scholarly journals The Potent Trypanocidal Effect of LQB303, a Novel Redox-Active Phenyl-Tert-Butyl-Nitrone Derivate That Causes Mitochondrial Collapse in Trypanosoma cruzi

2021 ◽  
Vol 12 ◽  
Author(s):  
Carolina Machado Macedo ◽  
Francis Monique de Souza Saraiva ◽  
Jéssica Isis Oliveira Paula ◽  
Suelen de Brito Nascimento ◽  
Débora de Souza dos Santos Costa ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Mitochondrial Metabolism ◽  
Host Cells ◽  
Trypanocidal Activity ◽  
Tert Butyl ◽  
Redox Active ◽  
Extracellular Amastigotes

Chagas disease, which is caused by Trypanosoma cruzi, establishes lifelong infections in humans and other mammals that lead to severe cardiac and gastrointestinal complications despite the competent immune response of the hosts. Furthermore, it is a neglected disease that affects 8 million people worldwide. The scenario is even more frustrating since the main chemotherapy is based on benznidazole, a drug that presents severe side effects and low efficacy in the chronic phase of the disease. Thus, the search for new therapeutic alternatives is urgent. In the present study, we investigated the activity of a novel phenyl-tert-butyl-nitrone (PBN) derivate, LQB303, against T. cruzi. LQB303 presented trypanocidal effect against intracellular [IC50/48 h = 2.6 μM] and extracellular amastigotes [IC50/24 h = 3.3 μM] in vitro, leading to parasite lysis; however, it does not present any toxicity to host cells. Despite emerging evidence that mitochondrial metabolism is essential for amastigotes to grow inside mammalian cells, the mechanism of redox-active molecules that target T. cruzi mitochondrion is still poorly explored. Therefore, we investigated if LQB303 trypanocidal activity was related to the impairment of the mitochondrial function of amastigotes. The investigation showed there was a significant decrease compared to the baseline oxygen consumption rate (OCR) of LQB303-treated extracellular amastigotes of T. cruzi, as well as reduction of “proton leak” (the depletion of proton motive force by the inhibition of F1Fo ATP synthase) and “ETS” (maximal oxygen consumption after uncoupling) oxygen consumption rates. Interestingly, the residual respiration (“ROX”) enhanced about three times in LQB303-treated amastigotes. The spare respiratory capacity ratio (SRC: cell ability to meet new energy demands) and the ATP-linked OCR were also impaired by LQB303 treatment, correlating the trypanocidal activity of LQB303 with the impairment of mitochondrial redox metabolism of amastigotes. Flow cytometric analysis demonstrated a significant reduction of the ΔΨm of treated amastigotes. LQB303 had no significant influence on the OCR of treated mammalian cells, evidencing its specificity against T. cruzi mitochondrial metabolism. Our results suggest a promising trypanocidal activity of LQB303, associated with parasite bioenergetic inefficiency, with no influence on the host energy metabolism, a fact that may point to an attractive alternative therapy for Chagas disease.

scholarly journals Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 4081-4087 ◽  
Author(s):  
Craig Weinkauf ◽  
Ryan Salvador ◽  
Mercio PereiraPerrin
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Neuronal Cell ◽  
Host Cells ◽  
Neurotrophin Receptor ◽  
Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

scholarly journals Purinergic Antagonist Suramin Aggravates Myocarditis and Increases Mortality by Enhancing Parasitism, Inflammation, and Reactive Tissue Damage in Trypanosoma cruzi-Infected Mice

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Rômulo D. Novaes ◽  
Eliziária C. Santos ◽  
Marli C. Cupertino ◽  
Daniel S. S. Bastos ◽  
Andréa A. S. Mendonça ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Tissue Damage ◽  
Mammalian Cells ◽  
Negative Impact ◽  
Purinergic Signaling ◽  
Host Cells ◽  
Antioxidant Defenses ◽  
The Impact

Suramin (Sur) acts as an ecto-NTPDase inhibitor in Trypanosoma cruzi and a P2-purinoceptor antagonist in mammalian cells. Although the potent antitrypanosomal effect of Sur has been shown in vitro, limited evidence in vivo suggests that this drug can be dangerous to T. cruzi-infected hosts. Therefore, we investigated the dose-dependent effect of Sur-based chemotherapy in a murine model of Chagas disease. Seventy uninfected and T. cruzi-infected male C57BL/6 mice were randomized into five groups: SAL = uninfected; INF = infected; SR5, SR10, and SR20 = infected treated with 5, 10, or 20 mg/kg Sur. In addition to its effect on blood and heart parasitism, the impact of Sur-based chemotherapy on leucocytes myocardial infiltration, cytokine levels, antioxidant defenses, reactive tissue damage, and mortality was analyzed. Our results indicated that animals treated with 10 and 20 mg/kg Sur were disproportionally susceptible to T. cruzi, exhibiting increased parasitemia and cardiac parasitism (amastigote nests and parasite load (T. cruzi DNA)), intense protein, lipid and DNA oxidation, marked myocarditis, and mortality. Animals treated with Sur also exhibited reduced levels of nonprotein antioxidants. However, the upregulation of catalase, superoxide dismutase, and glutathione-S-transferase was insufficient to counteract reactive tissue damage and pathological myocardial remodeling. It is still poorly understood whether Sur exerts a negative impact on the purinergic signaling of T. cruzi-infected host cells. However, our findings clearly demonstrated that through enhanced parasitism, inflammation, and reactive tissue damage, Sur-based chemotherapy contributes to aggravating myocarditis and increasing mortality rates in T. cruzi-infected mice, contradicting the supposed relevance attributed to this drug for the treatment of Chagas disease.

scholarly journals Amlodipine Increases the Therapeutic Potential of Ravuconazole upon Trypanosoma cruzi Infection

2020 ◽  
Vol 64 (8) ◽  
Author(s):  
Yara Almeida Machado ◽  
Maria Terezinha Bahia ◽  
Ivo Santana Caldas ◽  
Ana Lia Mazzeti ◽  
Rômulo Dias Novaes ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Therapeutic Potential ◽  
Combined Treatment ◽  
Host Cells ◽  
Ergosterol Biosynthesis ◽  
Antiparasitic Activity ◽  
Content Type ◽  
Trypanocidal Activity ◽  
Long Term Treatment

ABSTRACT Mining existing agents that enhance the therapeutic potential of ergosterol biosynthesis inhibitors (EBI) is a promising approach to improve Chagas disease chemotherapy. In this study, we evaluated the effect of ravuconazole, an EBI, combined with amlodipine, a calcium channel blocker, upon Trypanosoma cruzi experimental infection. In vitro assays confirmed the trypanocidal activity of both compounds in monotherapy and demonstrated an additive effect (sum of the fractional inhibitory concentration [ΣFIC] > 0.5) of the combined treatment without additional toxicity to host cells. In vivo experiments, using a murine model of the T. cruzi Y strain in a short-term protocol, demonstrated that amlodipine, although lacking trypanocidal activity, dramatically increased the antiparasitic activity of underdosing ravuconazole regimens. Additional analysis using long-term treatment (20 days) showed that parasitemia relapse until 60 days after treatment was significatively lower in mice treated with the combination (4 out of 14 mice) than ravuconazole monotherapy (10 out of 14 mice), even in the presence of immunosuppressant pressure. Furthermore, the combined therapy was well tolerated and protected the mice from mortality. The treatments also impacted on the cellular and humoral immune response of infected animals, inducing a reduction of serum cytokine levels in all ravuconazole-treated mice. Our findings demonstrate that amlodipine is efficacious in enhancing the antiparasitic activity of ravuconazole in an experimental model of T. cruzi infection and indicates a potential strategy to be explored in Chagas disease treatment.

scholarly journals Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

2005 ◽  
Vol 77 (1) ◽  
pp. 77-94 ◽  
Author(s):  
Renato A. Mortara ◽  
Walter K. Andreoli ◽  
Noemi N. Taniwaki ◽  
Adriana B. Fernandes ◽  
Claudio V. da Silva ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Mammalian Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Mammalian Host ◽  
Target Cells ◽  
Sylvatic Cycle ◽  
Final Destination ◽  
Different Strains

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

scholarly journals In Vitro and In Vivo Activities of E5700 and ER-119884, Two Novel Orally Active Squalene Synthase Inhibitors, against Trypanosoma cruzi

2004 ◽  
Vol 48 (7) ◽  
pp. 2379-2387 ◽  
Author(s):  
Julio A. Urbina ◽  
Juan Luis Concepcion ◽  
Aura Caldera ◽  
Gilberto Payares ◽  
Cristina Sanoja ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Public Health Problem ◽  
Squalene Synthase ◽  
Host Cells ◽  
In Vivo Studies ◽  
Inorganic Pyrophosphate ◽  
Orally Active

ABSTRACT Chagas' disease is a serious public health problem in Latin America, and no treatment is available for the prevalent chronic stage. Its causative agent, Trypanosoma cruzi, requires specific endogenous sterols for survival, and we have recently demonstrated that squalene synthase (SQS) is a promising target for antiparasitic chemotherapy. E5700 and ER-119884 are quinuclidine-based inhibitors of mammalian SQS that are currently in development as cholesterol- and triglyceride-lowering agents in humans. These compounds were found to be potent noncompetitive or mixed-type inhibitors of T. cruzi SQS with K i values in the low nanomolar to subnanomolar range in the absence or presence of 20 μM inorganic pyrophosphate. The antiproliferative 50% inhibitory concentrations of the compounds against extracellular epimastigotes and intracellular amastigotes were ca. 10 nM and 0.4 to 1.6 nM, respectively, with no effects on host cells. When treated with these compounds at the MIC, all of the parasite's sterols disappeared from the parasite cells. In vivo studies indicated that E5700 was able to provide full protection against death and completely arrested the development of parasitemia when given at a concentration of 50 mg/kg of body weight/day for 30 days, while ER-119884 provided only partial protection. This is the first report of an orally active SQS inhibitor that is capable of providing complete protection against fulminant, acute Chagas' disease.

scholarly journals Inhibition of Trypanosoma cruzi growth in mammalian cells by purine and pyrimidine analogs.

1996 ◽  
Vol 40 (11) ◽  
pp. 2455-2458 ◽  
Author(s):  
J Nakajima-Shimada ◽  
Y Hirota ◽  
T Aoki
Keyword(s):  
Trypanosoma Cruzi ◽  
Growth Inhibition ◽  
Host Cell ◽  
Culture System ◽  
Mammalian Cells ◽  
Screening Method ◽  
Host Cells ◽  
Mammalian Host ◽  
Dependent Manner ◽  
Pyrimidine Analogs

Trypanosoma cruzi, the causative agent of Chagas' disease, exhibits two different developmental stages in mammals, the amastigote, an intracellular form that proliferates in the cytoplasm of host cells, and the trypomastigote, an extracellular form that circulates in the bloodstream. We have already established an in vitro culture system using mammalian host cells (HeLa) infected with T. cruzi in which the time course of parasite growth is determined quantitatively. We adopted this system for the screening of anti-T. cruzi agents that would ideally prove to be effective against trypanosomes with no toxicity to the host cell. Of the purine analogs tested, allopurinol markedly inhibited the growth of amastigotes in a dose-dependent manner, with no lethal effect on trypomastigotes. 3'-Deoxyinosine and 3'-deoxyadenosine also suppressed T. cruzi growth inside the host cell, with the concentrations causing 50% growth inhibition being 10 and 5 microM, respectively, in contrast to a concentration causing 50% growth inhibition of 3 microM for allopurinol. Among the pyrimidine analogs examined, 3'-azido-3'-deoxythymidine (zidovudine) significantly reduced the growth of the parasite at concentrations as low as 1 microM. The anti-human immunodeficiency virus agents 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine caused a decrease in amastigote growth, while 2',3'-dideoxycytidine and 2',3'-dideoxyuridine had no inhibitory effect. When Swiss 3T3 fibroblasts were used as host cells, allopurinol, 3'-deoxyinosine, 3'-deoxyadenosine, and 3'-azid-3'-deoxythymidine also markedly inhibited T. cruzi proliferation. These results indicate that our culture system is useful as a primary screening method for candidate compounds against T. cruzi on the basis of two criteria, namely, intracellular replication by the parasite and host-cell infection rate.

scholarly journals Efficacy of Novel Pyrazolone Phosphodiesterase Inhibitors in Experimental Mouse Models of Trypanosoma cruzi

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Julianna Siciliano de Araújo ◽  
Cristiane França da Silva ◽  
Denise da Gama Jaén Batista ◽  
Aline Nefertiti ◽  
Ludmila Ferreira de Almeida Fiuza ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Phosphodiesterase Inhibitors ◽  
Biological Properties ◽  
Host Cells ◽  
Mammalian Host ◽  
Intracellular Camp ◽  
Additive Interaction ◽  
Content Type

ABSTRACT Pyrazolones are heterocyclic compounds with interesting biological properties. Some derivatives inhibit phosphodiesterases (PDEs) and thereby increase the cellular concentration of cyclic AMP (cAMP), which plays a vital role in the control of metabolism in eukaryotic cells, including the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease (CD), a major neglected tropical disease. In vitro phenotypic screening identified a 4-bromophenyl-dihydropyrazole dimer as an anti-T. cruzi hit and 17 novel pyrazolone analogues with variations on the phenyl ring were investigated in a panel of phenotypic laboratory models. Potent activity against the intracellular forms (Tulahuen and Y strains) was obtained with 50% effective concentration (EC50) values within the 0.17 to 3.3 μM range. Although most were not active against bloodstream trypomastigotes, an altered morphology and loss of infectivity were observed. Pretreatment of the mammalian host cells with pyrazolones did not interfere with infection and proliferation, showing that the drug activity was not the result of changes to host cell metabolism. The pyrazolone NPD-227 increased the intracellular cAMP levels and was able to sterilize T. cruzi-infected cell cultures. Thus, due to its high potency and selectivity in vitro, and its additive interaction with benznidazole (Bz), NPD-227 was next assessed in the acute mouse model. Oral dosing for 5 days of NPD-227 at 10 mg/kg + Bz at 10 mg/kg not only reduced parasitemia (>87%) but also protected against mortality (>83% survival), hence demonstrating superiority to the monotherapy schemes. These data support these pyrazolone molecules as potential novel therapeutic alternatives for Chagas disease.

scholarly journals Molecular, functional and structural properties of the prolyl oligopeptidase of Trypanosoma cruzi (POP Tc80), which is required for parasite entry into mammalian cells

2005 ◽  
Vol 388 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Izabela M. D. BASTOS ◽  
Philippe GRELLIER ◽  
Natalia F. MARTINS ◽  
Gloria CADAVID-RESTREPO ◽  
Marian R. de SOUZA-AULT ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Single Copy ◽  
Catalytic Triad ◽  
Host Cells ◽  
Mammalian Host ◽  
Kinetic Properties ◽  
Prolyl Oligopeptidase ◽  
Single Chromosome

We have demonstrated that the 80 kDa POP Tc80 (prolyl oligopeptidase of Trypanosoma cruzi) is involved in the process of cell invasion, since specific inhibitors block parasite entry into non-phagocytic mammalian host cells. In contrast with other POPs, POP Tc80 is capable of hydrolysing large substrates, such as fibronectin and native collagen. In this study, we present the cloning of the POPTc80 gene, whose deduced amino acid sequence shares considerable identity with other members of the POP family, mainly within its C-terminal portion that forms the catalytic domain. Southern-blot analysis indicated that POPTc80 is present as a single copy in the genome of the parasite. These results are consistent with mapping of POPTc80 to a single chromosome. The active recombinant protein (rPOP Tc80) displayed kinetic properties comparable with those of the native enzyme. Novel inhibitors were assayed with rPOP Tc80, and the most efficient ones presented values of inhibition coefficient Ki≤1.52 nM. Infective parasites treated with these specific POP Tc80 inhibitors attached to the surface of mammalian host cells, but were incapable of infecting them. Structural modelling of POP Tc80, based on the crystallized porcine POP, suggested that POP Tc80 is composed of an α/β-hydrolase domain containing the catalytic triad Ser548–Asp631–His667 and a seven-bladed β-propeller non-catalytic domain. Docking analysis suggests that triple-helical collagen access to the catalytic site of POP Tc80 occurs in the vicinity of the interface between the two domains.

scholarly journals Capsaicin inhibits arginine kinase and exerts anti-Trypanosoma cruzi activity

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.

scholarly journals Amaryllidaceae alkaloids with anti-Trypanosoma cruzi activity

2020 ◽  
Author(s):  
Nieves Martinez-Peinado ◽  
Nuria Cortes-Serra ◽  
Laura Torras-Claveria ◽  
Maria-Jesus Pinazo ◽  
Joaquim Gascon ◽  
...  
Keyword(s):  
Natural Products ◽  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
New Drugs ◽  
Host Cells ◽  
Amaryllidaceae Alkaloids ◽  
Cytotoxicity Assays ◽  
Limited Efficacy ◽  
Single Compound ◽  
New Compounds

Abstract Background: Chagas disease, caused by the protozoan Trypanosoma cruzi, is a neglected disease that affects ~7 million people worldwide. Development of new drugs to treat the infection remains a priority since those currently available have frequent side effects and limited efficacy at the chronic stage. Natural products provide a pool of diversity structures to lead the chemical synthesis of novel molecules for this purpose. Herein we analyzed the anti- T. cruzi activity of 9 alkaloids derived from plants of the Amaryllidaceae family. Methods: the activity of each alkaloid was assessed by means of a newly developed anti- T. cruzi phenotypic assay. We further evaluated the compounds that inhibited the parasite growth on two distinct cytotoxicity assays to discard those that were toxic to host cells and assure parasite selectivity. Results: we identified a single compound (hippeastrine 2 ) that was selectively active against the parasite yielding selectivity indexes of 12.7 and 35.2 against Vero and HepG2 cells, respectively. Conclusions: results reported here suggest that natural products are an interesting source of new compounds for the development of drugs against Chagas disease.

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