Identification of novel bromodomain inhibitors of Trypanosoma cruzi bromodomain factor 2 (TcBDF2) using a fluorescence polarization-based high-throughput assay.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors.

Immunoendocrine dysbalance during uncontrolled T. cruzi infection is associated with the acquisition of a Th-1-like phenotype by Foxp3(+) T cells.

We previously showed that Trypanosomacruzi infection in C57BL/6 mice results in a lethal infection linked to unbalanced pro- and anti-inflammatory mediators production. Here, we examined the dynamics of CD4(+)Foxp3(+) regulatory T (Treg) cells within this inflammatory and highly Th1-polarized environment. Treg cells showed a reduced proliferation rate and their frequency is progressively reduced along infection compared to effector T (Teff) cells. Also, a higher fraction of Treg cells showed a naïve phenotype, meanwhile Teff cells were mostly of the effector memory type. T. cruzi infection was associated with the production of pro- and anti-inflammatory cytokines, notably IL-27p28, and with the induction of T-bet and IFN-γ expression in Treg cells. Furthermore, endogenous glucocorticoids released in response to T. cruzi-driven immune activation were crucial to sustain the Treg/Teff cell balance. Notably, IL-2 plus dexamethasone combined treatment before infection was associated with increased Treg cell proliferation and expression of GATA-3, IL-4 and IL-10, and increased mice survival time. Overall, our results indicate that therapies aimed at specifically boosting Treg cells, which during T. cruzi infection are overwhelmed by the effector immune response, represent new opportunities for the treatment of Chagas disease, which is actually only based on parasite-targeted chemotherapy.

Reciprocal influences between leptin and glucocorticoids during acute Trypanosoma cruzi infection.

Leptin and glucocorticoids (GCs) are involved in metabolic functions, thymic homeostasis and immune activity through complex interactions. We recently showed that C57BL/6 mice infected with Trypanosoma cruzi revealed a fatal disease associated with a dysregulated immune-endocrine response characterized by weight loss, deleterious synthesis of pro-inflammatory cytokines and GCs-driven thymus atrophy. Extending this study, we now explored the relationship between leptin and GCs, in terms of infection outcome, thymic and metabolic changes. T. cruzi-infected mice showed a food intake reduction, together with hypoglycemia and lipolysis-related changes. Infected animals also displayed a reduction in systemic and adipose tissue levels of leptin, paralleled by a down-regulation of their receptor (ObR) in the hypothalamus. Studies in infected mice subjected to adrenalectomy (Adx) showed a worsened course of infection accompanied by even more diminished systemic and intrathymic leptin levels, for which GCs are necessary not only to decrease inflammation but also to sustain leptin secretion. Adx also protected from thymic atrophy, independently of the reduced leptin contents. Leptin administration to infected mice aggravated inflammation, lowered parasite burden and attenuated GCs release, but did not normalize thymic atrophy or metabolic parameters. Acute T. cruzi infection in C57BL/6 mice coexists with a dysregulation of leptin/hypothalamic ObR circuitry dissociated from body weight and food intake control. Endogenous GCs production attempted to reestablish systemic leptin concentrations, but failed to improve leptin-protective activities at the thymic level, suggesting that the leptin/GCs intrathymic relationship is also altered during this infection.

Downregulation of intestinal multidrug resistance transporter 1 in obese mice: Effect on its barrier function and role of TNF-α receptor 1 signaling.

OBJECTIVES: Multidrug resistance transporter 1 (Mdr-1) is a relevant component of the intestinal transcellular barrier that decreases absorption of oral drugs, thus modulating their bioavailability. Obese patients with metabolic disorders take medications that are subjected to intestinal metabolism and the Mdr-1-dependent barrier. This study evaluated the effect of a high-fat diet (HFD; 40% fat for 16 wk) on Mdr-1 expression and transport activity in C57BL/6 (C57) male mice. Comparable studies were performed in tumor necrosis factor α (TNF-α) receptor 1 knockout mice (R1KO) to delineate a possible role of TNF-α signaling. METHODS: mRNA expression was evaluated by real-time polymerase chain reaction and protein levels by western blotting and immunohistochemistry. Mdr-1 activity was assessed using the everted intestinal sac model, with rhodamine 123 as the substrate. Statistical comparisons were made using the Student t test or one-way analysis of variance followed by the post hoc Tukey test. RESULTS: Mdr-1 protein, as well as its corresponding Mdr1a and Mdr1b mRNA, was decreased in C57-HFD mice compared with controls. Immunohistochemical studies confirmed downregulation of Mdr-1 in situ. These results correlated with a 48% decrease in the basolateral to apical transport of rhodamine 123. In contrast, R1KO-HFD modified neither intestinal Mdr-1 mRNA nor its protein expression or activity. In addition, C57-HFD showed elevated intestinal TNF-α mRNA and protein (enzyme-linked immunosorbent assay) levels, whereas R1KO-HFD was undetectable or had a lower increase, respectively. CONCLUSIONS: This study demonstrated an impairment of the Mdr-1 intestinal barrier function induced by HFD as a consequence of downregulation of both Mdr-1 gene homologues, resulting in impaired Mdr-1 protein expression. Inflammatory response mediated by TNF-α receptor 1 signaling was likely involved.

In Vitro Drug Screening Against All Life Cycle Stages of Trypanosoma cruzi Using Parasites Expressing ß-galactosidase.

Trypanosoma cruzi is the causative agent of Chagas disease (ChD), an endemic disease of public health importance in Latin America that also affects many non-endemic countries due to the increase in migration. This disease affects nearly 8 million people, with new cases estimated at 50,000 per year. In the 1960s and 70s, two drugs for ChD treatment were introduced: nifurtimox and benznidazole (BZN). Both are effective in newborns and during the acute phase of the disease but not in the chronic phase, and their use is associated with important side effects. These facts underscore the urgent need to intensify the search for new drugs against T. cruzi. T. cruzi is transmitted through hematophagous insect vectors of the Reduviidae and Hemiptera families. Once in the mammalian host, it multiplies intracellularly as the non-flagellated amastigote form and differentiates into the trypomastigote, the bloodstream non-replicative infective form. Inside the insect vector, trypomastigotes transform into the epimastigote stage and multiply through binary fission. This paper describes an assay based on measuring the activity of the cytoplasmic ß-galactosidase released into the culture due to parasites lysis by using the substrate, chlorophenol red ß-D-galactopyranoside (CPRG). For this, the T. cruzi Dm28c strain was transfected with a ß-galactosidase-overexpressing plasmid and used for in vitro pharmacological screening in epimastigote, trypomastigote, and amastigote stages. This paper also describes how to measure the enzymatic activity in cultured epimastigotes, infected Vero cells with amastigotes, and trypomastigotes released from the cultured cells using the reference drug, benznidazole, as an example. This colorimetric assay is easily performed and can be scaled to a high-throughput format and applied to other T. cruzi strains.

Chagas' disease: an update on immune mechanisms and therapeutic strategies.

* Introduction * Chagas' disease * Chemotherapy * Immune response in experimental T. cruzi infection * Immune response in human beings infected with T. cruzi * Immune response in the treatment of chagasic infection * The need for new therapeutic alternatives for Chagas' disease * Conclusions The final decade of the 20th century was marked by an alarming resurgence in infectious diseases caused by tropical parasites belonging to the kinetoplastid protozoan order. Among the pathogenic trypanosomatids, some species are of particular interest due to their medical importance. These species include the agent responsible for Chagas' disease, Trypanosoma cruzi. Approximately 8 to 10 million people are infected in the Americas, and approximately 40 million are at risk. In the present review, we discuss in detail the immune mechanisms elicited during infection by T. cruzi and the effects of chemotherapy in controlling parasite proliferation and on the host immune system.

Trypanosoma cruzi High Mobility Group B (TcHMGB) can act as an inflammatory mediator on mammalian cells.

BACKGROUND: High Mobility Group B (HMGB) proteins are nuclear architectural factors involved in chromatin remodeling and important nuclear events. HMGBs also play key roles outside the cell acting as alarmins or Damage-associated Molecular Patterns (DAMPs). In response to a danger signal these proteins act as immune mediators in the extracellular milieu. Moreover, these molecules play a central role in the pathogenesis of many autoimmune and both infectious and sterile inflammatory chronic diseases. PRINCIPAL FINDINGS: We have previously identified a High mobility group B protein from Trypanosoma cruzi (TcHMGB) and showed that it has architectural properties interacting with DNA like HMGBs from other eukaryotes. Here we show that TcHMGB can be translocated to the cytoplasm and secreted out of the parasite, a process that seems to be stimulated by acetylation. We report that recombinant TcHMGB is able to induce an inflammatory response in vitro and in vivo, evidenced by the production of Nitric Oxide and induction of inflammatory cytokines like TNF-α, IL-1ß and IFN-γ gene expression. Also, TGF-ß and IL-10, which are not inflammatory cytokines but do play key roles in Chagas disease, were induced by rTcHMGB. CONCLUSIONS: These preliminary results suggest that TcHMGB can act as an exogenous immune mediator that may be important for both the control of parasite replication as the pathogenesis of Chagas disease and can be envisioned as a pathogen associated molecular pattern (PAMP) partially overlapping in function with the host DAMPs.

Overexpression of cytoplasmic TcSIR2RP1 and mitochondrial TcSIR2RP3 impacts on Trypanosoma cruzi growth and cell invasion.

BACKGROUND: Trypanosoma cruzi is a protozoan pathogen responsible for Chagas disease. Current therapies are inadequate because of their severe host toxicity and numerous side effects. The identification of new biotargets is essential for the development of more efficient therapeutic alternatives. Inhibition of sirtuins from Trypanosoma brucei and Leishmania ssp. showed promising results, indicating that these enzymes may be considered as targets for drug discovery in parasite infection. Here, we report the first characterization of the two sirtuins present in T. cruzi. METHODOLOGY: Dm28c epimastigotes that inducibly overexpress TcSIR2RP1 and TcSIR2RP3 were constructed and used to determine their localizations and functions. These transfected lines were tested regarding their acetylation levels, proliferation and metacyclogenesis rate, viability when treated with sirtuin inhibitors and in vitro infectivity. CONCLUSION: TcSIR2RP1 and TcSIR2RP3 are cytosolic and mitochondrial proteins respectively. Our data suggest that sirtuin activity is important for the proliferation of T. cruzi replicative forms, for the host cell-parasite interplay, and for differentiation among life-cycle stages; but each one performs different roles in most of these processes. Our results increase the knowledge on the localization and function of these enzymes, and the overexpressing T. cruzi strains we obtained can be useful tools for experimental screening of trypanosomatid sirtuin inhibitors.

Efficacy of novel benznidazole solutions during the experimental infection with Trypanosoma cruzi.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. About 8 million people throughout Latin America are infected causing approximately 10,000 deaths annually. Benznidazole, available as unique 100 mg tablets in many of the endemic countries, is currently the drug of choice for the specific treatment of this condition. Despite of the large number of pediatric patients infected, there are no commercial liquid dosage forms available to treat this trypanosomiasis. This work showed that novel benznidazole-water-polyethylene glycol 400 solutions are active against T. cruzi in a murine model of Chagas' disease. Present results constitute the first demonstration on the usefulness of benznidazole solutions in infected mice.

Tumor necrosis factor-α regulates glucocorticoid synthesis in the adrenal glands of Trypanosoma cruzi acutely-infected mice. the role of TNF-R1.

Adrenal steroidogenesis is under a complex regulation involving extrinsic and intrinsic adrenal factors. TNF-α is an inflammatory cytokine produced in response to tissue injury and several other stimuli. We have previously demonstrated that TNF-R1 knockout (TNF-R1(-/-)) mice have a dysregulated synthesis of glucocorticoids (GCs) during Trypanosoma cruzi acute infection. Since TNF-α may influence GCs production, not only through the hypothalamus-pituitary axis, but also at the adrenal level, we now investigated the role of this cytokine on the adrenal GCs production. Wild type (WT) and TNF-R1(-/-) mice undergoing acute infection (Tc-WT and Tc-TNF-R1(-/-) groups), displayed adrenal hyperplasia together with increased GCs levels. Notably, systemic ACTH remained unchanged in Tc-WT and Tc-TNF-R1(-/-) compared with uninfected mice, suggesting some degree of ACTH-independence of GCs synthesis. TNF-α expression was increased within the adrenal gland from both infected mouse groups, with Tc-WT mice showing an augmented TNF-R1 expression. Tc-WT mice showed increased levels of P-p38 and P-ERK compared to uninfected WT animals, whereas Tc-TNF-R1(-/-) mice had increased p38 and JNK phosphorylation respect to Tc-WT mice. Strikingly, adrenal NF-κB and AP-1 activation during infection was blunted in Tc-TNF-R1(-/-) mice. The accumulation of mRNAs for steroidogenic acute regulatory protein and cytochrome P450 were significantly increased in both Tc-WT and Tc-TNF-R1(-/-) mice; being much more augmented in the latter group, which also had remarkably increased GCs levels. TNF-α emerges as a potent modulator of steroidogenesis in adrenocortical cells during T. cruzi infection in which MAPK pathways, NF-κB and AP-1 seem to play a role in the adrenal synthesis of pro-inflammatory cytokines and enzymes regulating GCs synthesis. These results suggest the existence of an intrinsic immune-adrenal interaction involved in the dysregulated synthesis of GCs during murine Chagas disease.

Benznidazole treatment attenuates liver NF-κB activity and MAPK in a cecal ligation and puncture model of sepsis.

Recent studies have shown that Benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and nitric oxide (NO) release in activated macrophages by blocking NF-κB through inhibition of IKK in vitro. As so far, little is known about the mechanism by which BZL provokes the inhibition of inflammatory response in sepsis in vivo, we aimed to delineate the possible role of BZL as a modulator in liver inflammation in mice with sepsis induced by cecal ligation and puncture (CLP). Specifically, we analyzed leukocytes, liver production of TNF-α and NO and the intracellular pathways modulated by these mediators, including NF-κB and MAPKs, in the liver of mice 24 h post-CLP. Our results show that BZL reduces leukocytes in peripheral blood accompanied by an increase in peritoneal macrophages 24h after CLP. In the liver of these septic mice, BZL decreased expression of mRNA and protein for TNF-α and NOS-2 by inhibition of NF-κB and MAPK (p-38 and ERK). The body of evidence suggests that the immunomodulatory effects of BZL could act selectively, as it is able to decrease the systemic inflammatory reaction and the hepatic response but it can increase the number of cells in the site of infection.

Benznidazole blocks NF-kappaB activation but not AP-1 through inhibition of IKK.

Previously, we demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and NO release in macrophages by inhibiting NF-kappaB. We now proceeded to elucidate the molecular mechanisms by which BZL exerts its inhibitory action on NF-kappaB. We demonstrated that the inhibitory effect of BZL is not extended to other macrophage responses, since it did not inhibit other typical hallmarks of macrophage activation such as phagocytosis, MHC-II molecules expression or production of reactive oxygen species (ROS) by NADPH oxidase. BZL was able to interfere specifically with the activation of NF-kappaB pathway without affecting AP-1 activation in RAW 264.7 macrophages, not only in LPS-mediated activation, but also for other stimuli, such as pro-inflammatory cytokines (IL-1beta, TNF-alpha), PMA or H(2)O(2). Also, BZL delayed the activation of p38 MAPK, but not that of ERK1/2 and JNK. Finally, treatment with BZL inhibited IkappaBalpha phosporylation and hence its degradation, whereas it did not block IkappaB kinase (IKK) alpha/beta phosphorylation. Collectively, BZL behaves as a broad range specific inhibitor of NF-kappaB activation, independently of the stimuli tested.

Novel cytostatic activity of the trypanocidal drug Benznidazole.

We have shown that Benznidazole (BZL), a compound with well documented trypanocidal activity, possesses anti-inflammatory properties and inhibits the nuclear factor kappaB (NF-kappaB). Given the relationship between this transcription factor and cell growth, in this study we address the role of NF-kappaB blockade by BZL in the proliferation of different cell lines. Our studies demonstrate that this compound significantly reduced proliferation of RAW 264.7 macrophage cell line, as assessed by trypan blue exclusion, MTT reduction and [(3)H]-thymidine incorporation, at a concentration shown to inhibit NF-kappaB. Treatment with BZL also led to growth arrest in CHO, MDCK and HeLa cells. Interestingly, growth inhibition was found to be a reversible process, not accompanied by significant cell death, indicating that the drug behaves mainly as a cytostatic compound. As this effect might be related to NF-kappaB inhibition, we next evaluated whether other NF-kappaB inhibitors could induce growth arrest in RAW 264.7 and HeLa cells. We found that IKK inhibition led to growth arrest in both cell lines, indicating that NF-kappaB inhibition may be the potential mechanism by which BZL inhibits cell proliferation. To the best of our knowledge, this is the first report of an anti-proliferative activity of the trypanocidal drug against different cell lines and provides a mechanistic insight that may help understand some of the adverse effects associated with prolonged treatment.

Beneficial effects of benznidazole during an infectious-based situation of systemic inflammatory response: cecal ligation and puncture.

We have shown that benznidazole (BZL), a drug used to treat Chagas disease, markedly reduced the production of pro-inflammatory cytokines and NO-derived metabolites in experimentally Trypanosoma cruzi-infected rats. Treatment with BZL exerted beneficial effects in a model of inflammation-based pathology like murine experimental endotoxemia. Based on these findings, we wished to ascertain the effect of BZL in a closer situation to sepsis: the cecal ligation and puncture (CLP) model in C57BL/6 mice. We analyzed clinical course, survival, circulating levels of inflammation-related compounds (NO, tumor necrosis factor [TNF]-alpha), and bacteriemia. Recipients of BZL, 25 mg/kg, had an increased survival rate at 24 hours after CLP, showing a better clinical situation and a significant reduction of TNF-alpha levels and bacteriemia, with respect to the other groups. BZL failed to inhibit in vitro bacterial growth, suggesting that these effects may be partly caused by the immunomodulatory effects of BZL.