Arginine decarboxylase inhibitors reduce the capacity of Trypanosoma cruzi to infect and multiply in mammalian host cells.

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.11.2455 ◽

1996 ◽

Vol 40 (11) ◽

pp. 2455-2458 ◽

Cited By ~ 47

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.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00414-20 ◽

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.

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Anti-parasitic effect of novel amidines against Trypanosoma cruzi: phenotypic and in silico absorption, distribution, metabolism, excretion and toxicity analysis

Parasitology Open ◽

10.1017/pao.2017.5 ◽

2017 ◽

Vol 3 ◽

Cited By ~ 2

Author(s):

ALINE SILVA DA GAMA NEFERTITI ◽

MARCOS MEUSER BATISTA ◽

PATRÍCIA BERNARDINO DA SILVA ◽

EDUARDO CAIO TORRES-SANTOS ◽

EDEZIO F. CUNHA-JÚNIOR ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

In Silico ◽

Host Cells ◽

Mammalian Host ◽

Aromatic Molecules ◽

Toxicity Analysis ◽

Parasitic Effect ◽

The Many ◽

Discrete Typing Unit

SUMMARYNew more selective and potent drugs are urgently need to treat Chagas disease (CD). Among the many synthetic compounds evaluated againstTrypanosoma cruzi, aromatic amidines (AAs) and especially arylimidamides (AIAs) have potent activity against this parasite. Presently, the effect of four mono-amidines (DB2228, DB2229, DB2292 and DB2294), four diamidines (DB2232, DB2235, DB2251 and DB2253) and one AIA (DB2255) was screenedin vitroagainst different forms (bloodstream trypomastigotes – BT and intracellular forms) and strains from discrete typing unit (DTU) I and VI ofT. cruziand their cytotoxic profile on mammalian host cells. Except for DB2253, all molecules were as active as benznidazole (Bz), resulting in 50% of reduction in the number of alive BT, with EC50ranging from 2·7 to 10·1µmafter 24 h of incubation. DB2255 was also the most potent against amastigotes (Tulahuen strain) showing similar activity to that of Bz (3µm).In silicoabsorption, distribution, metabolism, excretion and toxicity analysis demonstrated probability of human intestinal adsorption, while mutagenicity and inhibition of hERG1 were not predicted, besides giving acceptable predicted volumes of distribution. Our findings contribute for better knowledge regarding the biological effect of this class of aromatic molecules againstT. cruziaiming to identify novel promising agent for CD therapy.

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

Biochemical Journal ◽

10.1042/bj20041049 ◽

2005 ◽

Vol 388 (1) ◽

pp. 29-38 ◽

Cited By ~ 53

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.

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Glutamine Analogues Impair Cell Proliferation, the Intracellular Cycle and Metacyclogenesis in Trypanosoma cruzi

Molecules ◽

10.3390/molecules25071628 ◽

2020 ◽

Vol 25 (7) ◽

pp. 1628

Author(s):

Rodolpho Ornitz Oliveira Souza ◽

Marcell Crispim ◽

Ariel Mariano Silber ◽

Flávia Silva Damasceno

Keyword(s):

Cell Proliferation ◽

Life Cycle ◽

Trypanosoma Cruzi ◽

Host Cells ◽

Glutamine Metabolism ◽

Complex Life Cycle ◽

Mammalian Host ◽

Developmental Cycle ◽

Parasite Life Cycle

Trypanosoma cruzi is the aetiologic agent of Chagas disease, which affects people in the Americas and worldwide. The parasite has a complex life cycle that alternates among mammalian hosts and insect vectors. During its life cycle, T. cruzi passes through different environments and faces nutrient shortages. It has been established that amino acids, such as proline, histidine, alanine, and glutamate, are crucial to T. cruzi survival. Recently, we described that T. cruzi can biosynthesize glutamine from glutamate and/or obtain it from the extracellular environment, and the role of glutamine in energetic metabolism and metacyclogenesis was demonstrated. In this study, we analysed the effect of glutamine analogues on the parasite life cycle. Here, we show that glutamine analogues impair cell proliferation, the developmental cycle during the infection of mammalian host cells and metacyclogenesis. Taken together, these results show that glutamine is an important metabolite for T. cruzi survival and suggest that glutamine analogues can be used as scaffolds for the development of new trypanocidal drugs. These data also reinforce the supposition that glutamine metabolism is an unexplored possible therapeutic target.

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Trypanosoma cruzi does not induce apoptosis in murine fibroblasts

Parasitology ◽

10.1017/s0031182098003631 ◽

1999 ◽

Vol 118 (2) ◽

pp. 167-175 ◽

Cited By ~ 13

Author(s):

R. K. CLARK ◽

R. E. KUHN

Keyword(s):

Trypanosoma Cruzi ◽

Host Cell ◽

Cell Apoptosis ◽

Membrane Integrity ◽

Host Cells ◽

Mammalian Host ◽

Host Cell Apoptosis ◽

Intracellular Parasites ◽

Murine Fibroblasts ◽

Infected Cells

The intracellular cycle of Trypanosoma cruzi in mammalian host cells involves the differentiation of dividing amastigote forms into flagellated trypomastigote forms. The mechanism(s) regulating the growth and differentiation of the intracellular parasites is (are) not known. The number of parasites in infected cells can be several hundred and may be enough to induce apoptosis, a suicide-like death programme, generating products (e.g. nuclear proteins) that could function as signals to initiate the differentiation of amastigotes into trypomastigotes. Murine fibroblasts infected with T. cruzi were examined during a 5-day course of infection for evidence of apoptosis. However, characteristics of apoptosis, including degeneration of nuclear structure, condensation of chromatin, loss of plasma membrane integrity, or the cleavage of DNA into nucleosomal fragments, were not observed. Therefore, it is unlikely that products resulting from host cell apoptosis function to induce parasite differentiation. The possibility that T. cruzi might inhibit host cell apoptosis by increasing intracellular levels of Bcl-2, an endogenous inhibitor of apoptosis, was then investigated. Analysis of infected cells by flow cytometry did not demonstrate a significant amount of intracellular Bcl-2. This suggests that if the parasite is inhibiting host cell apoptosis, it is by a method that does not involve increasing levels of Bcl-2.

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An aromatic imidazoline derived from chloroquinoline triggers cell cycle arrest and inhibits with high selectivity the Trypanosoma cruzi mammalian host-cells infection

PLoS Neglected Tropical Diseases ◽

10.1371/journal.pntd.0009994 ◽

2021 ◽

Vol 15 (11) ◽

pp. e0009994

Author(s):

Roberto I. Cuevas-Hernández ◽

Richard M. B. M. Girard ◽

Luka Krstulović ◽

Miroslav Bajić ◽

Ariel Mariano Silber

Keyword(s):

Cell Cycle ◽

Trypanosoma Cruzi ◽

Host Cells ◽

Mammalian Host ◽

Cycle Arrest ◽

Mitochondrial Inner Membrane ◽

Intracellular Ca ◽

Low Toxicity ◽

Plasma Membrane Permeabilization ◽

Chimeric Molecules

Trypanosoma cruzi is a hemoflagellated parasite causing Chagas disease, which affects 6–8 million people in the Americas. More than one hundred years after the description of this disease, the available drugs for treating the T. cruzi infection remain largely unsatisfactory. Chloroquinoline and arylamidine moieties are separately found in various compounds reported for their anti-trypanosoma activities. In this work we evaluate the anti-T. cruzi activity of a collection of 26 “chimeric” molecules combining choroquinoline and amidine structures. In a first screening using epimastigote forms of the parasite as a proxy for the clinically relevant stages, we selected the compound 7-chloro-4-[4-(4,5-dihydro-1H-imidazol-2-yl)phenoxy]quinoline (named here as A6) that performed better as an anti-T. cruzi compound (IC50 of 2.2 ± 0.3 μM) and showed a low toxicity for the mammalian cell CHO-K1 (CC50 of 137.9 ± 17.3 μM). We initially investigated the mechanism of death associated to the selected compound. The A6 did not trigger phosphatidylserine exposure or plasma membrane permeabilization. Further investigation led us to observe that under short-term incubations (until 6 hours), no alterations of mitochondrial function were observed. However, at longer incubation times (4 days), A6 was able to decrease the intracellular Ca2+, to diminish the intracellular ATP levels, and to collapse mitochondrial inner membrane potential. After analysing the cell cycle, we found as well that A6 produced an arrest in the S phase that impairs the parasite proliferation. Finally, A6 was effective against the infective forms of the parasite during the infection of the mammalian host cells at a nanomolar concentration (IC50(tryps) = 26.7 ± 3.7 nM), exhibiting a selectivity index (SI) of 5,170. Our data suggest that A6 is a promising hit against T. cruzi.

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