Cruzi Activity Research Articles

Diversity, distribution and phytotoxic and anti-Trypanosoma activities of cultivable fungi associated with Magellan sub-Antarctic strait and Maritime Antarctic macroalgae.

We isolated and characterized the community of cultivable fungi associated with marine macroalgae present in the Magellan sub-Antarctic straits and the South Shetland Islands, Maritime Antarctica, and evaluated their production of bioactive metabolites. A total of 201 filamentous fungal isolates were obtained. The genera Antarctomyces, Pseudogymnoascus, Microdochium, Trichoderma, Cladosporium, Penicillium, Neoascochyta, Entomortierella and Linnemannia were associated with Antarctic macroalgae, with Neoascochyta paspali being the most abundant taxon. In contrast, 12 taxa representing Cadophora, Microdochium, Penicillium, Pseudogymnoascus were associated with macroalgae from the Magellan sub-Antarctic, with Penicillium dominating the assemblages. The diversity indices of the fungal communities associated with macroalgae in the two regions were similar. Among 177 fungal extracts assessed for metabolite production, 31 (17.5%) showed strong phytotoxicactivity and 17 (9.6%) showed anti-Trypanosoma cruzi activity. Penicillium showed the highest phytotoxic and anti-Trypanosoma activity values. The detection of taxa in common between the polar and cold temperate zones reinforces the need for further investigations of the distribution of species in these distinct ecoregions. The detection of bioactive extracts produced particularly by Penicillium representatives reinforces the potential to obtain active molecules that can be explored as natural products or as sources of bioactive compounds with application in agriculture and biomedicine.

Mechanisms underlying molecules exhibiting anti-Trypanosoma cruzi activity in vitro

Benznidazol and nifurtimox are the unique drugs to have trypanocidal effects which are used to treat Chagasic patients although both of then exhibit considerable cytotoxicity. Therefore, there is a need for continued investigation into molecules that are more effective against T. cruzi and have fewer adverse effects. The aim of this study is to review the described mechanisms of action as well as the physicochemical properties of molecules with anti-Trypanosoma cruzi activity in vitro. Studies available on PubMed and LILACS, describing the mechanism of action of molecules tested in vitro against T. cruzi published from 2003 to May 2023, were evaluated. The Chemicalize.org platform was used to estimate the physicochemical characteristics of the molecules. A total of 188 molecules with described mechanisms of action was reviwed. Among these mechanisms, the primary one described was the alteration of oxidative metabolism, reported as both a direct action on the mitochondria and the inhibition of key enzymes in this process, such as trypanothione reductase, iron superoxide dismutase, and mitochondrial dehydrogenase. Other mechanisms identified included morphological alterations, inhibition of sterol synthesis, and DNA damage. Understanding the molecular pathways involved in drug action enables the targeted search for more effective and less cytotoxic molecules. The molecules studied have great potential for the development of new medicines, due to their promising chemical characteristics, highlighting the importance of continuing studies in this field.

Structure-Aided Computational Design of Triazole-Based Targeted Covalent Inhibitors of Cruzipain.

Cruzipain (CZP), the major cysteine protease present in T. cruzi, the ethiological agent of Chagas disease, has attracted particular attention as a therapeutic target for the development of targeted covalent inhibitors (TCI). The vast chemical space associated with the enormous molecular diversity feasible to explore by means of modern synthetic approaches allows the design of CZP inhibitors capable of exhibiting not only an efficient enzyme inhibition but also an adequate translation to anti-T. cruzi activity. In this work, a computer-aided design strategy was developed to combinatorially construct and screen large libraries of 1,4-disubstituted 1,2,3-triazole analogues, further identifying a selected set of candidates for advancement towards synthetic and biological activity evaluation stages. In this way, a virtual molecular library comprising more than 75 thousand diverse and synthetically feasible analogues was studied by means of molecular docking and molecular dynamic simulations in the search of potential TCI of CZP, guiding the synthetic efforts towards a subset of 48 candidates. These were synthesized by applying a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) centered synthetic scheme, resulting in moderate to good yields and leading to the identification of 12 hits selectively inhibiting CZP activity with IC50 in the low micromolar range. Furthermore, four triazole derivatives showed good anti-T. cruzi inhibition when studied at 50 μM; and Ald-6 excelled for its high antitrypanocidal activity and low cytotoxicity, exhibiting complete in vitro biological activity translation from CZP to T. cruzi. Overall, not only Ald-6 merits further advancement to preclinical in vivo studies, but these findings also shed light on a valuable chemical space where molecular diversity might be explored in the search for efficient triazole-based antichagasic agents.

Structure-Metabolism Relationships of Benzimidazole Derivatives with anti-Trypanosoma cruzi Activity for Chagas Disease.

This study introduces further insights from the hit-to-lead optimization process involving a series of benzimidazole derivatives acting as inhibitors of the cruzain enzyme, which targets Trypanosoma cruzi, the causative parasite of Chagas disease. Here, we present the design, synthesis and biological evaluation of 30 new compounds as a third generation of benzimidazole analogues with trypanocidal activity, aiming to enhance our understanding of their pharmacokinetic profiles and establish a structure-metabolism relationships within the series. The design of these new analogues was guided by the analysis of previous pharmacokinetic results, considering identified metabolic sites and biotransformation studies. This optimization resulted in the discovery of two compounds (42 e and 49 b) exhibiting enhanced metabolic stability, anti-Trypanosoma cruzi activity compared to benznidazole (the reference drug for Chagas disease), as well as being non-cruzain inhibitors, and demonstrating a satisfactory in vitro pharmacokinetic profile. These findings unveil a new subclass of aminobenzimidazole and rigid compounds, which offer potential for further exploration in the quest for discovering novel classes of antichagasic compounds.

In Vitro Antiprotozoal Activity of Schinus molle Extract, Partitions, and Fractions against Trypanosoma cruzi.

Chagas disease, caused by the protozoan Trypanosoma cruzi, represents an important and worldwide public health issue, particularly in Latin America. Limitations of conventional treatment with benznidazole and nifurtimox underscore the urgent need for new therapeutic strategies for this disease. Schinus molle, a tree used in traditional medicine for various ailments, has demonstrated promising antiparasitic activity. The in vitro anti-T. cruzi activity of Schinus molle crude methanol extract, partitions, and fractions, as well as their cytotoxicity in Vero cells and Artemia salina, and hemolytic activity in human erythrocytes were assessed. Most of the extracts possessed anti-T. cruzi effects, with Sm-CF3 being the fraction with the highest activity (IC50 = 19 µg/mL; SI = 6.8). Gas chromatography-mass spectrometry analysis identified 20 compounds, with fatty acyls comprising the predominant chemical class (55%). We also identified the antiparasitic compounds cis-5,8,11,14,17-eicosapentaenoic acid and trans-Z-α-bisabolene epoxide, suggesting their potential contribution to the observed anti-T. cruzi activity. In conclusion, our findings support the therapeutic potential of S. molle as a source of novel antiparasitic agents against T. cruzi.

Steam distillation, supercritical fluid extraction, and anti-Trypanosoma cruzi activity of compounds from pink pepper (Schinus terebinthifolius Raddi)

Anti-Trypanosoma cruzi activity of compounds from fruits of Schinus terebinthifolius Raddi (pink pepper) were evaluated, using sustainable techniques such as steam distillation (SD) and supercritical fluid extraction (SFE). SD was optimised using a design of experiment and SFE was carried out using supercritical CO2 solvent (300 bar and 60 °C). Results of the anti-T. cruzi activity showed that the essential oil presented high activity (IC50 = 4.5 ± 0.3 μg/mL), whereas the supercritical extract had a moderate effect (IC50 = 19.7 ± 2.9 μg/mL). The differences in the anti-T. cruzi activity can be attributed to the extraction of non-volatile compounds in the SFE, such as moronic and (Z)-masticadienoic acids. In contrast, SD extracted only volatile compounds such as monoterpenes and sesquiterpenes. Therefore, these results suggest that the volatile compounds from pink pepper are involved with the anti-T. cruzi activity.

Pt(II) and Pd(II) complexes with coumarin-thiosemicarbazone hybrid ligands and triphenylphosphine coligand as potential anti T. cruzi agents

Chagas disease is a neglected tropical disease caused by the parasite Trypanosoma cruzi. The disease is endemic in 21 Latin American countries, but it has also been found worldwide. Current treatments for Chagas disease are not effective in all stages of the disease and can have severe side effects. In recent years, there has been a growing interest in the development of new drugs for the treatment of Chagas disease, including metal complexes containing bioactive ligands. In this work, we designed and synthesized eight novel palladium and platinum complexes of the formula [M(L)(PPh3)] containing four different coumarin-thiosemicarbazone hybrid ligands (H2L) as bioactive ligands and triphenylphosphine as lipophilic co-ligand. Compounds were characterized in the solid state and in solution including the resolution of the crystal structure of two of them by single crystal X-ray diffraction. The activity of these hybrids and their corresponding complexes against T. cruzi, along with their unspecific cytotoxicity, was assessed. The results showed that the palladium complexes fairly improved the ligands’ anti-T. cruzi activity in vitro but also the cytotoxicity on some mammalian cell lines. The most active palladium compound decreased the rate of parasitic infection of Vero cells. Synergy studies with benznidazole were also performed for this compound. In addition, we explored mitochondrion disruption and ROS generation as potential modes of action for the obtained compounds. Further studies are needed to optimize the design of new compounds to attain less unspecific toxicity and higher anti T. cruzi activity.

Synthesis and Anti-Chagas Activity Profile of a Redox-Active Lead 3-Benzylmenadione Revealed by High-Content Imaging.

Chagas disease, or American trypanosomiasis, is a neglected tropical disease which is a top priority target of the World Health Organization. The disease, endemic mainly in Latin America, is caused by the protozoan Trypanosoma cruzi and has spread around the globe due to human migration. There are multiple transmission routes, including vectorial, congenital, oral, and iatrogenic. Less than 1% of patients have access to treatment, relying on two old redox-active drugs that show poor pharmacokinetics and severe adverse effects. Hence, the priorities for the next steps of R&D include (i) the discovery of novel drugs/chemical classes, (ii) filling the pipeline with drug candidates that have new mechanisms of action, and (iii) the pressing need for more research and access to new chemical entities. In the present work, we first identified a hit (4a) with a potent anti-T. cruzi activity from a library of 3-benzylmenadiones. We then designed a synthetic strategy to build a library of 49 3-(4-monoamino)benzylmenadione derivatives via reductive amination to obtain diazacyclic benz(o)ylmenadiones. Among them, we identified by high content imaging an anti-amastigote "early lead" 11b (henceforth called cruzidione) revealing optimized pharmacokinetic properties and enhanced specificity. Studies in a yeast model revealed that a cruzidione metabolite, the 3-benzoylmenadione (cruzidione oxide), enters redox cycling with the NADH-dehydrogenase, generating reactive oxygen species, as hypothesized for the early hit (4a).

Metallomics: An Essential Tool for the Study of Potential Antiparasitic Metallodrugs.

Metallomics is an emerging area of omics approaches that has grown enormously in the past few years. It integrates research related to metals in biological systems, in symbiosis with genomics and proteomics. These omics approaches can provide in-depth insights into the mechanisms of action of potential metallodrugs, including their physiological metabolism and their molecular targets. Herein, we review the most significant advances concerning cellular uptake and subcellular distribution assays of different potential metallodrugs with activity against Trypanosma cruzi, the protozoan parasite that causes Chagas disease, a pressing health problem in high-poverty areas of Latin America. Furthermore, the first multiomics approaches including metallomics, proteomics, and transcriptomics for the comprehensive study of potential metallodrugs with anti-Trypanosoma cruzi activity are described.

Structure-activity relationships of novel N-imidazoylpiperazines with potent anti-Trypanosoma cruzi activity.

Background: Chagas disease is caused by the parasite Trypanosoma cruzi, and the lack of effective and safe treatments makes identifying new classes of compounds with anti-T. cruzi activity of paramount importance. Methods: Hit-to-lead exploration of a metabolically stable N-imidazoylpiperazine was performed. Results: Compound 2, a piperazine derivative active against T. cruzi, was selected to perform the hit-to-lead exploration, which involved the design, synthesis and biological evaluation of 39 new derivatives. Conclusion: Compounds 6e and 10a were identified as optimized compounds with low micromolar in vitro activity, low cytotoxicity and suitable preliminary absorption, distribution, metabolism and excretion and physicochemical properties. Both compounds reduced parasitemia in mouse models of Chagas disease, providing a promising opportunity for further exploration of new antichagasic compounds.

RCB-4, a novel cyclic peptide, from Ricinus communis with anti-Trypanosoma cruzi activities

IntroductionChagas disease and toxoplasmosis caused by parasites Trypanosoma cruzi and Toxoplasma gondii affect more than 28 million people in poor areas of Latin America. There is an urgent need for therapeutic options as the treatments are related to efficacy, lower costs, and shorter administration time. Encouraged by the need to discover valid targets and new treatment options, we evaluated castor seed compounds against T. cruzi and T. gondii, considering their effects against proliferation, infection, and ultrastructure. Methodologycastor seed extract was fractionated by Gel-filtration chromatography using the Sephadex G-50 resin, and a fraction with MW <10 kDa, denoted LMWF, was isolated. The effect of this fraction against T. cruzi and T. gondii was investigated. LMWF was repurified by HPLC-Reverse Phase chromatography using C-18 column. The major isolated peptide, with antiprotozoal activity, denoted RCB-4, was characterized by SDS-PAGE, mass spectrometry, and “in silico” studies. To understand the mechanism of action underlying RCB-4 against T. cruzi activity, we investigated ultrastructural changes by optical and electron microscopy. ResultsRCB-4 is a cycle peptide with an MW of 2229.78 Dalton, and the sequence is “ARCCLVMPVPPFACVKFCSSA.” RCB-4 was resistant to trypsin, endo-Glu, and endo-Asp enzymes. It inhibited parasite growth by up to 50 % at 10 and 15 µg/mL (4.48 µM and 6.73 µM), inside and outside the cells, respectively. Ultrastructural studies showed that trypomastigotes incubated with natural or synthetic RCB-4 could impair T. cruzi proliferation and cause ultrastructural alterations, such as Golgi apparatus disorganization and mitochondrial swelling. ConclusionsCyclic peptides emerged as promising organic compounds with diverse pharmacological activities. They possess superior therapeutic value due to the particular structures conferring resistance to enzymatic degradation and higher bioavailability. Our data allow us to prospect using RCB-4 to combat the parasites T. cruzi and T. gondii.

In vitro anti-Trypanosoma cruzi activity of methanolic extract of Bidens pilosa and identification of active compounds by gas chromatography-mass spectrometry analysis.

Chagas disease, caused by Trypanosoma cruzi parasite, is a significant but neglected tropical public health issue in Latin America due to the diversity of its genotypes and pathogenic profiles. This complexity is compounded by the adverse effects of current treatments, underscoring the need for new therapeutic options that employ medicinal plant extracts without negative side effects. Our research aimed to evaluate the trypanocidal activity of Bidens pilosa fractions against epimastigote and trypomastigote stages of T. cruzi, specifically targeting the Brener and Nuevo León strains-the latter isolated from Triatoma gerstaeckeri in General Terán, Nuevo León, México. We processed the plant's aerial parts (stems, leaves, and flowers) to obtain a methanolic extract (Bp-mOH) and fractions with varying solvent polarities. These preparations inhibited more than 90% of growth at concentrations as low as 800 μg/ml for both parasite stages. The median lethal concentration (LC50) values for the Bp-mOH extract and its fractions were below 500 μg/ml. Tests for cytotoxicity using Artemia salina and Vero cells and hemolytic activity assays for the extract and its fractions yielded negative results. The methanol fraction (BPFC3MOH1) exhibited superior inhibitory activity. Its functional groups, identified as phenols, enols, alkaloids, carbohydrates, and proteins, include compounds such as 2-hydroxy-3-methylbenzaldehyde (50.9%), pentadecyl prop-2-enoate (22.1%), and linalool (15.4%). Eight compounds were identified, with a match confirmed by the National Institute of Standards and Technology (NIST-MS) software through mass spectrometry analysis.

Metallomics as an Essential Analytical Tool for the Development of Potential Metallodrugs

Metallomics as an Essential Analytical Tool for the Development of Potential Metallodrugs

Cyclodextrin-encapsulated new drug with promising anti-Trypanosoma cruzi activity

Cyclodextrin-encapsulated new drug with promising anti-Trypanosoma cruzi activity

1,3-Thiazole derivatives as privileged structures for anti-Trypanosoma cruzi activity: Rational design, synthesis, in silico and in vitro studies

Chagas disease is a deadly and centenary neglected disease that is recently surging as a potential global threat. Approximately 30% of infected individuals develop chronic Chagas cardiomyopathy and current treatment with the reference benznidazole (BZN) is ineffective for this stage. We presently report the structural planning, synthesis, characterization, molecular docking prediction, cytotoxicity, in vitro bioactivity and mechanistic studies on the anti-T. cruzi activity of a series of 16 novel 1,3-thiazoles (2–17) derived from thiosemicarbazones (1a, 1b) in a two-step and reproducible Hantzsch-based synthesis approach. The anti-T. cruzi activity was evaluated in vitro against the epimastigote, amastigote and trypomastigote forms of the parasite. In the bioactivity assays, all thiazoles were more potent than BZN against epimastigotes. We found that the compounds presented an overall increased anti-tripomastigote selectivity (Cpd 8 was 24-fold more selective) than BZN, and they mostly presented anti-amastigote activity at very low doses (from 3.65 μM, cpd 15). Mechanistic studies on cell death suggested that the series of 1,3-thiazole compounds herein reported cause parasite cell death through apoptosis, but without compromising the mitochondrial membrane potential. In silico prediction of physicochemical properties and pharmacokinetic parameters showed promising drug-like results, being all the reported compounds in compliance with Lipinski and Veber rules. In summary, our work contributes towards a more rational design of potent and selective antitripanosomal drugs, using affordable methodology to yield industrially viable drug candidates.

Piperazine amides with desirable solubility, physicochemical and drug-like properties: Synthesis and evaluation of the anti-Trypanosoma cruzi activity

The absence of effective chronic treatment, expansion to non-endemic countries and the significant burden in public health have stimulated the search for novel therapeutic options to treat Chagas disease, a protozoan disease caused by Trypanosoma cruzi. Despite current efforts, no new drug candidates were approved in clinical trials in the past five decades. Considering this, our group has focused on the expansion of a series (LINS03) with low micromolar activity against amastigotes, considering the optimization of pharmacokinetic properties through increasing drug-likeness and solubility. In this work, we report a new set of 13 compounds with modifications in both the arylpiperazine and the aromatic region linked by an amide group. Five analogues showed activity against intracellular amastigotes (IC50 17.8 to 35.9 µM) and no relevant cytotoxicity to mammalian cells (CC50 > 200 µM). Principal component analysis (PCA) was performed to identify structural features associated to improved activity. The data revealed that polarity, hydrogen bonding ability and flexibility were key properties that influenced the antiparasitic activity. In silico drug-likeness assessments indicated that compounds with the 4-methoxycinammyl (especially compound 2b) had the most prominent balance between properties and activity in the series, as confirmed by SAR analysis.

Candimine from Hippeastrum escoipense (Amaryllidaceae): Anti-Trypanosoma cruzi activity and synergistic effect with benznidazole

BackgroundChagas disease (CD), caused by Trypanosoma cruzi, represents a health threat to around 20 million people worldwide. Side effects of benznidazole (Bzn) cause 15–20% of patients to discontinue their treatment. Evidence has increased in favor of the use of drug combinations to improve the efficacy and tolerance of the treatment. Natural products are well known to provide structures that could serve as new drugs or scaffolds for CD treatment. Spp of the Amaryllidoideae sub family of Amaryllidaceae family are known by their bioactives alkaloids, which have been reported by their antiparasitic activities. PurposeTo evaluate the anti-T. cruzi activity of the isolated alkaloid candimine (Cnd) from Hippeastrum escoipense Slanis & Huaylla; and to assess the combination effect between Cnd and Bzn against different life stages of T. cruzi parasites. MethodsThe chemical profile of H. escoipense alkaloids extract (AE-H. escoipense), including quantitation of Cnd was performed through GC/MS and UPLC-MS/MS techniques. Subsequently, Cnd was isolated using Shephadex LH-20. Then, the AE-H. escoipense and Cnd were tested against T. cruzi, (epimastigotes, trypomastigotes, and amastigotes) by in vitro proliferation and viability assays. The cytotoxicity was evaluated against Vero and HepG2 mammalian cells. The ultrastructural analysis was perform by transmission electron microscopy (TEM) and mitochondrial activity was carried out by MTT assay. Drug combination assay between Cnd and Bzn was evaluated using the Chou-Talalay method. ResultsThe AE-H. escoipense and Cnd showed high and specific anti-T. cruzi activity, comparable to Bzn. Cnd induces ultrastructural changes in T. cruzi, such as vacuolization, membrane blebs, and increased mitochondrial activity. Regarding the interaction between Cnd and Bzn, it generates synergism in the combinations of 0.25×IC50 in epimastigotes, 2×IC50 in trypomastigotes+amastigotes, and 0.25, 2, and 4×IC50 in amastigotes. ConclusionThe synergism between Cnd and Bzn indicates that the combination at the concentration of 4×IC50 could be useful as an effective new therapy against CD in the chronic stage. Thus, Cnd isolated from the leaves of H. escoipense emerges as potential candidate for the development of a new drug for the treatment of CD.

Anti-Trypanosoma cruzi activity of Coptis rhizome extract and its constituents

BackgroundCurrent therapeutic agents, including nifurtimox and benznidazole, are not sufficiently effective in the chronic phase of Trypanosoma cruzi infection and are accompanied by various side effects. In this study, 120 kinds of extracts from medicinal herbs used for Kampo formulations and 94 kinds of compounds isolated from medicinal herbs for Kampo formulations were screened for anti-T. cruzi activity in vitro and in vivo.MethodsAs an experimental method, a recombinant protozoan cloned strain expressing luciferase, namely Luc2-Tulahuen, was used in the experiments. The in vitro anti-T. cruzi activity on epimastigote, trypomastigote, and amastigote forms was assessed by measuring luminescence intensity after treatment with the Kampo extracts or compounds. In addition, the cytotoxicity of compounds was tested using mouse and human feeder cell lines. The in vivo anti-T. cruzi activity was measured by a murine acute infection model using intraperitoneal injection of trypomastigotes followed by live bioluminescence imaging.ResultsAs a result, three protoberberine-type alkaloids, namely coptisine chloride, dehydrocorydaline nitrate, and palmatine chloride, showed strong anti-T. cruzi activities with low cytotoxicity. The IC50 values of these compounds differed depending on the side chain, and the most effective compound, coptisine chloride, showed a significant effect in the acute infection model.ConclusionsFor these reasons, coptisine chloride is a hit compound that can be a potential candidate for anti-Chagas disease drugs. In addition, it was expected that there would be room for further improvement by modifying the side chains of the basic skeleton.

Synthetic Analogues of Gibbilimbol B Induce Bioenergetic Damage and Calcium Imbalance in Trypanosoma cruzi

Chagas disease is an endemic tropical disease caused by the protozoan Trypanosoma cruzi, which affects around 7 million people worldwide, mostly in development countries. The treatment relies on only two available drugs, with severe adverse effects and a limited efficacy. Therefore, the search for new therapies is a legitimate need. Within this context, our group reported the anti-Trypanosoma cruzi activity of gibbilimbol B, a natural alkylphenol isolated from the plant Piper malacophyllum. Two synthetic derivatives, LINS03018 (1) and LINS03024 (2), demonstrated a higher antiparasitic potency and were selected for mechanism of action investigations. Our studies revealed no alterations in the plasma membrane potential, but a rapid alkalinization of the acidocalcisomes. Nevertheless, compound 1 exhibit a pronounced effect in the bioenergetics metabolism, with a mitochondrial impairment and consequent decrease in ATP and reactive oxygen species (ROS) levels. Compound 2 only depolarized the mitochondrial membrane potential, with no interferences in the respiratory chain. Additionally, no macrophages response of nitric oxide (NO) was observed in both compounds. Noteworthy, simple structure modifications in these derivatives induced significant differences in their lethal effects. Thus, this work reinforces the importance of the mechanism of action investigations at the early phases of drug discovery and support further developments of the series.

A functional assay using human whole blood and flow cytometry analysis to evaluate cytotoxicity and immunomodulatory effect of anti-Trypanosoma cruzi drugs

The discovery and development of new drugs for the treatment of Chagas disease is urgent due to the high toxicity and low cure efficacy, mainly during the chronic phase of this disease. Other chemotherapeutic approaches for Chagas disease treatment are being researched and require screening assays suitable for evaluating the effectivity of new biologically active compounds. This study aims to evaluate a functional assay using the internalization of epimastigotes forms of Trypanosoma cruzi by human peripheral blood leukocytes from healthy volunteers and analyses by flow cytometry of cytotoxicity, anti-T. cruzi activity, and immunomodulatory effect of benznidazole, ravuconazole, and posaconazole. The culture supernatant was used to measure cytokines (IL-1-β, IL-6, INF-γ, TNF and IL-10) and chemokines (MCP-1/CCL2, CCL5/RANTES and CXCL8/IL-8). The data showed a reduction in the internalization of T. cruzi epimastigote forms treated with ravuconazole, demonstrating its potential anti-T. cruzi activity. In addition, an increased amount of IL-10 and TNF cytokines was observed in the supernatant of cultures upon the addition of the drug, mainly IL-10 in the presence of benznidazole, ravuconazole and posaconazole, and TNF in the presence of ravuconazole and posaconazole. Moreover, the results revealed a decrease in the MCP-1/CCL2 index in cultures in the presence of benznidazole, ravuconazole, and posaconazole. A decrease in the CCL5/RANTES and CXCL8/IL-8 index in cultures with BZ, when compared to the culture without drugs, was also observed. In conclusion, the innovative functional test proposed in this study may be a valuable tool as a confirmatory test for selecting promising compounds identified in prospecting programs for new drugs for Chagas disease treatment.