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Infection with the protozoan parasite Trypanosoma cruzi causes human Chagas disease. Benznidazole (BNZ) and nifurtimox are the current drugs for the treatment; however, they induce severe adverse side effects in patients; therefore, there is a need to improve the treatment effectiveness and efficiency of these drugs for its safer use. Background/Objective: Glyburide, glipizide, and gliquidone, hypoglycemic drugs for diabetes treatment, were previously predicted to bind to dihydrofolate reductase-thymidylate synthase from T. cruzi by in silico docking analysis; they also showed antiproliferative effects against T. cruzi epimastigotes, the stage of the insect vector. In the present study, the potential parasiticidal effect of these antidiabetic drugs was tested in monotherapy and bi-therapy with BNZ in human cells in vitro and in animals. Methods: Evaluation was performed in (a) a model of in vitro infection of T. cruzi trypomastigotes using human fibroblasts as host cells and (b) in mice infected with T. cruzi. Results: The antidiabetic drugs in monotherapy showed antiparasitic effects in preventing infection progression (trypomastigotes release), with an IC50 of 8.4–14.3 µM in comparison to that of BNZ (0.26 µM) in vitro. However, in bi-therapy, the presence of just 0.5 or 1 µM of the antidiabetics decreased the BNZ IC50 by 5–10 times to 0.03–0.05 µM. Remarkably, the antidiabetic drugs in monotherapy decreased the infection in mice by 40–60% in a similar extent to BNZ (80%). In addition, the combination of BNZ plus antidiabetics perturbed the antioxidant metabolites in epimastigotes. Conclusions: These results identified antidiabetics as potential drugs in combination therapy with BNZ to treat T. cruzi infection. [ABSTRACT FROM AUTHOR]

In a previous phylogenetic study of the family of pyruvate kinase EC (2.7.1.40), a cluster with Glu117 and another with Lys117 were found (numbered according to the rabbit muscle enzyme). The sequences with Glu117 have been found to be K+-dependent, whereas those with Lys117 were K+-independent. Interestingly, only γ-proteobacteria exhibit sequences in both branches of the tree. In this context, it was explored whether these phylogenetically distinct pyruvate kinases were both expressed and contribute to the pyruvate kinase activity in Vibrio cholerae. The main findings of this work showed that the isozyme with Glu117 is an active K+-dependent enzyme. At the same substrate concentration, its Vmax in the absence of fructose 1,6 bisphosphate was 80% of that with its effector. This result is in accordance with the non-essential activation described by allosteric ligands for most pyruvate kinases. In contrast, the pyruvate kinase with Lys117 was a K+-independent enzyme displaying an allosteric activation by ribose 5-phosphate. At the same substrate concentration, its activity without the effector was 0.5% of the one obtained in the presence of ribose 5-phosphate, indicating that this sugar monophosphate is a strong activator of this enzyme. This absolute allosteric dependence is a novel feature of pyruvate kinase activity. Interestingly, in the K+-independent enzyme, Mn2+ may “mimic” the allosteric effect of Rib 5-P. Despite their different allosteric behavior, both isozymes display a rapid equilibrium random order kinetic mechanism. The intracellular concentrations of fructose 1,6-bisphosphate and ribose 5-phosphate in Vibrio cholerae have been experimentally verified to be sufficient to induce maximal activation of both enzymes. In addition, Western blot analysis indicated that both enzymes were co-expressed. Therefore, it is concluded that VcIPK and VcIIPK contribute to the activity of pyruvate kinase in this γ-proteobacterium. [ABSTRACT FROM AUTHOR]

In the validation of therapeutic targets on the intermediary metabolism of protozoan parasites (and in general of pathological cells to determine the essentiality of a protein) the gene expression silencing method is commonly applied. The results of these analyses generally conclude that ALL silenced metabolic enzymes are essential for parasite survival or infectivity. Therefore, it is necessary to apply additional strategies to identify those enzymes that, in addition to being essential, when inhibited at a lower percentage than those obtained by genetic or pharmacological methods, have negative effects on the parameters of antiparasiticidal activity being sought. In the study of the mechanisms that regulate and control the metabolic pathways of a cell, Metabolic Control Analysis (MCA) and kinetic modeling (a Systems Biology approach that consists of building computational models of cell metabolism) are strategies that allow us to quantify the degree of control that an enzyme has over the fluxes of the metabolic pathway to which it belongs. By means of these strategies it is possible to identify those enzymes that have the greatest control of the pathway, which from the metabolic point of view are the sites with the greatest therapeutic potential. These strategies were applied to trypanothione-dependent antioxidant metabolism and cysteine metabolism in the Trypanosoma cruzi parasite. In silico, in vitro and in vivo results indicate that mild/moderate inhibition of enzymes/transporters that have the greatest degree of control of a metabolic pathway have a greater negative effect on the parasite than inhibition of an enzyme with little control. Therefore, MCA and computational modeling of metabolic pathways are strategies to help prioritize sites for therapeutic intervention. These approaches can direct basic research for drug design or drug repositioning against validated therapeutic targets. [ABSTRACT FROM AUTHOR]

Adhesion to cells, cytotoxicity and proteolysis are functions required for virulence and pathogenicity of E ntamoeba histolytica. However, there was no correlation between these in vitro functions and the early elimination of non-pathogenic E . dispar and non-virulent E . histolytica ( nv Eh) in experimental amoebic liver abscesses developed in hamsters. Thus, additional functions may be involved in amoebic pathogenicity and virulence. In the present study, an integral experimental assessment, including innovative technologies for analyses of amoebal pathophysiology, cell biology, biochemistry and transcriptomics, was carried out to elucidate whether other cellular processes are involved in amoebal pathogenicity and virulence. In comparison with virulent E . histolytica, the data indicated that the main reasons for the early clearance of nv Eh from hamster liver are decreased intracellular H2O2 detoxification rate and deficient heat shock protein expression, whereas for E . dispar, it is a relatively lower capacity for O2 reduction. Therefore, maintenance of an intracellular hypoxic environment combined with the induction of an adequate parasite response to oxidative stress are essential requirements for E ntamoeba survival in the liver, and therefore for pathogenicity. [ABSTRACT FROM AUTHOR]

The synthesis of ATP in the human parasiteEntamoeba histolyticais carried out solely by the glycolytic pathway. Little kinetic and structural information is available for most of the pathway enzymes. We report here the gene cloning, overexpression and purification of hexokinase, hexose-6-phosphate isomerase, inorganic pyrophosphate-dependent phosphofructokinase, fructose-1,6 bisphosphate aldolase (ALDO), triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase, phosphoglycerate mutase (PGAM), enolase, and pyruvate phosphate dikinase (PPDK) enzymes fromE. histolytica.Kinetic characterization of these 10 recombinant enzymes was made, establishing the kinetic constants at optimal and physiological pH values, analyzing the effect of activators and inhibitors, and investigating the storage stability and oligomeric state. Determination of the catalytic efficiencies at the pH optimum and at pH values that resemble those of the amoebal trophozoites was performed for each enzyme to identify possible controlling steps. This analysis suggested that PGAM, ALDO, GAPDH, and PPDK might be flux control steps, as they showed the lowest catalytic efficiencies. Anin vitroreconstruction of the final stages of glycolysiswas made to determine their flux control coefficients. Our results indicate that PGAM and PPDK exhibit high control coefficient values at physiological pH. [ABSTRACT FROM AUTHOR]