Apis mellifera venom induces different cell death pathways in Trypanosoma cruzi

SUMMARYChagas 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.

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

Toxicon ◽

10.1016/j.toxicon.2013.03.011 ◽

2013 ◽

Vol 69 ◽

pp. 227-239 ◽

Cited By ~ 39

Author(s):

Camila M. Adade ◽

Isabelle R.S. Oliveira ◽

Joana A.R. Pais ◽

Thaïs Souto-Padrón

Keyword(s):

Cell Death ◽

Trypanosoma Cruzi ◽

Cell Death Pathways

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Capsaicin inhibits arginine kinase and exerts anti-Trypanosoma cruzi activity

10.1101/2020.06.23.167346 ◽

2020 ◽

Author(s):

Edward A. Valera-Vera ◽

Chantal Reigada ◽

Melisa Sayé ◽

Fabio A. Digirolamo ◽

Mariana R. Miranda ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Stress Responses ◽

Mammalian Cells ◽

Arginine Kinase ◽

Chronic Phase ◽

New Drugs ◽

World Health ◽

Neglected Diseases ◽

Health Organization

ABSTRACTTrypanosoma cruzi is the causative agent of Chagas disease, considered within the list of twenty neglected diseases according to the World Health Organization. There are only two therapeutic drugs for Chagas disease, both of them unsuitable for the chronic phase, therefore the development of new drugs is a priority.T. cruzi arginine kinase (TcAK) is a promising drug target since it is absent in humans and it is involved in cellular stress responses. In a previous study from our laboratory, possible TcAK inhibitors were identified through computer simulations, resulting in the best-scoring compounds cyanidin derivatives and capsaicin. Considering these results, in this work we evaluate the effect of capsaicin on TcAK activity and its trypanocidal effect. Although capsaicin produced a weak inhibition on the recombinant TcAK activity (IC50 ≈ 800 µM), it had a strong trypanocidal effect on epimastigotes and trypomastigotes (IC50 = 6.26 µM and 0.26 µM, respectively) being 20-fold more active on trypomastigotes than mammalian cells. Epimastigotes that overexpress TcAK were 37% more resistant to capsaicin than wild type parasites, suggesting that trypanocidal activity could be due, in part, to the enzyme inhibition. However, the difference between the concentrations at which the enzyme is inhibited and the parasite death is caused implies the presence of other targets. In this sense, the prohibitin-2 and calmodulin were identified as other possible capsaicin targets. Capsaicin is a strong and selective trypanocidal agent active in nanomolar concentrations, with an IC50 57-fold lower than benznidazole, the drug currently used for treating Chagas disease.

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Synthesis of New Thiosemicarbazones and Semicarbazones Containing the 1,2,3-1H-triazole-isatin Scaffold: Trypanocidal, Cytotoxicity, Electrochemical Assays, and Molecular Docking

Medicinal Chemistry ◽

10.2174/1573406414666180912120502 ◽

2019 ◽

Vol 15 (3) ◽

pp. 240-256 ◽

Cited By ~ 1

Author(s):

Bianca N.M. Silva ◽

Policarpo A. Sales Junior ◽

Alvaro J. Romanha ◽

Silvane M.F. Murta ◽

Camilo H.S. Lima ◽

...

Keyword(s):

Molecular Docking ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Docking Studies ◽

World Health ◽

Neglected Diseases ◽

Alkyl Aryl ◽

Development Of Resistance ◽

Health Organization

Background: Chagas disease, also known as American trypanosomiasis, is classified as one of the 17 most important neglected diseases by the World Health Organization. The only drugs with proven efficacy against Chagas disease are benznidazole and nifurtimox, however both show adverse effects, poor clinical efficacy, and development of resistance. For these reasons, the search for new effective chemical entities is a challenge to research groups and the pharmaceutical industry. Objective: Synthesis and evaluation of antitrypanosomal activities of a series of thiosemicarbazones and semicarbazones containing 1,2,3-1H triazole isatin scaffold. Method: 5&'-(4-alkyl/aryl)-1H-1,2,3-triazole-isatins were prepared by Huisgen 1,3-dipolar cycloaddition and the thiosemicarbazones and semicarbazones were obtained by the 1:1 reactions of the carbonylated derivatives with thiosemicarbazide and semicarbazide hydrochloride, respectively, in methanol, using conventional reflux or microwave heating. The compounds were assayed for in vitro trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Beyond the thio/semicarbazone derivatives, isatin and triazole synthetic intermediates were also evaluated for comparison. Results: A series of compounds were prepared in good yields. Among the 37 compounds evaluated, 18 were found to be active, in particular thiosemicarbazones containing a non-polar saturated alkyl chain (IC50 = 24.1, 38.6, and 83.2 &µM; SI = 11.6, 11.8, and 14.0, respectively). To further elucidate the mechanism of action of these new compounds, the redox behaviour of some active and inactive derivatives was studied by cyclic voltammetry. Molecular docking studies were also performed in two validated protein targets of Trypanosoma cruzi, i.e., cruzipain (CRZ) and phosphodiesterase C (TcrPDEC). Conclusion: A class of thio/semicarbazones structurally simple and easily accessible was synthesized. Compounds containing thiosemicarbazone moieties showed the best results in the series, being more active than the corresponding semicarbazones. Our results indicated that the activity of these compounds does not originate from an oxidation-reduction pathway but probably from the interactions with trypanosomal enzymes.

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Proinflammatory and Cytotoxic Effects of Hexadecylphosphocholine (Miltefosine) against Drug-Resistant Strains of Trypanosoma cruzi

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.11.3472-3477.2002 ◽

2002 ◽

Vol 46 (11) ◽

pp. 3472-3477 ◽

Cited By ~ 37

Author(s):

Victor B. Saraiva ◽

Daniel Gibaldi ◽

José O. Previato ◽

Lucia Mendonça-Previato ◽

Marcelo T. Bozza ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Resistant Strain ◽

Ether Lipid ◽

In Vivo Studies ◽

No Production ◽

Dependent Manner ◽

Response Curves ◽

Intracellular Amastigotes ◽

Resistant Strains

ABSTRACT The increased resistance of the protozoan parasite Trypanosoma cruzi to nitro derivatives is one of the major problems for the successful treatment of Chagas' disease. In the present study, we have tested the effects of 1-O-hexadecylphosphocholine (miltefosine) against strains of T. cruzi that are partially resistant (strain Y) and highly resistant (strain Colombiana) to the drugs in clinical use. As expected, epimastigotes of strain Colombiana showed higher levels of resistance to benznidazole than those of strain Y. However, the level of resistance to miltefosine was the same for both strains. This alkylphospholipid was also extremely toxic against intracellular amastigotes of both strains. This ether-lipid analogue induced in a dose-dependent manner the production of tumor necrosis factor alpha and nitric oxide (NO) radicals by infected and noninfected macrophages, suggesting that miltefosine may activate macrophages in vitro. Nevertheless, the cytotoxic effect of miltefosine against intracellular amastigotes was independent of the amount of NO produced by the infected macrophages since the same dose-response curves for miltefosine were observed when the NO production was blocked by the NO synthase inhibitor N G-monomethyl-l-arginine monoacetate. Preliminary in vivo studies with BALB/c mice infected with strain Y indicated that oral miltefosine promoted survival and reduced the parasitemia to levels comparable to those observed when benznidazole was used. Four months after treatment, no parasites were detected in the blood or spleen tissue sections maintained in culture. Together, these results support the hypothesis that miltefosine may be used for the treatment of Chagas' disease, including cases caused by resistant strains of T. cruzi.

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Standardization of Trypanosoma cruzi DNA extraction and purification protocol from samples collected on Whatman 903 filter paper to Chagas disease diagnosis.

10.1101/2020.07.16.20153916 ◽

2020 ◽

Author(s):

Laura Smeldy Jurado Medina ◽

Griselda Ballering ◽

Margarita Bisio ◽

Rodrigo Ojeda ◽

Jaime M Altcheh

Keyword(s):

Trypanosoma Cruzi ◽

Dna Extraction ◽

Chagas Disease ◽

Filter Paper ◽

Metabolic Diseases ◽

Vero Cells ◽

Disease Diagnosis ◽

Neglected Diseases ◽

Sample Collection ◽

Serological Tests

Chagas disease (CD) caused by the parasite Trypanosoma cruzi, belongs to the so-called neglected diseases group. In Argentina about 1,500 children are born with congenital Chagas disease per year. The diagnosis of CD in the newborn relies on the ability to detect parasites in the blood by microscopic observation, as the serological tests are ruled out because of the presence of maternal antibodies. CD treatment is more effective during the acute phase of infection. Early diagnosis and treatment of the disease is thus very important. The Argentinian National Program for early detection of metabolic diseases uses Whatman903 filter paper for blood sampling. This type of sample collection presents many advantages as the use of low blood volumes, minimal biological risk, and easy storage and transportation. The objective of the study was to evaluate the conservation efficiency of blood samples on filter paper in order to access good sensitivity on qPCR results for the detection of T. cruzi. To standardize the procedure, negative samples of blood were infected artificially with serial dilutions of trypomastigotes forms of T. cruzi from the TcVI strain obtained by cell culture in Vero cells. Concentrations between 50000 and 5 parasites/mL were prepared and loaded in filter paper for analysis. DNA extraction was conducted by the QIAamp DNA Mini Kit from QIAGEN. For qPCR, a method based on TaqMan technology was used, with a multiplex reaction for quantification of T. cruzi satellite DNA and an internal amplification control (IAC). The detection limit found from our results was 400 parasites/mL, demonstrating that this method could be a reliable option for the diagnosis of congenital CD by the detection of T. cruzi in blood collected in filter paper.

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Synergic Effect of Allopurinol in Combination with Nitroheterocyclic Compounds against Trypanosoma cruzi

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02264-18 ◽

2019 ◽

Vol 63 (6) ◽

Cited By ~ 7

Author(s):

Ana Lia Mazzeti ◽

Lívia de F. Diniz ◽

Karolina R. Gonçalves ◽

Ruan Schott WonDollinger ◽

Tassiane Assíria ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Parasite Load ◽

H9c2 Cells ◽

Cure Rate ◽

Positive Interaction ◽

Content Type ◽

Intracellular Amastigotes ◽

Subsequent Relapse

ABSTRACT Combination therapy has gained attention as a possible strategy for overcoming the limitations of the present therapeutic arsenal for Chagas disease. The aim of this study was to evaluate the effect of allopurinol in association with nitroheterocyclic compounds on infection with the Y strain of Trypanosoma cruzi. The in vitro effect of allopurinol plus benznidazole or nifurtimox on intracellular amastigotes in infected H9c2 cells was assessed in a 72-h assay. The interactions were classified as synergic for both allopurinol-nifurtimox (sums of fractional inhibitory concentrations [∑FICs] = 0.49 ± 0.08) and allopurinol-benznidazole (∑FICs = 0.48 ± 0.09). In the next step, infected Swiss mice were treated with allopurinol at 30, 60, and 90 mg/kg of body weight and with benznidazole at 25, 50, and 75 mg/kg in monotherapy and in combination at the same doses; as a reference treatment, another group of animals received benznidazole at 100 mg/kg. Allopurinol in monotherapy led to a smaller or nil effect in the reduction of parasite load and mortality rate. Treatment with benznidazole at suboptimal doses induced a transient suppression of parasitaemia with subsequent relapse in all animals treated with 25 and 50 mg/kg and in 80% of those that received 75 mg/kg. Administration of the drugs in combination significantly increased the cure rate to 60 to 100% among mice treated with benznidazole at 75 mg/kg plus 30, 60, or 90 mg/kg of allopurinol. These results show a positive interaction between allopurinol and benznidazole, and since both drugs are commercially available, their use in combination may be considered for the assessment in the treatment of Chagas disease patients.

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Repurposing strategies for Chagas disease therapy: the effect of imatinib and derivatives against Trypanosoma cruzi

Parasitology ◽

10.1017/s0031182019000234 ◽

2019 ◽

Vol 146 (8) ◽

pp. 1006-1012 ◽

Cited By ~ 7

Author(s):

M. R. Simões-Silva ◽

J. S. De Araújo ◽

R. B. Peres ◽

P. B. Da Silva ◽

M. M. Batista ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Kinase Inhibitor ◽

Fixed Ratio ◽

Chronic Phase ◽

Host Cells ◽

Public Health Issue ◽

Mammalian Host ◽

Neglected Diseases

AbstractChagas disease (CD) is a neglected parasitic condition endemic in the Americas caused by Trypanosoma cruzi. Patients present an acute phase that may or not be symptomatic, followed by lifelong chronic stage, mostly indeterminate, or with cardiac and/or digestive progressive lesions. Benznidazole (BZ) and nifurtimox are the only drugs approved for treatment but not effective in the late chronic phase and many strains of the parasite are naturally resistant. New alternative therapy is required to address this serious public health issue. Repositioning and combination represent faster, and cheaper trial strategies encouraged for neglected diseases. The effect of imatinib (IMB), a tyrosine kinase inhibitor designed for use in neoplasias, was assessed in vitro on T. cruzi and mammalian host cells. In comparison with BZ, IMB was moderately active against different strains and forms of the parasite. The combination IMB + BZ in fixed-ratio proportions was additive. Novel 14 derivatives of IMB were screened and a 3,2-difluoro-2-phenylacetamide (3e) was as potent as BZ on T. cruzi but had low selectivity index. The results demonstrate the importance of phenotypic assays, encourage the improvement of IMB derivatives to reach selectivity and testify to the use of repurposing and combination in drug screening for CD.

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Antiparasitic Effect of Stilbene and Terphenyl Compounds against Trypanosoma cruzi Parasites

Pharmaceuticals ◽

10.3390/ph14111199 ◽

2021 ◽

Vol 14 (11) ◽

pp. 1199

Author(s):

Federica Bruno ◽

Germano Castelli ◽

Fabrizio Vitale ◽

Simone Catanzaro ◽

Valeria Vitale Badaco ◽

...

Keyword(s):

Natural Products ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Flow Cytometric Analysis ◽

New Drugs ◽

Normal Cells ◽

Antiparasitic Activity ◽

Intracellular Amastigotes ◽

Caspase 1

Background: Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi. No progress in the treatment of this pathology has been made since Nifurtimox was introduced more than fifty years ago, and this drug is considered very aggressive and may cause several adverse effects. This drug currently has severe limitations, including a high frequency of undesirable side effects and limited efficacy and availability, so research to discover new drugs for the treatment of Chagas disease is imperative. Many drugs available on the market are natural products as found in nature or compounds designed based on the structure and activity of these natural products. Methods: This study evaluated the in vitro antiparasitic activity of a series of previously synthesized stilbene and terphenyl compounds in T. cruzi epimastigotes and intracellular amastigotes. The action of the most selective compounds was investigated by flow cytometric analysis to evaluate the mechanism of cell death. The ability to induce apoptosis or caspase-1 inflammasomes was assayed in macrophages infected with T. cruzi after treatment, comparing it with that of Nifurtimox. Results: The stilbene ST18 was the most potent compound of the series. It was slightly less active than Nifurtimox in epimastigotes but most active in intracellular amastigotes. Compared to Nifurtimox, it was markedly less cytotoxic when tested in vitro on normal cells. ST18 was able to induce a marked increase in parasites positive for Annexin V and monodansylcadaverine. Moreover, ST18 induced the activation, in infected macrophages, of caspase-1, a conserved enzyme that plays a major role in controlling parasitemia, host survival and the onset of the adaptive immune response in Trypanosoma infection. Conclusions: The antiparasitic activity of ST18 together with its ability to activate caspase-1 in infected macrophages and its low toxicity toward normal cells makes this compound interesting for further clinical investigation.

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Pep5, a Fragment of Cyclin D2, Shows Antiparasitic Effects in Different Stages of the Trypanosoma cruzi Life Cycle and Blocks Parasite Infectivity

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01806-18 ◽

2019 ◽

Vol 63 (5) ◽

Cited By ~ 2

Author(s):

Christiane Bezerra de Araujo ◽

Loyze Paola de Lima ◽

Simone Guedes Calderano ◽

Flávia Silva Damasceno ◽

Ariel M. Silber ◽

...

Keyword(s):

Cell Death ◽

Trypanosoma Cruzi ◽

Chagas Disease ◽

Fold Increase ◽

Host Cells ◽

Public Health Issue ◽

Mammalian Host ◽

Cyclin D2 ◽

Content Type

ABSTRACT Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cell-penetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells. Additionally, Pep5-cpp reduces the in vivo tumor volume of rat glioblastoma. Chagas disease, which is caused by the flagellated parasite Trypanosoma cruzi, is a neglected disease that occurs mainly in the Americas, where it is considered an important public health issue. Given that there are only two options for treating the disease, it is exceptionally crucial to search for new molecules with potential pharmacological action against the parasites. In this study, we demonstrate that Pep5-cpp induces cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi. The Pep5-cpp peptide was also able to decrease the percentage of infected cells without causing any detectable toxic effects in mammalian host cells. The infective, i.e., trypomastigote form of T. cruzi pretreated with Pep5-cpp was unable to infect LLC-MK2 monkey kidney cells. Also, Pep5-binding proteins were identified by mass spectrometry, including calmodulin-ubiquitin-associated protein, which is related to the virulence and parasitemia of T. cruzi. Taken together, these data suggest that Pep5 can be used as a novel alternative for the treatment of Chagas disease.

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