Epimastigote Proliferation Research Articles

Synthesis of Naphthofuranquinones with Activity Against Trypanosoma cruzi.

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Selective activity of polyene macrolides produced by genetically modified Streptomyces on Trypanosoma cruzi

The growth inhibitory effects on Trypanosoma cruzi of several natural tetraene macrolides and their derivatives were studied and compared with that of amphotericin B. All tetraenes strongly inhibited in vitro multiplication. Proliferation of epimastigotes was arrested by all these drugs at ≤3.6 μM, which were also active on amastigotes proliferating in fibroblasts. Compared with amphotericin B, the compounds were less effective but also less toxic, showing no effect on the proliferation of J774 and NCTC 929 mammalian cells at concentrations active against the parasites. CE-108B (a polyene amide) appeared to be an especially potent trypanocidal compound, with strong in vivo trypanocidal activity and very low or no toxic side effects, and thus should be considered for further studies.

Synthesis of naphthofuranquinones with activity against Trypanosoma cruzi

Four new naphthofuranquinones, obtained from 2-hydroxy-3-allyl-naphthoquinone ( 1) and nor-lapachol ( 2), have their structures established by physical and X-ray analysis and their activity evaluated against Trypanosoma cruzi. Compounds 3 and 4 were obtained by addition of iodine to 1 followed by cyclization generating a furan ring. Compound 5 was obtained through the acid-catalyzed reaction by dissolution of 1 in sulfuric acid. Compound 6 was synthesized by addition of bromine and aniline to 2. The IC 50/24 h for 3– 6 in assays with T. cruzi trypomastigotes was between 157 and 640 μM, while the value for crystal violet was 536.0 ± 3.0 μM. Compounds 3– 5 also inhibited epimastigote proliferation. The trypanocidal activity of the new naphthofuranquinones endowed with redox properties reinforces a rational approach in the chemotherapy of Chagas' disease.

In vitro anti-parasitic activity of Cyclosporin A analogs on Trypanosoma cruzi

Cyclosporin A (CsA) nonimmunosuppressive analogs were evaluated against Trypanosoma cruzi and on TcCyP19, a cyclophilin of 19 kDa. Two out of eight CsA analogs, H-7-94 and F-7-62 showed the best anti-parasitic effects on all in vitro assays. Their IC 50 values were 0.82 and 3.41 μM, respectively, compared to CsA IC 50 value 5.39 μM on epimastigote proliferation; and on trypomastigote lysis their IC 50 values were 0.97 and 2.66 μM compared to CsA IC 50 value 7.19 μM. H-7-94 and F-7-62 were also more effective than CsA in inhibiting trypomastigote infection. The enzymatic activity of TcCyP19 was inhibited by all CsA derivatives, suggesting this target is involved in the trypanocidal effects observed.

Effects of fluorinated inositols on the phosphoinositide metabolism and the proliferation of Trypanosoma cruzi.

Inositol has been cited as being essential for the growth of micro-organisms and animals. Its action rests mostly in the formation of a set of inositol-containing lipids, including phosphatidylinositol and its phosphorylated derivatives. To evaluate the functional responses coupled to the phosphoinositide metabolism in Trypanosoma cruzi we used myo-inositol and its six fluorinated analogues. Their uptake into epimastigotes was characterised using tritium-labeled myo-inositol and monodeoxyfluoro-myo-inositols. The analogues were tested for their ability to inhibit [3H]-myo-inositol incorporation into phosphoinositides and the proliferation of epimastigotes and amastigotes. The results showed differences between T. cruzi and mammalian systems in the responses to the fluorinated analogues. We found that the 3-, 5- and 6-fluoro analogues did not enter the cells but had an inhibitory effect on the incorporation of the radioactive inositol into lipids and on the amastigotes' and epimastigotes' replication. The most effective inhibitor, 1-D-6-deoxy-6-myo-inositol, had no effect on mammalian cell division.

Inhibition of phosphatidylcholine biosynthesis and cell proliferation in trypanosomacruzi by ajoene, an antiplatelet compound isolated from garlic

Ajoene [( E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide], a potent antiplatelet compound derived from garlic, inhibits the proliferation of both epimastigotes and amastigotes of Trypanosoma cruzi, the causative agent of Chagas' disease. The growth of the epimastigote form was immediately arrested by 80 μM ajoene, while 100 μM induced cell lysis in 24 hr. In the amastigote form proliferating inside VERO cells, 40 μM ajoene was sufficient to eradicate the parasite from the host cells in 96 hr. Growth inhibition of the epimastigotes was accompanied by a gross alteration of the phospholipid composition of the treated cells in which phosphatidylcholine (PC), the major phospholipid class present in control cells, dropped to the least abundant phospholipid in cells treated with 60 μM ajoene for 96 hr, while its immediate precursor, phosphatidylethanolamine (PE), became the predominant species; this was correlated with a marked drop in the incorporation of [ 14C-U]acetate in PC and a corresponding increase in PE. Concomitant with the change in the phospholipid headgroup composition of the cells, the fatty acids esterified to this lipid fraction underwent a dramatic alteration due to the increase in the content of saturated fatty acids and a marked reduction in the content of linoleic (18:2) acid, which is the predominant fatty acid in control cells. We also found that ajoene inhibited the de novo synthesis of neutral lipids and, in particular, of sterols in the epimastigotes, but the resultant changes in the sterol composition were not sufficient to explain the antiproliferative effects of the drug. Electron-microscopy showed a concentration-dependent alteration of intracellular membranous structures, particularly the mitochondrion and endoplasmatic reticulum. The results suggest that one important factor associated with the antiproliferative effects of ajoene against T. cruzi is its specific alteration of the phospholipid composition of these cells.

The challenge of Chagas' disease chemotherapy: An update of drugs assayed against Trypanosoma cruzi

The chemotherapy of Chagas' disease remains an unsolved problem, and the search for alternative drugs continues. Only two nitroheterocyclic drugs are in clinical use at the present time, and these have severely restricted applicability for chronic patients, as well as being highly toxic. This review covers drugs tested in the last 12 years. A large number of different compounds have been assayed in a variety of ways, most commonly in terms of their capacity to inhibit epimastigote proliferation. Allopurinol has emerged for the treatment of chronic cases. However, only with greater knowledge of the biochemistry of the parasite and in particular of its peculiarities, will it be possible to shift the emphasis of drug research away from random screenings onto a more rational footing. This is exemplified by recent studies carried out using purine derivatives and trypanothione reductase inhibitors.

Mevinolin (lovastatin) potentiates the antiproliferative effects of ketoconazole and terbinafine against Trypanosoma (Schizotrypanum) cruzi: in vitro and in vivo studies

We have studied the antiproliferative effects of mevinolin (lovastatin), an inhibitor of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase, on the protozoan parasite Trypanosoma (Schizotrypanum) cruzi and its ability to potentiate the action of specific ergosterol biosynthesis inhibitors, such as ketoconazole and terbinafine, both in vitro and in vivo. Against the epimastigote form in vitro, mevinolin produced a dose-dependent reduction of the growth rate up to 25 microM, but at 50 and 75 microM, complete growth arrest and cell lysis took place after 144 and 96 h, respectively. A systematic study of the effects of mevinolin combined with ketoconazole and terbinafine, which act at different points in the ergosterol biosynthesis pathway, on the proliferation of epimastigotes indicated a synergic action, as shown by concave isobolograms and fractional inhibitory concentration indexes ranging from 0.17 to 0.54. Analysis of the sterol composition and de novo sterol synthesis in control and treated cells by thin-layer and gas-liquid chromatographies showed that the antiproliferative effects of the drug alone and in combination were correlated with the depletion of the endogenous ergosterol pool and particularly with a critical (exogenous) cholesterol/endogenous 4-desmethyl sterol ratio in the cells. When we studied the effects of mevinolin on the amastigote form proliferating inside Vero cells in vitro, only very modest effects on the parasites were observed up to 0.75 microM; above this concentration, significant deleterious effects on the host cells were found. However, when the same concentration of the drug was combined with ketoconazole, it was able to reduce by a factor of 10 the concentration of the azole required to eradicate the parasite (from 10 to 1 nM), again indicating a synergic action. On the other hand, a combination of mevinolin and terbinafine had only additive effects on amastigotes, but a ternary combination of mevinolin, ketoconazole, and terbinafine was again clearly synergistic. In vivo studies with a murine model of Chagas' disease showed that mevinolin can also potentiate the therapeutic effects of ketoconazole in this system; combined treatment with the two drugs at doses that alone offered only limited protection against the parasite was able to essentially eliminate circulating parasites and produce complete protection against death. These results confirm the synergic action against the proliferative stages of T. cruzi both in vitro and in vivo and in vivo of combined ergosterol biosynthesis inhibitors that act at different points in the pathway and suggest that mevinolin combined with azoles, such as ketoconazole, can be used in the treatment of human Chagas' disease.

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.