Macromolecular Targets of Antiparasitic Germacranolide Sesquiterpenoids: An In Silico Investigation.

BACKGROUND: The parasitic protozoal infections leishmaniasis, human African trypanosomiasis, and Chagas disease are neglected tropical diseases that pose serious health risks for much of the world's population. Current treatment options suffer from limitations, but plantderived natural products may provide economically advantageous therapeutic alternatives. Several germacranolide sesquiterpenoids have shown promising antiparasitic activities, but the mechanisms of activity have not been clearly established. OBJECTIVE: The objective is to use in silico screening of known antiparasitic germacranolides against recognized protozoal protein targets in order to provide insight into the molecular mechanisms of activity of these natural products. METHODS: Conformational analyses of the germacranolides were carried out using density functional theory, followed by molecular docking. A total of 88 Leishmania protein structures, 86 T. brucei protein structures, and 50 T. cruzi protein structures were screened against 27 antiparasitic germacranolides. RESULTS: The in-silico screening has revealed which of the protein targets of Leishmania spp., Trypanosoma brucei, and Trypanosoma cruzi are preferred by the sesquiterpenoid ligands.

Trypanosoma cruzi: analysis of two different strains after piplartine treatment.

The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

Trypanosoma cruzi: analysis of two different strains after piplartine treatment

ABSTRACT The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

Computational and Investigative Study of Flavonoids Active Against Typanosoma cruzi and Leishmania spp.

Flavonoid compounds active against Trypanosoma cruzi and Leishmania species were submitted to several methodologies in silico: docking with the enzymes cruzain and trypanothione reductase (from T. cruzi), and N-myristoyltransferase, dihydroorotate dehydrogenase, and trypanothiona reductase (from Leishmania spp). Molecular maps of the complexes and the ligands were calculated. In order to compare and evaluate the antioxidant activity of the flavonoids with their antiprotozoal activity, quantum parameters were calculated. Considering the energies, interactions, and hydrophobic surfaces calculated, the flavonoids chrysin dimethyl ether against T. cruzi, and ladanein against Leishmania sp. presented the best results. The antioxidant activity did not show any correlation with anti-parasitic activity; only chrysin and its dimethyl ether showed favorable anti-parasitic results. This study hopes to contribute to existing research on these natural products against these tropical parasites.

Antiprotozoal activity of (8-hydroxymethylen)-trieicosanyl acetate isolated from Senna villosa.

A white solid compound was isolated from the chloroform extract of the leaves of Senna villosa. The material was identified by (1)H-NMR, (13)C-NMR, IR and EM methods as (8-hydroxymethylen)-trieicosanyl acetate, a new compound with biological activity, which was tested in vitro at concentrations of 1.65, 3.3 and 6.6 microg/ml for inhibition of the growth of Trypanosoma cruzi epimastigotes and tripomastigotes. We observed inhibition of growth at all concentrations tested, and the effect at concentrations of 3.3 and 6.6 microg/ml was greater than that of gentian violet (positive control). At the concentration of 6.6 microg/ml, the compound showed the greatest inhibitory effect against the growth of both forms of the parasite.

Effects of the isolation methodology on protein profiles of blood trypomastigotes of Trypanosoma cruzi.

Blood trypomastigotes of Trypanosoma cruzi were isolated from infected athymic rnu/rnu rats and purified by an improved procedure of DEAE-Sephacel ion-exchange chromatography. Elution into a buffer supplemented with bovine serum albumin avoided column-induced changes on the surface of the parasites. Biotin-labelled bovine serum albumin, fluorescence microscopy, flow cytometry and Western blot analysis revealed a very intense binding of albumin to the parasite. Incubation and washing of cells without protein supplementation did not result in any damage or lysis of parasites but it did cause extensive shedding of cellular and surface proteins into the supernatant which could be prevented by using the protein-supplemented buffer. A decreasing yield of high molecular weight cellular proteins in relation to centrifugal force was a general phenomenon observed in scanning densitometry of SDS gels after isolation in either protein-supplemented buffer or protein-free buffer. The quantity of shed cellular components increased with increasing centrifugal force. In contrast, quantities of high molecular weight, biotin-labelled surface proteins increased with greater centrifugal force, indicating labelling of otherwise inaccessible residues. These data emphasize the importance of protein supplementation of buffers with proteins and of choosing low centrifugation forces (<400 g) during investigations of T. cruzi.

Targeted reduction in expression of Trypanosoma cruzi surface glycoprotein gp90 increases parasite infectivity.

A previous study had shown that the expression of gp90, a stage-specific surface glycoprotein of Trypanosoma cruzi metacyclic trypomastigotes, is inversely correlated with the parasite's ability to invade mammalian cells. By using antisense oligonucleotides complementary to a region of the gp90 gene implicated in host cell adhesion, we investigated whether the selective inhibition of gp90 synthesis affected the capacity of metacyclic forms to enter target cells. Parasites were incubated for 24 h with 20 microM PS1, a phosphorothioate oligonucleotide based on a sequence of the gp90 coding strand; PS2, the antisense counterpart of PS1; or PO2, the unmodified version of PS2 containing phosphodiester linkages, and the expression of surface molecules was analyzed by flow cytometry and immunoblotting using specific monoclonal antibodies. PS2 but not PS1 or PO2 inhibited the expression of gp90. Inhibition by PS2 was dose dependent. Northern blot analysis revealed that steady-state gp90 mRNA levels were diminished in PS2-treated parasites compared to untreated controls. Treatment with PS2 did not affect the expression of other metacyclic stage surface glycoproteins involved in parasite-host cell interaction, such as gp82 and the mucin-like gp35/50. Expression of gp90 was also inhibited by other phosphorothioate oligonucleotides targeted to the gp90 gene (PS4, PS5, PS6, and PS7) but not by PS3, with the same base composition as PS2 but a mismatched sequence. Parasites treated with PS2, PS4, or PS5 entered HeLa cells in significantly higher numbers than untreated controls, whereas the invasive capacity of PS1- and PS3-treated parasites was unchanged, confirming the inverse association between infectivity and gp90 expression.

Molecular cloning and characterization of two iron superoxide dismutase cDNAs from Trypanosoma cruzi.

Two cDNAs (FeSODA and FeSODB cDNAs) corresponding to superoxide dismutase (1.15.1.1., SOD) were isolated from a Trypanosoma cruzi cDNA library. Comparison of the deduced amino acid sequences with previously reported SOD protein sequences revealed that the T. cruzi open reading frames had considerable homology with FeSODs. The coding region of the T. cruzi FeSODB cDNA has been expressed in fusion with glutathione-S-transferase using an Escherichia coli mutant QC779, lacking both MnSOD and FeSOD genes (sodA sodB). Staining of native polyacrylamide gels for SOD activity of T cruzi crude lysate and the recombinant SOD suggests that this protein is an FeSOD. The recombinant enzyme also protected the E. coli mutant QC779 from paraquat toxicity. Northern blot analysis showed that FeSODB is differentially expressed, showing a higher level at the epimastigote stage of T. cruzi development; whereas, FeSODA is constitutively expressed at a lower level in all developmental stages. Furthermore, Southern hybridization shows that both FeSODA and FeSODB genes appear to be present in the T. cruzi genome as multiple repeating units (multi-copy gene family).

Fetal bovine serum induces changes in fatty acid composition of Trypanosoma cruzi phosphoinositides.

Fetal bovine serum (FBS) is a necessary constituent of the culture media employed to foster the growth of Trypanosoma cruzi epimastigote forms. In different laboratories, the serum is used at final concentrations of 5 or 10%. We have normally supplemented the complex medium with 10% FBS. Under this condition we have described the fatty acid composition of the total lipids and of the phosphoinositide fractions. Additionally, we have reported the increase of polyphosphoinositides and phosphatidic acid after cholinergic stimulation. Since further attempts to reproduce these results with 5% FBS in the culture medium were not successful, the effect of the FBS concentration on the fatty acid composition of phospholipids from the T. cruzi epimastigote forms was thoroughly examined. This work showed that when the FBS concentration supplementing the culture medium was reduced from 10 to 5%, the fatty acid composition of the phosphoinositides was altered while the other major phospholipids were not significantly affected. The most relevant result was the decrease in the content of linoleic acid (18:2) and the increase of palmitoleic acid (16:1) in phosphatidylinositol 4,5-bisphosphate. Phosphatidylinositol (PI) and phosphatidylinositol phosphate also exhibited similar changes in the same fatty acids. The C2 fatty acid composition of the phosphoinositides, under the same conditions, is also reported here for the first time.