Chagas' disease are partially effective in the acute phase but ineffective in the chronic phase of the disease. The aim of the present study was to evaluate the antitrypanosomal activity and morphological, ultrastructural and biochemical alterations induced by a new molecule, 4-nitrobenzaldehyde thiosemicarbazone (BZTS), derived from S-(-)-limonene against epimastigote, trypomastigote and intracellular amastigote forms of T. cruzi. BZTS inhibited the growth of epimastigotes (IC50 = 9·2 µ m), intracellular amastigotes (IC50 = 3·23 µ m) and inhibited the viability of trypomastigotes (EC50 = 1·43 µ m). BZTS had a CC50 of 37·45 µ m in LLCMK2 cells. BZTS induced rounding and distortion of the cell body and severely damaged parasite mitochondria, reflected by extensive swelling and disorganization in the inner mitochondrial membrane and the presence of concentric membrane structures inside the organelle. Cytoplasmic vacuolization, endoplasmic reticulum that surrounded organelles, the loss of mitochondrial membrane potential, and increased mitochondrial O2 •- production were also observed. Our results suggest that BZTS alters the ultrastructure and physiology of mitochondria, which could be closely related to parasite death.

JVG9, a benzimidazole derivative, alters the surface and cytoskeleton of Trypanosoma cruzi bloodstream trypomastigotes

Trypanosoma cruzi has a particular cytoskeleton that consists of a subpellicular network of microtubules and actin microfilaments. Therefore, it is an excellent target for the development of new anti-parasitic drugs. Benzimidazole 2-carbamates, a class of well-known broad-spectrum anthelmintics, have been shown to inhibit the in vitro growth of many protozoa. Therefore, to find efficient anti-trypanosomal (trypanocidal) drugs, our group has designed and synthesised several benzimidazole derivatives. One, named JVG9 (5-chloro-1H-benzimidazole-2-thiol), has been found to be effective against T. cruzi bloodstream trypomastigotes under both in vitro and in vivo conditions. Here, we present the in vitro effects observed by laser scanning confocal and scanning electron microscopy on T. cruzi trypomastigotes. Changes in the surface and the distribution of the cytoskeletal proteins are consistent with the hypothesis that the trypanocidal activity of JVG9 involves the cytoskeleton as a target.

Effects of camptothecin derivatives and topoisomerase dual inhibitors on Trypanosoma cruzi growth and ultrastructure.

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas' disease that is an endemic disease in Latin America and affects about 8 million people. This parasite belongs to the Trypanosomatidae family which contains a single mitochondrion with an enlarged region, named kinetoplast that harbors the mitochondrial DNA (kDNA). The kinetoplast and the nucleus present a great variety of essential enzymes involved in DNA replication and topology, including DNA topoisomerases. Such enzymes are considered to be promising molecular targets for cancer treatment and for antiparasitic chemotherapy. In this work, the proliferation and ultrastructure of T. cruzi epimastigotes were evaluated after treatment with eukaryotic topoisomerase I inhibitors, such as topotecan and irinotecan, as well as with dual inhibitors (compounds that block eukaryotic topoisomerase I and topoisomerase II activities), such as baicalein, luteolin and evodiamine. Previous studies have shown that such inhibitors were able to block the growth of tumor cells, however most of them have never been tested on trypanosomatids. RESULTS: Considering the effects of topoisomerase I inhibitors, our results showed that topotecan decreased cell proliferation and caused unpacking of nuclear heterochromatin, however none of these alterations were observed after treatment with irinotecan. The dual inhibitors baicalein and evodiamine decreased cell growth; however the nuclear and kinetoplast ultrastructures were not affected. CONCLUSIONS: Taken together, our data showed that camptothecin is more efficient than its derivatives in decreasing T. cruzi proliferation. Furthermore, we conclude that drugs pertaining to a certain class of topoisomerase inhibitors may present different efficiencies as chemotherapeutical agents.

Cellular response to Trypanosoma cruzi infection induces secretion of defensin α-1, which damages the flagellum, neutralizes trypanosome motility, and inhibits infection.

Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that colonic epithelial model HCT116 cells respond to Trypanosoma cruzi infection by secreting defensin α-1, which reduces infection. We also report the early effects of defensin α-1 on invasive trypomastigotes that involve damage of the flagellar structure to inhibit parasite motility and reduce cellular infection. Short exposure of defensin α-1 to trypomastigotes shows that defensin α-1 binds to the flagellum, resulting in flagellar membrane and axoneme alterations, followed by breaking of the flagellar membrane connected to the trypanosome body, leading to detachment and release of the parasite flagellum. In addition, defensin α-1 induces a significant reduction in parasite motility in a peptide concentration-dependent manner, which is abrogated by anti-defensin α-1 IgG. Preincubation of trypomastigotes with a concentration of defensin α-1 that inhibits 50% trypanosome motility significantly reduced cellular infection by 80%. Thus, human defensin α-1 is an innate immune molecule that is secreted by HCT116 cells in response to T. cruzi infection, inhibits T. cruzi motility, and plays an important role in reducing cellular infection. This is the first report showing a novel cellular innate immune response to a human parasite by secretion of defensin α-1, which neutralizes the motility of a human parasite to reduce cellular infection. The mode of activity of human defensin α-1 against T. cruzi and its function may provide insights for the development of new antiparasitic strategies.

Melittin peptide kills Trypanosoma cruzi parasites by inducing different cell death pathways.

Antimicrobial peptides (AMPs) are components of the innate immune response that represent desirable alternatives to conventional pharmaceuticals, as they have a fast mode of action, a low likelihood of resistance development and can act in conjunction with existing drug regimens. AMPs exhibit strong inhibitory activity against both Gram-positive and Gram-negative bacteria, fungi, viruses, metazoans and other parasites, such as the protozoan Leishmania. Melittin is a naturally occurring AMP, which comprises 40-50% of the dry weight of Apis mellifera venom. Our group has recently shown that crude A. mellifera venom is lethal to Trypanosoma cruzi, the Chagas disease etiologic agent, and generates a variety of cell death phenotypes among treated parasites. Here, we demonstrate that the melittin affected all of T. cruzi developmental forms, including the intracellular amastigotes. The ultrastructural changes induced by melittin suggested the occurrence of different programmed cell death pathways, as was observed in A. mellifera-treated parasites. Autophagic cell death appeared to be the main death mechanism in epimastigotes. In contrast, melittin-treated trypomastigotes appeared to be dying via an apoptotic mechanism. Our findings confirm the great potential of AMPs, including melittin, as a potential source of new drugs for the treatment of neglected diseases, such as Chagas disease.

Estudo da atividade biológica de adutos Morita-Baylis-Hillman sobre Trypanosoma cruzi / Study of the biological activity of Morita-Baylis-Hillman adducts against Trypanosoma cruzi

A doença de Chagas, causada pelo protozoário hemoflagelado Trypanosoma cruzi, consiste em um grave problema de saúde pública na América Latina, com cerca de 7 a 8 milhões de pessoas infectadas. O benznidazol é atualmente o único fármaco disponível para o tratamento, apresentando boa atividade na fase aguda, mas com eficácia questionada na fase crônica, além de apresentar severos efeitos colaterais e longo período de tratamento. Tendo em vista a sua fácil síntese e baixo custo, adutos aromáticos oriundos da reação Morita-Baylis-Hillman têm sido considerados promissores como quimioterápicos. Neste sentido, foi avaliada a atividade tripanocida e citotóxica de seis adutos Morita-Baylis-Hillman. Para analisar possíveis efeitos dos adutos sobre estruturas específicas do protozoário foi utilizado a microscopia confocal a laser, através da marcação com Rodamina 123, MitoSOXT, Laranja de Acridina e Kit Live/Dead(R). As alterações morfológicas em parasitas submetidos ao tratamento foram acompanhadas por microscopia eletrônica de transmissão. Nossos resultados mostraram que todos os compostos foram capazes de inibir o crescimento de formas epimastigotas e causaram uma diminuição da viabilidade em formas tripomastigotas, apresentando uma moderada citotoxicidade para células de mamíferos. Os adutos MBH1, MBH2, MBH5 e MBH7 também foram efetivos em inibir a infecção de macrófagos e diminuir a viabilidade das formas amastigotas. Os compostos não foram capazes de alterar os níveis de produção do óxido nítrico em macrófagos. Análises pela microscopia confocal e microscopia eletrônica de transmissão (MET) apontam a mitocôndria como principal alvo intracelular destes compostos. Alterações compatíveis com a perda da viabilidade e morte celular por necrose e autofagia foram observadas por MET. Os resultados obtidos neste trabalho colocam os adutos Morita -Baylis-Hillman em evidência como agentes promissores para o tratamento da tripanossomíase americana.

Apis mellifera venom induces different cell death pathways in Trypanosoma cruzi.

Chagas disease chemotherapy is based on drugs that exhibit toxic effects and have limited efficacy, such as Benznidazole. Therefore, research into new chemotherapeutic agents from natural sources needs to be exploited. Apis mellifera venom consists of many biologically active molecules and has been reported to exhibit remarkable anti-cancer effects, often promoting an apoptosis-like death phenotype. This study demonstrates that A. mellifera venom can affect the growth, viability and ultrastructure of all Trypanosoma cruzi developmental forms, including intracellular amastigotes, at concentrations 15- to 100-fold lower than those required to cause toxic effects in mammalian cells. The ultrastructural changes induced by the venom in the different developmental forms led us to hypothesize the occurrence of different programmed cell death pathways. Autophagic cell death, characterized by the presence of autophagosomes-like organelles and a strong monodansyl cadaverine labelling, appears to be the main death mechanism in epimastigotes. In contrast, increased TUNEL staining, abnormal nuclear chromatin condensation and kDNA disorganization was observed in venom-treated trypomastigotes, suggesting cell death by an apoptotic mechanism. On the other hand, intracellular amastigotes presented a heterogeneous cell death phenotype profile, where apoptosis-like death seemed to be predominant. Our findings confirm the great potential of A. mellifera venom as a source for the development of new drugs for the treatment of neglected diseases such as Chagas disease.

Antimicrobial activity of synthetic bornyl benzoates against Trypanosoma cruzi.

We report here for the first time the in vitro effects of (1S,2R,4S)-1,7,7-trimethyl-bicyclo[2·2·1]heptan-2-yl-3',4',5'-trimethoxy benzoate (1) and (1S,2R,4S)-1,7,7-trimethyl-bicyclo[2·2·1]heptan-2-yl benzoate (2) on the growth and ultrastructure of Trypanosoma cruzi. These two synthetic compounds exerted an antiproliferative effect on the epimastigote forms of the parasite. The ICs(50/72h) of two synthetic L-bornyl benzoates, 1 and 2, was 10·1 and 12·8 µg/ml, respectively. Both compounds were more selective against epimastigotes than HEp-2 cells. Ultrastructural analysis revealed intense cytoplasmic vacuolization and the appearance of cytoplasmic materials surrounded by membranes. The treatment of peritoneal macrophages with compounds 1 and 2 caused a significant decrease in the number of T. cruzi-infected cells. L-Bornyl benzoate derivatives may serve as a potential source for the development of more effective and safer chemotherapeutic agents against T. cruzi infections.

Differential trypanocidal activity of novel macrolide antibiotics; correlation to genetic lineage.

Here we report the systematic study of the anti-trypanocidal activity of some new products derived from S. diastatus on 14 different T. cruzi strains spanning the six genetic lineages of T. cruzi. As the traditional growth inhibition curves giving similar IC(50) showed great differences on antibiotic and lineage tested, we decided to preserve the wealth of information derived from each inhibition curve and used an algorithm related to potency of the drugs, combined in a matrix data set used to generate a cluster tree. The cluster thus generated based just on drug susceptibility data closely resembles the phylogenies of the lineages derived from genetic data and provides a novel approach to correlate genetic data with phenotypes related to pathogenesis of Chagas disease. Furthermore we provide clues on the drugs mechanism of action.

Expression profile and subcellular localization of HslV, the proteasome related protease from Trypanosoma cruzi.

Trypanosoma cruzi is a rare example of an eukaryote that has genes for two threonine proteases: HslVU complex and 20S proteasome. HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. In this study, we expressed and obtained specific antibodies to HslU and HslV recombinant proteins and demonstrated the interaction between HslU/HslV by coimmunoprecipitation. To evaluate the intracellular distribution of HslV in T. cruzi we used an immunofluorescence assay and ultrastructural localization by transmission electron microscopy. Both techniques demonstrated that HslV was localized in the kinetoplast of epimastigotes. We also analyzed the HslV/20S proteasome co-expression in Y, Berenice 62 (Be-62) and Berenice 78 (Be-78) T. cruzi strains. Our results showed that HslV and 20S proteasome are differently expressed in these strains. To investigate whether a proteasome inhibitor could modulate HslV and proteasome expressions, epimastigotes from T. cruzi were grown in the presence of PSI, a classical proteasome inhibitor. This result showed that while the level of expression of HslV/20S proteasome is not affected in Be-78 strain, in Y and Be-62 strains the presence of PSI induced a significantly increase in Hslv/20S proteasome expression. Together, these results suggest the coexistence of the protease HslVU and 20S proteasome in T. cruzi, reinforcing the hypothesis that non-lysosomal degradation pathways have an important role in T. cruzi biology.

Novas perspectivas de fármacos com propriedade tripanocida: estudos biológicos de compostos heterocíclicos / Novel perspectives of drugs with tripanocidal properties: biological study of heterocyclic compounds

Os compostos mesoiônicos são moléculas não aromáticas planas, pertencentes à classe das betaínas, os quais apresentam um grande número de atividades biológicas incluindo antibacteriana, anti-inflamatória e anti-protozoária. A despeito do potencial quimoterápico destes compostos, estudos destas moléculas contra o Trypanosoma cruzi, agente etiológico da doença de Chagas, ainda são incipientes. No presente trabalho nós avaliamos as atividades tripanocida e citotóxica dos mesoiônicos MECPYR-04, MEC-02, MC-01 e MC-02. Nossos resultados demonstraram que todos os compostos inibiram o crescimento de formas epimastigotas e causaram decréscimo da viabilidade celular em tripomastigotas. Estes compostos também foram efetivos em inibir a infecção de macrófagos e diminuir a viabilidade das formas amastigotas. MECPYR-04 foi o mais efetivo contra todas as formas evolutivas do parasito, entretanto também apresentou alta toxicidade para células de mamíferos, enquanto MEC02, MC-01 e MC-02 apresentaram baixa toxidade em células de mamíferos e alta seletividade para os parasitas. Apenas MEC-02 e MECPYR-04 aumentaram a produção de NO em macrófagos quando comparados com o controle, sugerindo um potencial imunomodulatório destes compostos. Com objetivo de analisar os efeitos de MEC-02, MC-01 e MC-02 sobre a integridade da membrana plasmática e o potencial de membrana mitocondrial, células tratadas e controles foram submetidas a marcação com iodeto de propidio e rodamina 123 respectivamente, e analisadas por microscopia confocal. Nenhuma diferença significativa na integridade da membrana foi observada em células tratadas. Estes achados foram confirmados pela microscopia eletrônica de varredura. Apenas MC-01 e MC-02 foram capazes de induzir alterações no potencial de membrana mitocondrial como observado por microscopia confocal e citometria de fluxo. Análise ultraestrutural revelou apenas pequenas alterações em formas tripomastigotas tratadas com MEC-02. Por outro lado, células tratadas com MC-01 e MC-02 apresentaram drástico inchaço da mitocôndria com desorganização ou perda do material da matriz mitocondrial, sugerindo que esta estrutura seja o alvo de ação de MC-01 e MC-02. Todos estes resultados apontam MEC-02, MC-01 e MC-02 como candidatos a fármacos contra a doença de Chagas.