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Chagas disease is a parasitic infection caused by the intracellular protozoan Trypanosoma cruzi . Chronic Chagas cardiomyopathy (CCC) is the most severe manifestation of the disease, developed by approximately 20-40% of patients and characterized by occurrence of arrhythmias, heart failure and death. Despite having more than 100 years of discovery, Chagas disease remains without an effective treatment, especially for patients with CCC. Since the pathogenesis of CCC depends on a parasite-driven systemic inflammatory profile that leads to cardiac tissue damage, the use of immunomodulators has become a rational alternative for the treatment of CCC. In this context, different classes of drugs, cell therapies with dendritic cells or stem cells and gene therapy have shown potential to modulate systemic inflammation and myocarditis in CCC models. Based on that, the present review provides an overview of current reports regarding the use of immunomodulatory agents in treatment of CCC, bringing the challenges and future directions in this field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Santos, Silva, Reis, Barreto, Cardoso, Ribeiro dos Santos, Meira and Soares.)

Chronic Chagas disease cardiomyopathy (CCC) is the most frequent and severe form of this parasitic disease. CCC is caused by a progressive inflammation in the heart, resulting in alterations that can culminate in heart failure and death. The use of dendritic cells (DCs) appears as an option for the development of treatments due to their important role in regulating immune responses. Here, we investigated whether tolerogenic cells (tDCs) could interfere with the progression of CCC in an experimental model of Chagas disease. The tDCs were generated and characterized as CD11b + CD11c + cells, low expression of MHC-II, CD86, CD80, and CD40, and increased expression of PD-L. These cells produced low levels of IL-6 and IL-12p70 and higher levels of IL-10, compared to mature DCs (mDCs). Interestingly, tDCs inhibited lymphoproliferation and markedly increased the population of FoxP3 + Treg cells in vitro , compared to mature DCs. In a mouse model of CCC, treatment with tDCs reduced heart inflammation and fibrosis. Furthermore, tDCs treatment reduced the gene expression of pro-inflammatory cytokines ( Ifng and Il12 ) and of genes related to cardiac remodeling ( Col1a2 and Lgals3 ), while increasing the gene expression of IL-10. Finally, administration of tDCs, increased the percentage of Treg cells in the hearts and spleens of chagasic mice. Ours results show that tolerogenic dendritic cells have therapeutic potential on CCC, inhibiting disease progression., (Copyright © 2020 Santos, Aragão-França, Meira, Cerqueira, Vasconcelos, Nonaka, Pontes-de-Carvalho and Soares.)

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi. The current chemotherapy is based on benznidazole, and, in some countries, Nifurtimox, which is effective in the acute phase of the disease, but its efficacy in the chronic phase remains controversial. It can also cause serious side effects that lead sufferers to abandon treatment. In the present work, is reported the synthesis and trypanocidal activity of new 2-(phenylthio)ethylidene thiosemicarbazones (4-15) and 1,3-thiazoles (16-26). The cyclization of thiosemicarbazones into 1,3-thiazoles presents an improvement in the cytotoxic profile for T. cruzi parasite, denoting selective compounds. Compound 18 was identified as the most promising of all compounds tested, showing an IC 50 of 2.6 μM for the trypomastigote form and a non-cytotoxic effect on mouse spleen cells, reaching a selective index of 95.1. Among the 22 compounds tested, six compounds present a better trypanocidal activity, and five compounds have an equipotent activity compared to benznidazole. Flow cytometry and ultrastructural analysis were performed and indicate that compound 18 causes parasite cell death through apoptosis and acts via an autophagic pathway., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Chronic Chagas disease cardiomyopathy (CCC) is a major cause of heart disease in Latin America and treatment for this condition is unsatisfactory. Here we investigated the effects of BA5, an amide semi-synthetic derivative betulinic acid, in a model of CCC. Mice chronically infected with T. cruzi were treated orally with BA5 (10 or 1 mg/Kg), three times per week, for 2 months. BA5 treatment decreased inflammation and fibrosis in heart sections but did not improve exercise capacity or ameliorate cardiac electric disturbances in infected mice. Serum concentrations of TNF-α, IFN-γ, and IL-1β, as well as cardiac gene expression of pro-inflammatory mediators, were reduced after BA5 treatment. In contrast, a significant increase in the anti-inflammatory cytokine IL-10 concentration was observed in BA5-treated mice in both tested doses compared to vehicle-treated mice. Moreover, polarization to anti-inflammatory/M2 macrophage phenotype was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers, such as Arg1 and CHI3 in mice treated with BA5. In conclusion, BA5 had a potent anti-inflammatory activity on a model of parasite-driven heart disease related to IL-10 production and a switch from M1 to M2 subset of macrophages.

Current treatment for combatting Chagas disease, a life-threatening illness caused by the kinetoplastid protozoan parasite Trypanosoma cruzi is inadequate, and thus the discovery of new antiparasitic compounds is of prime importance. Previous studies identified the indirubins, a class of ATP kinase inhibitors, as potent growth inhibitors of the related kinetoplastid Leishmania. Herein, we evaluated the inhibitory activity of a series of 69 indirubin analogues screened against T. cruzi trypomastigotes and intracellular amastigotes. Seven indirubins were identified as potent T. cruzi inhibitors (low μΜ, nM range). Cell death analysis of specific compounds [3'oxime-6-bromoindirubin(6-BIO) analogues 10, 11 and 17, bearing a bulky extension on the oxime moiety and one 7 substituted analogue 32], as evaluated by electron microscopy and flow cytometry, showed a different mode of action between compound 32 compared to the three 6-BIO oxime- substituted indirubins, suggesting that indirubins may kill the parasite by different mechanisms dependent on their substitution. Moreover, the efficacy of four compounds that show the most potent anti-parasitic effect in both trypomastigotes and intracellular amastigotes (10, 11, 17, 32), was evaluated in a mouse model of T. cruzi infection. Compound 11 (3'piperazine-6-BIO) displayed the best in vivo efficacy (1/6 mortality, 94.5% blood parasitaemia reduction, 12 dpi) at a dose five times reduced over the reference drug benznidazole (20 mg/kg vs100 mg/kg). We propose 3'piperazine-6-BIO as a potential lead for the development of new treatments of Chagas disease.

Chagas disease is one of the most significant health problems in the American continent. benznidazole (BDZ) and nifurtimox (NFX) are the only drugs approved for treatment and exhibit strong side effects and ineffectiveness in the chronic stage, besides different susceptibility among T. cruzi DTUs (Discrete Typing Units). Therefore, new drugs to treat this disease are necessary. Thiazole compounds have been described as potent trypanocidal agents. Here we report the structural planning, synthesis and anti-T. cruzi evaluation of a new series of 1,3-thiazoles (7-28), which were designed by placing this heterocycle instead of thiazolidin-4-one ring. The synthesis was conducted in an ultrasonic bath with 2-propanol as solvent at room temperature. By varying substituents attached to the phenyl and thiazole rings, substituents were observed to retain, enhance or greatly increase their anti-T. cruzi activity. In some cases, methyl at position 5 of the thiazole (compounds 9, 12 and 23) increased trypanocidal property. The exchange of phenyl for pyridinyl heterocycle resulted in increased activity, giving rise to the most potent compound against the trypomasigote form (14, IC 50trypo  = 0.37 μM). Importantly, these new thiazoles were toxic for trypomastigotes without affecting macrophages and cardiomyoblast viability. The compounds were also evaluated against cruzain, and five of the most active compounds against trypomastigotes (7, 9, 12, 16 and 23) inhibited more than 70% of enzymatic activity at 10 μM, among which compound 7 had an IC 50 in the submicromolar range, suggesting a possible mechanism of action. In addition, examination of T. cruzi cell death showed that compound 14 induces apoptosis. We also examined the activity against intracellular parasites, revealing that compound 14 inhibited T. cruzi infection with potency similar to benznidazole. The antiparasitic effect of 14 and benznidazole in combination was also investigated against trypomastigotes and revealed that they have synergistic effects, showing a promising profile for drug combination. Finally, in mice acutely-infected with T. cruzi,14 treatment significanty reduced the blood parasitaemia and had a protective effect on mortality. In conclusion, we report the identification of compounds (7), (12), (15), (23) and (26) with similar trypanocidal activity of benznidazole; compounds (9) and (21) as trypanocidal agents equipotent with BDZ, and compound 14 with potency 28 times better than the reference drug without affecting macrophages and cardiomyoblast viability. Mechanistically, the compounds inhibit cruzain, and 14 induces T. cruzi cell death by an apoptotic process, being considered a good starting point for the development of new anti-Chagas drug candidates., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Betulinic acid is a pentacyclic triterpenoid with several biological properties already described, including antiparasitic activity. Here, the anti-Trypanosoma cruzi activity of betulinic acid and its semi-synthetic amide derivatives (BA1-BA8) was investigated. The anti-Trypanosoma cruzi activity and selectivity were enhanced in semi-synthetic derivatives, specially on derivatives BA5, BA6 and BA8. To understand the mechanism of action underlying betulinic acid anti-T. cruzi activity, we investigated ultrastructural changes by electron microscopy. Ultrastructural studies showed that trypomastigotes incubated with BA5 had membrane blebling, flagella retraction, atypical cytoplasmic vacuoles and Golgi cisternae dilatation. Flow cytometry analysis showed that parasite death is mainly caused by necrosis. Treatment with derivatives BA5, BA6 or BA8 reduced the invasion process, as well as intracellular parasite development in host cells, with a potency and selectivity similar to that observed in benznidazole-treated cells. More importantly, the combination of BA5 and benznidazole revealed synergistic effects on trypomastigote and amastigote forms of T. cruzi. In conclusion, we demonstrated that BA5 compound is an effective and selective anti-T. cruzi agent., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Chagas disease is an infection caused by protozoan Trypanosoma cruzi, which affects approximately 8-10million people worldwide. Benznidazole is the only drug approved for treatment during the acute and asymptomatic chronic phases of Chagas disease; however, it has poor efficacy during the symptomatic chronic phase. Therefore, the development of new pharmaceuticals is needed. Here, we employed the bioisosterism to modify a potent antiparasitic and cruzain-inhibitor aryl thiosemicarbazone (4) into 4-thiazolidinones (7-21). Compounds (7-21) were prepared by using a straightforward synthesis and enabled good to excellent yields. As a chemical elucidation tool, X-ray diffraction of compound (10) revealed the geometry and conformation of this class compounds. The screening against cruzain showed that 4-thiazolidinones were less active than thiosemicarbazone (4). However, the antiparasitic activity in Y strain trypomastigotes and host cell cytotoxicity in J774 macrophages revealed that compounds (10 and 18-21) are stronger and more selective antiparasitic agents than thiosemicarbazone (4). Specifically, compounds (18-20), which carry a phenyl at position N3 of heterocyclic ring, were the most active ones, suggesting that this is a structural determinant for activity. In infected macrophages, compounds (18-20) reduced intracellular amastigotes, whereas Benznidazole did not. In T. cruzi-infected mice treated orally with 100mg/kg of compound (20), a decreased of parasitemia was observed. In conclusion, we demonstrated that the conversation of thiosemicarbazones into 4-thiazolidinones retains pharmacological property while enhances selectivity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: The current treatment of Chagas disease, endemic in Latin America and emerging in several countries, is limited by the frequent side effects and variable efficacy of benznidazole. Natural products are an important source for the search for new drugs., Aim/hypothesis: Considering the great potential of natural products as antiparasitic agents, we investigated the anti-Trypanosoma cruzi activity of a concentrated ethanolic extract of Physalis angulata (EEPA)., Methods: Cytotoxicity to mammalian cells was determined using mouse peritoneal macrophages. The antiparasitic activity was evaluated against axenic epimastigote and bloodstream trypomastigote forms of T. cruzi, and against amastigote forms using T. cruzi-infected macrophages. Cell death mechanism was determined in trypomastigotes by flow cytometry analysis after annexin V and propidium iodide staining. The efficacy of EEPA was examined in vivo in an acute model of infection by monitoring blood parasitaemia and survival rate 30 days after treatment. The effect against trypomastigotes of EEPA and benznidazole acting in combination was evaluated., Results: EEPA effectively inhibits the epimastigote growth (IC50 2.9 ± 0.1 µM) and reduces bloodstream trypomastigote viability (EC50 1.7 ± 0.5 µM). It causes parasite cell death by necrosis. EEPA impairs parasite infectivity as well as amastigote development in concentrations noncytotoxic to mammalian cells. In mice acutely-infected with T. cruzi, EEPA reduced the blood parasitaemia in 72.7%. When combined with benznidazole, EEPA showed a synergistic anti-T. cruzi activity, displaying CI values of 0.8 ± 0.07 at EC50 and 0.83 ± 0.1 at EC90., Conclusion: EEPA has antiparasitic activity against T. cruzi, causing cell death by necrosis and showing synergistic activity with benznidazole. These findings were reinforced by the observed efficacy of EEPA in reducing parasite load in T. cruzi-mice. Therefore, this represents an important source of antiparasitic natural products., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The nonpolar fraction of an aqueous ethanol extract of the roots of Arrabidaea brachypoda, a Brazilian medicinal plant, demonstrated significant in vitro activity against Trypanosoma cruzi, the parasite responsible for Chagas disease. Targeted isolation of the active constituents led to the isolation of three new dimeric flavonoids (1-3), and their structures were elucidated using UV, NMR, and HRMS analysis, as well as by chemical derivatization. The anti-T. cruzi activity and cytotoxicity toward mammalian cells were determined for these substances. Compound 1 exhibited no activity toward T. cruzi, while flavonoids 2 and 3 exhibited selective activity against these trypomastigotes. Compounds 2 and 3 inhibited the parasite invasion process and its intracellular development in host cells with similar potencies to benznidazole. In addition, compound 2 reduced the blood parasitemia of T. cruzi-infected mice. This study has revealed that these two dimeric flavonoids represent potential anti-T. cruzi lead compounds for further drug development.

Investigation by GC-FID and GC-MS of the essential oil (LPOE) from the leaves of Lippia pedunculosa revealed, as the major compounds, the monoterpenes rotundifolone (71.7%) and (R)-limonene (21.8%). These two compounds and the minor constituent piperitenone (1.2%) were also isolated from the leaves and identified by spectrometric analysis. LPOE and isolated compounds were evaluated for their trypanocidal activity against epimastigote and trypomastigote forms of Trypanosoma cruzi. Significant results with IC50 values lower than 34.0 microg.mL(-1) were observed against these forms of T. cruzi for LPOE and isolated compounds. Rotundifolone was the most active compound with an ICso lower than 10.0 ig.mL"' for both forms of T. cruzi. The effects of LPOE and isolated compounds were also evaluated in cultures of macrophages infected with T. cruzi. Treatment with (R)-limonene and rotundifolone caused a moderate reduction in the percentage of macrophages infected by T. cruzi and in the number of intracellular parasites at concentrations non-toxic to macrophages.

We modified the thiazolidinic ring at positions N3, C4, and C5, yielding compounds 6-24. Compounds with a phenyl at position N3, 15-19, 22-24, exhibited better inhibitory properties for cruzain and against the parasite than 2-iminothiazolidin-4-one 5. We were able to identify one high-efficacy trypanocidal compound, 2-minothiazolidin-4-one 18, which inhibited the activity of cruzain and the proliferation of epimastigotes and was cidal for trypomastigotes but was not toxic for splenocytes. Having located some of the structural determinants of the trypanocidal properties, we subsequently wished to determine if the exchange of the thiazolidine for a thiazole ring leaves the functional properties unaffected. We therefore tested thiazoles 26-45 and observed that they did not inhibit cruzain, but they exhibited trypanocidal effects. Parasite development was severely impaired when treated with 18, thus reinforcing the notion that this class of heterocycles can lead to useful cidal agents for Chagas disease.

We recently showed that oxadiazoles have anti-Trypanosoma cruzi activity at micromolar concentrations. These compounds are easy to synthesize and show a number of clear and interpretable structure-activity relationships (SAR), features that make them attractive to pursue potency enhancement. We present here the structural design, synthesis, and anti-T. cruzi evaluation of new oxadiazoles denoted 5a-h and 6a-h. The design of these compounds was based on a previous model of computational docking of oxadiazoles on the T. cruzi protease cruzain. We tested the ability of these compounds to inhibit catalytic activity of cruzain, but we found no correlation between the enzyme inhibition and the antiparasitic activity of the compounds. However, we found reliable SAR data when we tested these compounds against the whole parasite. While none of these oxadiazoles showed toxicity for mammalian cells, oxadiazoles 6c (fluorine), 6d (chlorine), and 6e (bromine) reduced epimastigote proliferation and were cidal for trypomastigotes of T. cruzi Y strain. Oxadiazoles 6c and 6d have IC(50) of 9.5 ± 2.8 and 3.5 ± 1.8 μM for trypomastigotes, while Benznidazole, which is the currently used drug for Chagas disease treatment, showed an IC(50) of 11.3 ± 2.8 μM. Compounds 6c and 6d impair trypomastigote development and invasion in macrophages, and also induce ultrastructural alterations in trypomastigotes. Finally, compound 6d given orally at 50mg/kg substantially reduces the parasitemia in T. cruzi-infected BALB/c mice. Our drug design resulted in potency enhancement of oxadiazoles as anti-Chagas disease agents, and culminated with the identification of oxadiazole 6d, a trypanosomicidal compound in an animal model of infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)