Aromatic diamidines represent a class of DNA minor groove-binding ligands that exhibit high levels of antiparasitic activity. Since the chemotherapy for Chagas' disease is still an unsolved problem and previous reports on diamidines and related analogues show that they have high levels of activity against Trypanosoma cruzi infection both in vitro and in vivo, our present aim was to evaluate the cellular effects in vitro of three reversed amidines (DB889, DB702, and DB786) and one diguanidine (DB711) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas ' disease. Our data show that the reversed amidines have higher levels of activity than the diguanidine, with the order of trypanocidal activities being as follows: DB889 > DB702 > DB786 > DB711. Transmission electron microscopy analysis showed that the reversed amidines induced many alterations in the nuclear morphology, swelling of the endoplasmic reticulum and Golgi structures, and consistent damage in the mitochondria and kinetoplasts of the parasites. Interestingly, in trypomastigotes treated with the reversed amidine DB889, multiple axoneme structures (flagellar microtubules) were noted. Flow cytometry analysis confirmed that the treated parasites presented an important loss of the mitochondrial membrane potential, as revealed by a decrease in rhodamine 123 fluorescence. Our results show that the reversed amidines have promising activities against the relevant mammalian forms of T. cruzi and display high trypanocidal effects at very low doses. This is especially the case for DB889, which merits further in vivo evaluation.Aromatic diamidines are DNA minor groove-binding ligands (MGBLs), which present striking broad-spectrum antimicrobial effects (28). Although this class of compounds displays significant in vitro and in vivo activities against fungi, amoeba, bacteria, and especially protozoan parasites, certain structures can show toxicity toward mammalian cells (23). In addition, aromatic diamidines in general lack oral bioavailability, which limits their use (28). To overcome these limitations, prodrugs such as the methamidoxime prodrug of furamidine (DB289), which is currently undergoing phase III clinical trials for the treatment of human African trypanosomiasis, have been developed (29). Trypanosoma cruzi is the etiological agent of Chagas' disease, a zoonosis considered a major public health problem in the developing countries of Central and South America (14). The disease is widespread in areas of endemicity in Latin America, and it has been estimated that the overall prevalence of human infection is about 17 million cases and that approximately 120 million people are at risk of contracting the infection (30). However, up to now there has been neither an effective vaccine nor a satisfactory treatment for the disease. Drug therapy depends mostly upon nitrofurans and nitroimidazoles, such as nifurtimox and benznidazole (4,26,27).Our previous studies revealed that furamidine and its Nphenyl-substituted analo...

The antiinflammatory cytokine transforming growth factor ␤ (TGF-␤) plays an important role in Chagas disease, a parasitic infection caused by the protozoan Trypanosoma cruzi. In the present study, we show that SB-431542, an inhibitor of the TGF-␤ type I receptor (ALK5), inhibits T. cruzi-induced activation of the TGF-␤ pathway in epithelial cells and in cardiomyocytes. Further, we demonstrate that addition of SB-431542 greatly reduces cardiomyocyte invasion by T. cruzi. Finally, SB-431542 treatment significantly reduces the number of parasites per infected cell and trypomastigote differentiation and release. Taken together, these data further confirm the major role of the TGF-␤ signaling pathway in both T. cruzi infection and T. cruzi cell cycle completion. Our present data demonstrate that small inhibitors of the TGF-␤ signaling pathway might be potential pharmacological tools for the treatment of Chagas disease.

The lack of translation between preclinical assays and clinical trials for novel therapies for Chagas disease (CD) indicates a need for more feasible and standardized protocols and experimental models. Here, we investigated the effects of treatment with benznidazole (Bz) and with the potent experimental T. cruzi CYP51 inhibitor VNI in mouse models of Chagas disease by using different animal genders and parasite strains and employing distinct types of therapeutic schemes. Our findings confirm that female mice are less vulnerable to the infection than males, show that male models are less susceptible to treatment with both Bz and VNI, and thus suggest that male models are much more suitable for selection of the most promising antichagasic agents. Additionally, we have found that preventive protocols (compound given at 1 dpi) result in higher treatment success rates, which also should be avoided during advanced steps of in vivo trials of novel anti-T. cruzi drug candidates. Another consideration is the relevance of immunosuppression methods in order to verify the therapeutic profile of novel compounds, besides the usefulness of molecular diagnostic tools (quantitative PCR) to ascertain compound efficacy in experimental animals. Our study aims to contribute to the development of more reliable methods and decision gates for in vivo assays of novel antiparasitic compounds in order to move them from preclinical to clinical trials for CD. (1), is a neglected pathology caused by the obligately intracellular protozoan parasite Trypanosoma cruzi. The disease is endemic in 21 countries of Central and South America, where about 8 million people are infected and more than 12,000 die annually (http: //www.dndial.org). The treatment for CD is limited to the use of two nitroderivatives (benznidazole [Bz] and nifurtimox [Nf]), which are largely unsatisfactory, indicating a need for novel, safer, and more efficient therapies (2). Although a large number of in vitro and in vivo studies have been performed on experimental chemotherapy of novel drug candidates for CD, besides Bz and Nf, very few compounds have moved to clinical trials (3). C hagas disease (CD), discovered by Carlos ChagasRecently, two antifungal drugs, posaconazole and E1224 (the prodrug of ravuconazole), which are inhibitors of fungal sterol 14a-demethylase (CYP51), were evaluated as potential antichagasic drugs on chronic patients, but unfortunately both displayed rather high (70 to 80%) rates of therapeutic failure (4, 5). It has been suggested that at least part of this unexpected failure could be due to the lack of translation from in vitro and in vivo models to the clinic and that a redesign of the current screening strategy during the drug discovery process should be considered (5). On the other hand, recent data demonstrated the potency and selectivity of a novel experimental inhibitor of T. cruzi CYP51, the VNI molecule, which yielded promising in vivo findings even with highly resistant T. cruzi strains (6). In this vein, we evaluated the effects and outco...

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Leishmania are protozoa that invade mononuclear phagocytes with the involvement of different ligand-receptor systems, including mannose receptors. Until now, scant data are available concerning the mechanisms that govern the infection of Leishmania in other host cell types such as fibroblasts. Our aim was to analyze the expression of mannose receptors in primary cultures of skin fibroblasts (SF) further characterizing their role during the invasion of promastigotes of Leishmania (L.) amazonensis. Both fluorescent, light, and electron microscopy assays revealed that SF have mannose receptors since they bound and internalized mannosylated ligands in addition to being positively labeled by fuc-BSA-FITC probes. d-mannose competition assays revealed the participation of mannose receptors during the parasite association with SF presenting upregulated receptor expression during the initial steps of the infection. After longer periods of Leishmania:fibroblasts contact, the modulation noted in the host mannose receptors was reverted concomitantly to the infection control, suggesting that the parasites were required for the alteration maintenance and providing evidences that the SF may display microbicidal mechanisms to control the Leishmania infection.

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f Fifteen novel arylimidamides (AIAs) (6 bis-amidino and 9 mono-amidino analogues) were assayed against Trypanosoma cruzi in vitro and in vivo. All the bis-AIAs were more effective than the mono-AIAs, and two analogues, DB1967 and DB1989, were further evaluated in vivo. Although both of them reduced parasitemia, protection against mortality was not achieved. Our results show that the number of amidino-terminal units affects the efficacy of arylimidamides against T. cruzi.

Two Trypanosoma cruzi Z3 strains, designated as 3663 and 4167, were previously isolated from insect vectors captured in the Brazilian Amazon region. These strains exhibited different infection patterns in Vero, C6/36, RAW 264.7 and HEp-2 cell lineages, in which 3663 trypomastigote form was much less infective than 4167 ones. A proteomic approach was applied to investigate the differences in the global patterns of protein expression in these two Z3 strains. Two-dimensional (2D) protein maps were generated and certain spots were identified by mass spectrometry (MS). Our analyses revealed a significant difference in the expression profile of different proteins between strains 3663 and 4167. Among them, cruzipain, an important regulator of infectivity. This data was corroborated by flow cytometry analysis using anti-cruzipain antibody. This difference could contribute to the infectivity profiles observed for each strain by in vitro assay using different cell lines.