Benzoic acid and pyridine derivatives as inhibitors of Trypanosoma cruzi trans-sialidase

Benzoic acid and pyridine derivatives inhibit recombinant trans-sialidase from Trypanosoma cruzi with I50 values between 0.4 and 1mM. The best compounds, 4-acetylamino-3-hydroxymethylbenzoic acid and 5-acetylamino-6-aminopyridine-2-carboxylic acid, provide new leads to inhibitors not containing the synthetically complex sialic acid structure. The weak inhibition by such compounds contrasts with their much stronger inhibition of neuraminidase from Influenza virus.     

Trypanosoma cruzi: inhibition of metacyclogenesis by mannose.

Metacyclogenesis of Trypanosoma cruzi epimastigotes was evaluated in a medium supplemented with Triatoma infestans intestinal homogenate in the presence of sugars and derivates as are mannose, galactose, fucose, N-acetylglucosamine, mannose 6-P, and fructose 1,6-P at a concentration of 25 mM. Only mannose significantly inhibited metacyclogenesis. Sodium metaperiodate and trypsin treatment of the intestinal homogenate also inhibited differentiation. In our opinion there exists a proteinic factor in the intestine of the vector that promotes metacyclogenesis and is incorporated by the parasite. Treatment of the intestinal homogenate with alkaline phosphatase had no effect. Instead, high ionic strength in the medium (0.4 M NaCl) strongly inhibited metacyclogenesis indicating that, in these conditions, the possible binding of the differentiation factor to the parasite surface was inhibited.     

Selective inhibition of thromboxane synthetase by pyridine and its derivatives

Pyridine and some derivatives inhibit the conversion of prostaglandin endoperoxide to thromboxane catalyzed by thromboxane synthetase of human platelet microsomes. The structure-activity relationship of pyridine derivatives was investigated. Substitution of pyridine at position 2 either by an alkyl or an aryl group abolishes the inhibitory power of pyridine. Substitution at position 3 or 4 with a hydrophobic chain was found to increase the inhibitory potency, with 3-substituted pyridines being the most potent inhibitors. Inhibition by pyridine and its active derivatives appears to be selective for thromboxane synthetase since other enzymes in the arachidonic acid catabolic pathway were not affected. Kinetic studies indicate that inhibition as examined with hexylnicotinate is of the noncompetitive type.     

Trypanosoma cruzi glycoprotein of Mr 56000: characterization and assessment of its potential to protect against fatal parasite infections

A ∼ 56 000 Da membrane glycoprotein purified from epimastigotes of Trypanosoma cruzi was characterized biochemically and tested for its efficacy to induce protection in mice from a lethal challenge with this protozoan parasite. Immunofluorescence assays with live and formalin-fixed epimastigotes and trypomastigotes localized the glycoprotein to the flagellum, the body of the parasite, and the cell membrane. Immunoblotting demonstrated the glyco-protein's presence in nearly equal amounts in all developmental stages of several T. cruzi isolates. Mice immunized with the purified glycoprotein and challenged with 10000 infectious trypomastigote forms of isolate Y survived the controls by up to four days. This significant protection makes this antigen a potential candidate for a multi-subunit vaccine against 7. cruzi.     

Antiegressin acts late in the intracellular growth cycle of Trypanosoma cruzi to inhibit parasite egress

Trypanosoma cruzi is the causative agent of Chagas disease, which is characterized by acute and chronic phases. During the former, parasitemia rises dramatically, then decreases significantly during the chronic phase. Immune mechanisms responsible for the parasitemia reduction have not been thoroughly elucidated. The goal of the present study was to further characterize the immune response during chronic infection. Previously, we described antiegressin, an antibody in sera from chronically infected mice. The in vitro presence of antiegressin inhibits parasite egress from infected host cells. Antiegressin appears by day 14 of an in vivo infection and is maintained through at least day 280 postinfection. The in vitro functional activity of antiegressin is initiated late in the 4-6 days intracellular growth cycle of T. cruzi; antiegressin may be added at day 4, inhibiting parasite release at day 5. Immunocytochemical staining using antineuraminidase demonstrates the presence of mature parasites inside host BALB/c fibroblasts grown in the presence of antiegressin. These results demonstrate the ability of antiegressin to inhibit emergence of developmentally mature trypomastigotes from infected host cells late in their intracellular growth cycle. We believe this antibody plays an important and novel role in achieving the low-parasitemia characteristic of chronic Chagas disease.     

Novel drug design for Chagas disease via targeting Trypanosoma cruzi tubulin: Homology modeling and binding pocket prediction on Trypanosoma cruzi tubulin polymerization inhibition by naphthoquinone derivatives

Chagas disease, also called American trypanosomiasis, is a parasitic disease caused by Trypanosoma cruzi (T. cruzi). Recent findings have underscored the abundance of the causative organism, (T. cruzi), especially in the southern tier states of the US and the risk burden for the rural farming communities there. Due to a lack of safe and effective drugs, there is an urgent need for novel therapeutic options for treating Chagas disease. We report here our first scientific effort to pursue a novel drug design for treating Chagas disease via the targeting of T. cruzi tubulin. First, the anti T. cruzi tubulin activities of five naphthoquinone derivatives were determined and correlated to their anti-trypanosomal activities. The correlation between the ligand activities against the T. cruzi organism and their tubulin inhibitory activities was very strong with a Pearson’s r value of 0.88 (P value <0.05), indicating that this class of compounds could inhibit the activity of the trypanosome organism via T. cruzi tubulin polymerization inhibition. Subsequent molecular modeling studies were carried out to understand the mechanisms of the anti-tubulin activities, wherein, the homology model of T. cruzi tubulin dimer was generated and the putative binding site of naphthoquinone derivatives was predicted. The correlation coefficient for ligand anti-tubulin activities and their binding energies at the putative pocket was found to be r = 0.79, a high correlation efficiency that was not replicated in contiguous candidate pockets. The homology model of T. cruzi tubulin and the identification of its putative binding site lay a solid ground for further structure based drug design, including molecular docking and pharmacophore analysis. This study presents a new opportunity for designing potent and selective drugs for Chagas disease.     

Trypanosoma cruzi: inhibition of protein synthesis by nitrofuran SQ 18,506

SQ 18,506 is a nitrofuran compound related to the trypanocide Lampit. In vitro, radiolabeled leucine, uridine, and thymidine were incorporated into macromolecular protein, RNA, and DNA in order to study growth inhibition of Trypanosoma cruzi. Our findings suggest that the primary effect of the drug is on protein synthesis and not mediated solely by inhibition of RNA synthesis as indicated by prior studies. The drug was also found to reduce markedly the uptake of uridine into the nucleotide precursor pool but to affect only slightly the formation of aminoacyl-tRNA.     

Trypanosoma cruzi: effect of phenothiazines on the parasite and its interaction with host cells.

Phenothiazines were observed to have a direct effect on Trypanosoma cruzi and on its in vitro interaction with host cells. They caused lysis of trypomastigotes (50 uM/24 h) and, in axenic medium, dose-dependent inhibition of amastigote and, to a lesser extent, epimastigote proliferation. Treatment of infected peritoneal macrophages with 12.5 uM chlorpromazine or triflupromazine inhibited the infection; this effect was found to be partially reversible if the drugs were removed after 24 h of treatment. At 60 uM, the drugs caused damage to amastigotes interiorized in heart muscle cells. However, the narrow margin of toxicity between antitrypanosomal activity and damage to host cells mitigates against in vivo investigation at the present time. Possible hypotheses for the mechanism of action of phenothiazines are discussed.     

Trypanosoma cruzi I and Trypanosoma cruzi II: recognition of sugar structures by Arachis hypogaea (peanut agglutinin) lectin

Epimastigote culture forms of different isolates of Trypanosoma cruzi from different mammal hosts, humans, and vectors were tested with FITC-conjugated peanut agglutinin lectin (PNA-FITC). The parasites maintained in axenic medium, liver infusion tryptose. were evaluated by flow cytometric analyses; whereas T. cruzi I (Tcl), which is associated with the sylvatic transmission cycle, was labeled in high percentages with PNA (88-99.2%), T. cruzi II (TcII) (parasites associated with domiciliar cycle) and T. cruzi, zymodeme 3 (Tc/Z3) (also associated with the sylvatic cycle) were labeled in low percentages (TcII, 0-26% and Tc/Z3, 0-12.6%). It was demonstrated that it is possible to differentiate the 2 main T. cruzi subpopulations, TcI and TcII, using Arachis hypogaea. These results also showed a higher variability in TcII in terms of PNA binding.     

Trypanosoma cruzi: inhibition of intracellular and extracellular differentiation by ADP-ribosyl transferase antagonists

Both the intracellular and the extracellular differentiation of Trypanosoma cruzi amastigotes was studied. Intracellular differentiation was monitored during the parasite's cycle of infection in mammalian cells, and extracellular differentiation was monitored after transfer of the parasites to Warren's medium at 27 C. Several different chemical antagonists of ADP-ribosyl transferase inhibited parasite differentiation in both systems. This inhibition was mediated by a specific effect on the differentiation process and could not be ascribed to interference with simple proliferation of the parasite. The effect is strikingly similar to that observed in studies of the cell differentiation of several higher animals and suggests that ADP-ribosyl transferase frequently constitutes an important element in the mechanism of eukaryotic cell differentiation.     

Trypanosoma cruzi: serum antibody reactivity to the parasite antigens in susceptible and resistant mice

The specific antibody responses were compared among susceptible (A/Sn), moderately susceptible (Balb/c) and resistant (C57 BL/10J) mice infected with Trypanosoma cruzi (Y strain). Sera obtained during the second week of infection recognized a surface trypomastigote antigen of apparent Mr 80 kDa while displaying complex reactivity to surface epimastigote antigens. Complex trypomastigote antigens recognition was detected around the middle of the third week of infection. No major differences were observed along the infection, among the three strains of mice, neither in the patterns of surface antigen recognition by sera, nor in the titres of antibodies against blood trypomastigotes (lytic antibodies), tissue culture trypomastigotes or epimastigotes. On immunoblot analysis, however, IgG of the resistant strain displayed the most complex array of specificities against both trypo and epimastigote antigens, followed by the susceptible strain. IgM antibodies exhibited a more restricted antigen reactivity, in the three mouse strains studied. Balb/c sera (IgG and IgM) showed the least complex patterns of reactivity to antigens in the range of 30 kDa to 80 kDa. The onset of reactivity in the serum to trypomastigote surface antigens was also dependent on the parasite load to which the experimental animal was subjected.     

Trans-sialidase and mucins of Trypanosoma cruzi: an important interplay for the parasite

A dense glycocalix covers the surface of Trypanosoma cruzi, the agent of Chagas disease. Sialic acid in the surface of the parasite plays an important role in the infectious process, however, T. cruzi is unable to synthesize sialic acid or the usual donor CMP-sialic acid. Instead, T. cruzi expresses a unique enzyme, the trans-sialidase (TcTS) involved in the transfer of sialic acid from host glycoconjugates to mucins of the parasite. The mucins are the major glycoproteins in the insect stage epimastigotes and in the infective trypomastigotes. Both, the mucins and the TcTS are anchored to the plasma membrane by a glycosylphosphatidylinositol anchor. Thus, TcTS may be shed into the bloodstream of the mammal host by the action of a parasite phosphatidylinositol-phospholipase C, affecting the immune system. The composition and structure of the sugars in the parasite mucins is characteristic of each differentiation stage, also, interstrain variations were described for epimastigote mucins. This review focus on the characteristics of the interplay between the trans-sialidase and the mucins of T. cruzi and summarizes the known carbohydrate structures of the mucins.     

Inhibition of Trypanosoma cruzi growth and sterol biosynthesis by lovastatin

We have studied the effects of lovastatin, an inhibitor of hydroxy-methylglutaryl-CoA-reductase, on cultures of Trypanosoma cruzi epimastigotes. It inhibits growth at 10 and 30 micrograms per ml; these effects are reverted by 100 microM of squalene, but not by 100 microM of cholesterol. Lovastatin at the same amounts inhibits [14C]acetate incorporatin into sterols, isolated either by digitonin precipitation or thin layer chromatography. At 50 micrograms per ml it kills most of the trypanosomes. These concentrations are below reported toxic levels for mammals; this drug and its analogs should, therefore, be tested as chemotherapeutic agents against Chagas' disease.     

Trypanosoma cruzi: possible control of parasite transmission by blood transfusion using amphiphilic cationic drugs

About 200 clinically used amphiphilic cationic drugs have been shown to be active in vitro against Trypanosoma cruzi at concentrations of less than or equal to 1 mM. Activity against epimastigote and trypomastigote forms was similar, and in both cases the most potent drugs were litracene, maprotiline, thioproperazine, and the acridines: acranil, aminacrine, and mepacrine. Fluorescence microscopy demonstrated that epimastigotes rapidly accumulate acridines initially in discrete subcellular organelles. The amount of drug incorporated during 15 min of incubation was sufficient to produce subsequent lysis of both trypomastigotes and epimastigotes within 24 hr at 4 C. Trypanocidal activity was dependent on the extracellular pH (optimum greater than or equal to 8) and drug exposure time, but was independent of red blood cell density, serum dilution, and temperature (4 to 37 C). Despite their trypanocidal activity, amphiphilic cationic drugs appear to have no significant effect on the energy state of red blood cells at a concentration of 1 mM. These drugs have a possible role in the prevention of Chagas' disease by blood transfusion.     

Effects of human colony-stimulating factor on the uptake and destruction of a pathogenic parasite (Trypanosoma cruzi) by human neutrophils

The ability of granulocyte-macrophage colony-stimulating factor (CSF-H) to modulate human neutrophil functions was studied by using an in vitro system in which this cell type interacted with intracellular (amastigote [AMA]) forms of Trypanosoma cruzi. The presence of CSF-H during the 30-min period of neutrophil incubation with the AMA markedly enhanced parasite internalization. This effect was evidenced by significant increases in both the percentage of neutrophils incorporating AMA and the average number of AMA per 100 neutrophils with respect to mock-treated neutrophils. Pretreatment of the neutrophils with CSF-H reproduced the enhancement effect, whereas pretreatment of the AMA had no detectable consequence. The minimal neutrophil CSF-H pretreatment period required to significantly increase the number of AMA per 100 neutrophils was 20 min--suggesting that CSF-H induced time-dependent events ultimately leading to the manifestation of the noted effect--but neutrophil treatment with CSF-H for longer periods of time (up to 60 min) caused a much greater enhancement. Consistent with the notion of a regulatory action of CSF-H on neutrophils was the fact that the enhancing effect subsided gradually after removal of the factor and was no longer detectable after 16 hr. When 3H-labeled AMA were used, CSF-H-treated neutrophils released greater amounts of radiolabeled substances than mock-treated cells, indicating a stimulatory effect of CSF-H on the killing capacity of neutrophils. This was confirmed by the fact that untreated neutrophils that had internalized 3H-AMA killed the parasites at a faster rate when subsequently incubated with CSF-H. Catalase, but not superoxide dismutase, mannitol, benzoate, or histidine, inhibited neutrophil killing of the 3H-AMA whether the granulocytes had been exposed to CSF-H or not. This indicated that the cytotoxic mechanism involved the production of hydrogen peroxide in both cases, but possibly at a higher rate in the CSF-H-treated neutrophils. These results point to a regulatory effect of CSF-H on neutrophils that promotes cellular activities that might be relevant to the mechanisms of clearance of T. cruzi in vivo.     

Trypanosoma cruzi: early parasite proliferation and host resistance in inbred strains of mice

The extent of parasite proliferation following completion of the first cycle of intracellular replication was significantly higher in CD-1 nu/nu mice and in irradiated mice compared to other, including highly susceptible, mouse strains. A control of parasite proliferation thus occurs in normal mice as early as the first cycle of intracellular replication. The thymus dependency and radiation sensitivity of the early control of proliferation of Trypanosoma cruzi suggest that an immune response to the parasite is involved in the early control of proliferation. The BXH-2 recombinant inbred strain demonstrated an inability to control early proliferation and, 4-5 days after infection, had parasitemias several times higher than those observed in susceptible mouse strains. The BXH-2 strain appears to lack the early control mechanism. When the extent of proliferation of T. cruzi at completion of the first cycle of intracellular replication was compared in inbred strains of mice having varying levels of resistance to the parasite, the extent of proliferation correlated with host resistance, being lowest in the most resistant strains (C57BL/6, SJL) and highest in the most susceptible strains (C3H, A). It is suggested that the mechanism(s) controlling early parasite proliferation may be of primary importance as the basis for host resistance.