A new chemotype with promise against Trypanosoma cruzi

Pyridyl benzamide 2 is a potent inhibitor of Trypanosoma cruzi, but not other protozoan parasites, and had a selectivity-index of ≥10. The initial structure–activity relationship (SAR) indicates that benzamide and sulfonamide functional groups, and N-methylpiperazine and sterically unhindered 3-pyridyl substructures are required for high activity against T. cruzi. Compound 2 and its active analogs had low to moderate metabolic stabilities in human and mouse liver microsomes.     

Inducible suicide vector systems for Trypanosoma cruzi

Chagas disease caused by Trypanosoma cruzi is a major neglected tropical parasitic disease. The pathogenesis of this infection remains disputable. There is no suitable vaccine for the prevention. Attenuated live vaccines can provide strong protection against infection; however, there are the concerns about latent infection or reversion to virulence in such attenuated strains. A method to induce T. cruzi death would provide a critical tool for research into the pathophysiological mechanisms and provide a novel design of safe live attenuated vaccines. We established effective inducible systems for T. cruzi employing the degradation domain based on the Escherichia coli dihydrofolate reductase (ecDHFR). The DHFR degradation domain (DDD) can be stabilized by trimethoprim-lactate and can be used to express detrimental or toxic proteins. T. cruzi lines with Alpha-toxin, Cecropin A and GFP under the control of DDD with a hemagglutinin tag (HA) were developed. Interestingly, amastigotes bearing GFP-DDDHA, Alpha-toxin-DDDHA, Cecropin A-DDDHA and DDDHA all resulted in inducible cell death with these fusions, indicating that DDDHA protein is also detrimental to amastigotes. Furthermore, these strains were attenuated in mouse experiments producing no pathological changes and inoculation with these DDDHA strains in mice provided strong protection against lethal wild type infection.     

Role of dystrophin in acute Trypanosoma cruzi infection

Previous studies have demonstrated loss/reduction of dystrophin in cardiomyocytes in both acute and chronic stages of experimental Trypanosoma cruzi (T. cruzi) infection in mice. The mechanisms responsible for dystrophin disruption in the hearts of mice acutely infected with T. cruzi are not completely understood. The present in vivo and in vitro studies were undertaken to evaluate the role of inflammation in dystrophin disruption and its correlation with the high mortality rate during acute infection. C57BL/6 mice were infected with T. cruzi and killed 14, 20 and 26 days post infection (dpi). The intensity of inflammation, cardiac expression of dystrophin, calpain-1, NF-κB, TNF-α, and sarcolemmal permeability were evaluated. Cultured neonatal murine cardiomyocytes were incubated with serum, collected at the peak of cytokine production and free of parasites, from T. cruzi-infected mice and dystrophin, calpain-1, and NF-κB expression analyzed. Dystrophin disruption occurs at the peak of mortality and inflammation and is associated with increased expression of calpain-1, TNF-α, NF-κB, and increased sarcolemmal permeability in the heart of T. cruzi-infected mice at 20 dpi confirmed by in vitro studies. The peak of mortality occurred only when significant loss of dystrophin in the hearts of infected animals occurred, highlighting the correlation between inflammation, dystrophin loss and mortality.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Aromatic glycosyl disulfide derivatives: Evaluation of their inhibitory activities against Trypanosoma cruzi

Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(β-d-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC₅₀ values ranging from 4 to 11 μM at 37 °C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4 °C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well.     

Trypanosoma cruzi Proline Transport Presents a Cell Density‐dependent Regulation

Trypanosoma cruzi, the etiological agent of Chagas disease, uses proline as its main carbon source, essential for parasite growth and stage differentiation in epimastigotes and amastigotes. Since proline is mainly obtained from extracellular medium by transport proteins, in this work we studied the regulation of the T. cruzi proline transporter TcAAAP069. Proline uptake and intracellular concentration presented oscillations during epimastigote growth phases, increasing during the early exponential phase (322 pmol/min) and decreasing to undetectable levels during the late exponential phase. Transporter expression rate correlated with proline uptake, and its subcellular localization alternated from both, the plasma membrane and close to the flagellar pocket, when the transport is higher, to only the flagellar pocket region, when the transport decreased until proline uptake and TcAAAP069 protein became undetectable at the end of the growth curve. Interestingly, when parasites were treated with conditioned medium or were concentrated to artificially increase the culture density, the proline transport was completely abolished resembling the effects observed in late exponential phase. These data highlight for the first time the existence of a density‐associated regulation of relevant physiological processes such as proline metabolism.     

Characterization of a Trypanosoma cruzi antigen with homology to intracellular mammalian lectins

Two cDNAs, isolated from a Trypanosoma cruzi amastigote library immunoscreened with sera from patients with Chagas disease, encode proteins with sequence homology to eukaryotic components of the cellular sorting and recycling machinery. These proteins, denominated TcAGL, present an N-terminal lectin domain and a C-terminal region containing repetitive amino acids and a poly-glutamine tract. They are products of polymorphic alleles of a single copy gene constitutively expressed during the parasite life cycle. Polyclonal antibodies obtained from mice immunized with the recombinant antigen recognize proteins with apparent molecular weight ranging from 95 to 120 kDa in cell lysates from all three life stages and in various strains of the parasite. Sera from Chagas disease patients recognize the recombinant antigen in ELISA and immunoprecipitation assays but not in Western blot assays under denaturing conditions. Consistent with its proposed role in the glycoprotein secreting pathway, immunofluorescence analyses and expression of a green fluorescent protein-tagged TcAGL protein indicate a sub-cellular localization in the vicinity of the flagellar pocket membrane and the Golgi complex of the parasite.     

The pentose phosphate pathway in Trypanosoma cruzi

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).     

Steroidogenic capacity of Trypanosoma cruzi trypomastigotes

American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi (T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T. cruzi capacity to synthesize steroid hormones has not been investigated. Therefore, the aim of this work was to investigate the capacity of T. cruzi trypomastigotes to transform tritiated steroid precursors into androgens and estrogens. The T. cruzi Tulahuén strain was obtained from mice blood. The trypomastigotes were cultured for 6 and 24h in Dulbbeco's modified Eagle's medium plus FCS and antibiotics. Tritiated dehydroepiandrosterone or androstendione were added to the culture media and parasites were incubated for 6 or 24h. The cultures were centrifuged and ether extracted. The steroids were analyzed by thin layer chromatography (TLC) in two solvent systems. After incubation with (3)H-androstenedione, T. cruzi trypomastigotes synthesized (3)H-testosterone (T), (3)H-17beta-estradiol (E(2)) and (3)H-estrone (E(1)). Metabolism of (3)H-DHEA by the parasites yielded (3)H-androstendione and (3)H-androstendiol at 6h of incubation. The recrystallization procedure further demonstrated the (3)H-androstendiol and (3)H-17beta-estradiol syntheses. Results indicate for the first time that T. cruzi trypomastigotes produce androgens and estrogens when incubated in the presence of steroid precursors and suggest the presence of active parasite steroidogenic enzymes.     

Synthesis and tripanocidal activity of ferrocenyl and benzyl diamines against Trypanosoma brucei and Trypanosoma cruzi

Trypanosoma brucei and Trypanosoma cruzi are the etiologic agents of sleeping sickness and Chagas disease, respectively, two of the 17 preventable tropical infectious diseases (NTD) which have been neglected by governments and organizations working in the health sector, as well as pharmaceutical industries. High toxicity and resistance are problems of the conventional drugs employed against trypanosomiasis, hence the need for the development of new drugs with trypanocidal activity. In this work we have evaluated the trypanocidal activity of a series of N1,N2-dibenzylethane-1,2-diamine hydrochlorides (benzyl diamines) and N1-benzyl,N2-methyferrocenylethane-1,2-diamine hydrochlorides (ferrocenyl diamines) against T. brucei and T. cruzi parasite strains. We show that incorporation of the ferrocenyl group into the benzyl diamines increases the trypanocidal activity. The molecules exhibit potential trypanocidal activity in vitro against all parasite strains. Cytotoxicity assay was also carried out to evaluate the toxicity in HepG2 cells.     

Discovery of antichagasic inhibitors by high-throughput screening with Trypanosoma cruzi glucokinase

A high-throughput screening (HTS) campaign was carried out for Trypanosoma cruzi glucokinase (TcGlcK), a potential drug-target of the pathogenic protozoan parasite. Glycolysis and the pentose phosphate pathway (PPP) are important metabolic pathways for T. cruzi and the inhibition of the glucose kinases (i.e. glucokinase and hexokinase) may be a strategic approach for drug discovery. Glucose kinases phosphorylate d-glucose with co-substrate ATP to yield G6P, and moreover, the produced G6P enters both pathways for catabolism. The TcGlcK – HTS campaign revealed 25 novel enzyme inhibitors that were distributed in nine chemical classes and were discovered from a primary screen of 13,040 compounds. Thirteen of these compounds were found to have low micromolar IC₅₀ enzyme – inhibition values; strikingly, four of those compounds exhibited low toxicity towards NIH-3T3 murine host cells and notable in vitro trypanocidal activity. These compounds were of three chemical classes: (a) the 3-nitro-2-phenyl-2H-chromene scaffold, (b) the N-phenyl-benzenesulfonamide scaffold, and (c) the gossypol scaffold. Two compounds from the 3-nitro-2-phenyl-2H-chromene scaffold were determined to be hit-to-lead candidates that can proceed further down the early-stage drug discovery process.     

Retrospective distribution of Trypanosoma cruzi I genotypes in Colombia

Trypanosoma cruzi is the aetiological agent of Chagas disease, which affects approximately eight million people in the Americas. This parasite exhibits genetic variability, with at least six discrete typing units broadly distributed in the American continent. T. cruzi I (TcI) shows remarkable genetic diversity; a genotype linked to human infections and a domestic cycle of transmission have recently been identified, hence, this strain was named TcIDom. The aim of this work was to describe the spatiotemporal distribution of TcI subpopulations across humans, insect vectors and mammalian reservoirs in Colombia by means of molecular typing targeting the spliced leader intergenic region of mini-exon gene. We analysed 101 TcI isolates and observed a distribution of sylvatic TcI in 70% and TcIDom in 30%. In humans, the ratio was sylvatic TcI in 60% and TcIDom in 40%. In mammal reservoirs, the distribution corresponded to sylvatic TcI in 96% and TcIDom in 4%. Among insect vectors, sylvatic TcI was observed in 48% and TcIDom in 52%. In conclusion, the circulation of TcIDom is emerging in Colombia and this genotype is still adapting to the domestic cycle of transmission. The epidemiological and clinical implications of these findings are discussed herein.     

Trypanosoma cruzi: phosphatidylinositol 3-kinase and protein kinase B activation is associated with parasite invasion

Multiple signal transduction events are triggered in the host cell during invasion by the protozoan parasite Trypanosoma cruzi. Here, we report the regulation of host cell phosphatydilinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt) activities by T. cruzi during parasite-host cell interaction. Treatment of nonphagocytic cells (Vero, L(6)E(9), and NIH 3T3) and phagocytic cells (human and J774 murine macrophages) with the selective PI3K inhibitors Wortmannin and LY294002 significantly impaired parasite invasion in a dose-dependent fashion. A strong activation of PI3K and PKB/Akt activities in Vero cells was detected when these cells were incubated with trypomastigotes or their isolated membranes. Consistently, we were unable to detect activation of PI3K or PKB/Akt activities in host cells during epimastigote (noninfective) membrane-host cell interaction. Infection of transiently transfected cells containing an inactive mutant PKB showed a significant inhibition of invasion compared with the active mutant-transfected cells. T. cruzi PI3K-like activity was also required in host cell invasion since treatment of trypomastigotes with PI3K inhibitors prior to infection reduced parasite entry. Taken together, these results indicate that PI3K and PKB/Akt activation in parasites, as in host cells induced by T. cruzi, is an early invasion signal required for successful trypomastigote internalization.     

Biochemical Characterization of Branched Chain Amino Acids Uptake in Trypanosoma cruzi

Trypanosoma cruzi is the etiological agent of Chagas disease. During its life cycle, it alternates among vertebrate and invertebrate hosts. Metabolic flexibility is a main biochemical characteristic of this parasite, which is able to obtain energy by oxidizing a variety of nutrients that can be transported from the extracellular medium. Moreover, several of these metabolites, more specifically amino acids, have a variety of functions beyond being sources of energy. Branched chain amino acids (BCAA), beyond their role in ATP production, are involved in sterol biosynthesis; for example, leucine is involved as a negative regulator of the parasite differentiation process occurring in the insect midgut. BCAA are essential metabolites in most nonphotosynthetic eukaryotes, including trypanosomes. In view of this, the metabolism of BCAA in T. cruzi depends mainly on their transport into the cell. In this work, we kinetically characterized the BCAA transport in T. cruzi epimastigotes. Our data point to BCAA as being transported by a single saturable transport system able to recognize leucine, isoleucine and valine. In view of this, we used leucine to further characterize this system. The transport increased linearly with temperature from 10 to 45 °C, allowing the calculation of an activation energy of 51.30 kJ/mol. Leucine uptake was an active process depending on ATP production and a H⁺ gradient, but not on a Na⁺ or K⁺ gradient at the cytoplasmic membrane level.     

Structure-activity relationship of 4-azaindole-2-piperidine derivatives as agents against Trypanosoma cruzi

The structure-activity relationship of a 4-Azaindole-2-piperidine compound selected from GlaxoSmithKline’s recently disclosed open-resource “Chagas box” and possessing moderate activity against Trypanosoma cruzi, the parasite responsible for Chagas disease, is presented. Despite considerable medicinal chemistry efforts, a suitably potent and metabolically stable compound could not be identified to advance the series into in vivo studies. This research should be of interest to those in the area of neglected diseases and in particular anti-kinetoplastid drug discovery.     

Trypanosoma cruzi: allopurinol in the treatment of mice with experimental acute Chagas disease

The therapeutic effect of allopurinol was studied in an experimental Trypanosoma cruzi infection (Chagas disease) in outbred IVIC-NMRI and inbred C57B1/6J mice intraperitoneally inoculated with the parasites 2–6 days before drug treatment. Allopurinol protected against T. cruzi infection. This effect was evidenced by highly significant reductions in both parasitemias and mortality rates and increased survival time in allopurinol-treated animals compared with untreated infected mice. Allopurinol protected effectively when administered in 10 daily doses of 32–64 mg/kg body wt/day injected intraperitoneally. Using direct methods, parasitemia remained undetectable for at least 310 days. An indirect method, subinoculation to susceptible mice, showed a few circulating trypanosomes which decreased greatly in number after a second schedule of allopurinol treatment; finally no trypanosomes were detectable 275 days after treatment initiation. Allopurinol also induced a strong trypanostatic effect when tested in vitro on five different Trypanosoma cruzi strains (optimal inhibitory concentration: 3 μg/ml). These results suggest that allopurinol protects mice with acute Chagas infection by a direct trypanostatic effect. The low toxicity of this drug suggests its use in more chronic experimental Chagas infections.     

Phosphoproteomic analysis of the human pathogen Trypanosoma cruzi at the epimastigote stage

Trypanosoma cruzi is the etiologic agent of Chagas disease, which affects millions of people in Latin America and has become a public health concern in the United States and areas of Europe. The possibility that kinase inhibitors represent novel anti‐parasitic agents is currently being explored. However, fundamental understanding of the cell‐signaling networks requires the detailed analysis of the involved phosphorylated proteins. Here, we have performed a comprehensive MS‐based phosphorylation mapping of phosphoproteins from T. cruzi epimastigote forms. Our LC‐MS/MS, dual‐stage fragmentation, and multistage activation analysis has identified 237 phosphopeptides from 119 distinct proteins. Furthermore, 220 phosphorylation sites were unambiguously mapped: 148 on serine, 57 on threonine, and 8 on tyrosine. In addition, immunoprecipitation and Western blotting analysis confirmed the presence of at least seven tyrosine‐phosphorylated proteins in T. cruzi. The identified phosphoproteins were subjected to Gene Ontology, InterPro, and BLAST analysis, and categorized based on their role in cell structure, motility, transportation, metabolism, pathogenesis, DNA/RNA/protein turnover, and signaling. Taken together, our phosphoproteomic data provide new insights into the molecular mechanisms governed by protein kinases and phosphatases in T. cruzi. We discuss the potential roles of the identified phosphoproteins in parasite physiology and drug development.     

Nanoemulsions of sulfonamide carbonic anhydrase inhibitors strongly inhibit the growth of Trypanosoma cruzi

Sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors targeting the α-class enzyme from the protozoan pathogen Trypanosoma cruzi, responsible of Chagas disease, were recently reported. Although many such derivatives showed low nanomolar activity in vitro, they were inefficient anti-T. cruzi agents in vivo. Here, we show that by formulating such sulfonamides as nanoemulsions in clove (Eugenia caryophyllus) oil, highly efficient anti-protozoan effects are observed against two different strains of T. cruzi. These effects are probably due to an enhanced permeation of the enzyme inhibitor through the nanoemulsion formulation, interfering in this way with the life cycle of the pathogen either by inhibiting pH regulation or carboxylating reactions in which bicarbonate/CO₂ are involved. This type of formulation of sulfonamides with T. cruzi CA inhibitory effects may lead to novel therapeutic approaches against this orphan disease.