scholarly journals Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing beta-galactosidase.

1996 ◽  
Vol 40 (11) ◽  
pp. 2592-2597 ◽  
Author(s):  
F S Buckner ◽  
C L Verlinde ◽  
A C La Flamme ◽  
W C Van Voorhis
Keyword(s):  
Trypanosoma Cruzi ◽  
Screening Method ◽  
Host Cells ◽  
Enzyme Linked Immunosorbent Assay ◽  
Selective Toxicity ◽  
Microtiter Plates ◽  
Chlorophenol Red ◽  
New Compounds ◽  
Beta Galactosidase

A new drug screening method was devised utilizing Trypanosoma cruzi cells that express the Escherichia coli beta-galactosidase gene. Transfected parasites catalyze a colorimetric reaction with chlorophenol red beta-D-galactopyranoside as substrate. Parasite growth in the presence of drugs in microtiter plates was quantitated with an enzyme-linked immunosorbent assay reader. The assay was performed with the mammalian form of T. cruzi that requires intracellular growth on a monolayer of fibroblast cells. To determine if selective toxicity to the parasites was occurring, the viability of the host cells in the drug was assayed with AlamarBlue. The drugs benznidazole, fluconazole, and amphotericin B were shown to inhibit the parasites at concentrations similar to those previously reported. Several compounds were tested that are inhibitors of glyceraldehyde-3-phosphate dehydrogenase of the related organisms Leishmania mexicana and Trypanosoma brucei. One of these compounds, 2-guanidino-benzimidazole, had an 50% inhibitory concentration of 10 microM in our assay. Two derivatives of this compound were identified with in vitro activity at even lower concentrations. In addition, the assay was modified for testing compounds for lytic activity against the bloodstream form of the parasite under conditions used for storing blood products. Thus, an assay with beta-galactosidase-expressing T. cruzi greatly simplifies screening drugs for selective anti-T. cruzi activity, and three promising new compounds have been identified.

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 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 The inhibitory effects of toothpaste and mouthwash ingredients on the interaction between the SARS-CoV-2 spike protein and ACE2, and the protease activity of TMPRSS2, in vitro

2021 ◽  
Author(s):  
Riho Tateyama-Makino ◽  
Mari Abe-Yutori ◽  
Taku Iwamoto ◽  
Kota Tsutsumi ◽  
Motonori Tsuji ◽  
...  
Keyword(s):  
Serine Protease ◽  
Protease Activity ◽  
Oral Care ◽  
Sodium Dodecyl ◽  
Host Cells ◽  
Spike Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inhibitory Effects ◽  
Docking Simulations

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters host cells when the viral spike protein is cleaved by transmembrane protease serine 2 (TMPRSS2) after binding to the host angiotensin-converting enzyme 2 (ACE2). Since ACE2 and TMPRSS2 are expressed in the mucosa of the tongue and gingiva, the oral cavity seems like it is an entry point for SARS-CoV-2. Daily oral care using mouthwash seems to play an important role in preventing SARS-CoV-2 infection. However, the relationship between daily oral care and the mechanisms of virus entry into host cells is unclear. In this study, we evaluated the inhibitory effects of ingredients that are generally contained in toothpaste and mouthwash on the interaction between the spike protein and ACE2 and on the serine protease activity of TMPRSS2 using an enzyme-linked immunosorbent assay and in vitro enzyme assay, respectively. Both assays detected inhibitory effects of sodium tetradecene sulfonate, sodium N-lauroyl-N-methyltaurate, sodium N-lauroylsarcosinate, sodium dodecyl sulfate, and copper gluconate. Molecular docking simulations suggested that these ingredients could bind to the inhibitor-binding site of ACE2. In addition, tranexamic acid and 6-aminohexanoic acid, which act as serine protease inhibitors, exerted inhibitory effects on TMPRSS2 protease activity. Further experimental and clinical studies are needed to further elucidate these mechanisms. Our findings support the possibility that toothpaste and mouthwash contain ingredients that inhibit SARS-CoV-2 infection.

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.

Could angiotensin-modulating drugs be relevant for the treatment of Trypanosoma cruzi infection? A systematic review of preclinical and clinical evidence

Parasitology ◽  
2019 ◽  
Vol 146 (07) ◽  
pp. 914-927 ◽  
Author(s):  
Thaiany G. Souza-Silva ◽  
Lívia F. Diniz ◽  
Ana Lia Mazzeti ◽  
Andrea A. S. Mendonça ◽  
Reggiani V. Gonçalves ◽  
...  
Keyword(s):  
Systematic Review ◽  
Trypanosoma Cruzi ◽  
Clinical Evidence ◽  
Renin Angiotensin System ◽  
Risk Of Bias ◽  
Host Cells ◽  
Angiotensin System ◽  
Preclinical And Clinical Studies ◽  
Cruzi Infection

AbstractAlthough leucocytes are targets of renin-angiotensin system (RAS) effector molecules and RAS-modulating drugs exert immunomodulatory effects, their impact onTrypanosoma cruziinfection remains poorly understood. By using the framework of a systematic review, we integrated the preclinical and clinical evidence to investigate the relevance of angiotensin-inhibiting drugs onT. cruziinfections. From a comprehensive and structured search in biomedical databases, only original studies were analysed. In preclinical and clinical studies, captopril, enalapril and losartan were RAS-modulating drugs used. The mainin vitrofindings indicated that these drugs increased parasite uptake per host cells, IL-12 expression by infected dendritic cells and IFN-γby T lymphocytes, in addition to attenuating IL-10 and IL-17 production by CD8 + T cells. In animal models, reduced parasitaemia, tissue parasitism, leucocytes infiltration and mortality were often observed inT. cruzi-infected animals receiving RAS-modulating drugs. In patients with Chagas’ disease, these drugs exerted a controversial impact on cytokine and hormone levels, and a limited effect on cardiovascular function. Considering a detailed evaluation of reporting and methodological quality, the current preclinical and clinical evidence is at high risk of bias, and we hope that our critical analysis will be useful in mitigating the risk of bias in further studies.

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

2017 ◽  
Vol 3 ◽  
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.

scholarly journals Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Fernando J Sánchez-Valdéz ◽  
Angel Padilla ◽  
Wei Wang ◽  
Dylan Orr ◽  
Rick L Tarleton
Keyword(s):  
Trypanosoma Cruzi ◽  
Drug Treatment ◽  
Drug Exposure ◽  
Host Cells ◽  
New Host ◽  
Cytotoxic Compounds ◽  
Drug Treatments ◽  
Disease Agent

The ability of the Chagas disease agent Trypanosoma cruzi to resist extended in vivo exposure to highly effective trypanocidal compounds prompted us to explore the potential for dormancy and its contribution to failed drug treatments in this infection. We document the development of non-proliferating intracellular amastigotes in vivo and in vitro in the absence of drug treatment. Non-proliferative amastigotes ultimately converted to trypomastigotes and established infections in new host cells. Most significantly, dormant amastigotes were uniquely resistant to extended drug treatment in vivo and in vitro and could re-establish a flourishing infection after as many as 30 days of drug exposure. These results demonstrate a dormancy state in T. cruzi that accounts for the failure of highly cytotoxic compounds to completely resolve the infection. The ability of T. cruzi to establish dormancy throws into question current methods for identifying curative drugs but also suggests alternative therapeutic approaches.

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.

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 ◽  
Specific Activity ◽  
New Drugs ◽  
Host Cells ◽  
Amastigote Stage ◽  
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 an 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. Moreover, it showed specific activity against the amastigote stage (IC 50 = 3.31 μM). 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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