2016 Conference on Computational Modelling with COPASI
Manchester Institute of Biotechnology, 12th – 13th May, 2016
1 - Instituto Nacional de Cardiologia, Mexico
Keywords: antioxidant metabolism, glutathione, trypanothione, parasite, drug target, Trypanosoma
BACKGROUND: Trypanosoma cruzi is the protist parasite that causes human American trypanosomiasis, a disease endemic of Latin American countries. In trypanosomatids, the trypanothione, a conjugate of two glutathione and one polyamine moieties, is the main antioxidant metabolite; hence the trypanothione-based antioxidant enzymatic machinery replaces the function of the glutathione system in mammals. The aim of this work is to identify the enzymes that mainly control the trypanothione metabolism in Trypanosoma cruzi by applying the quantitative analyses of kinetic modeling and the fundamentals of Metabolic Control Analysis.
METHODS: Kinetic models of the trypanothione-synthesis and trypanothione-dependent peroxide detoxification pathways were constructed using the GEPASI/COPASI platform using (i) the kinetic parameters of the recombinant pathway enzymes determined under near-physiological conditions, and (ii) the enzyme activities in the cells. Model validation was established by its ability to simulate the metabolite concentrations and fluxes of the in vivo pathways. The in silico predictions regarding the pathway flux-control distribution were evaluated in the parasites in supplementation experiments with thiol-molecules and polyamines.
RESULTS: The models could robustly simulate the fluxes and metabolite concentrations found in the parasites. The models indicated that gamma-glutamylcysteine synthase > trypanothione synthase >>> polyamine supply were the main controlling steps of trypanothione synthesis. Supplementation of parasites with cysteine and GSH, but not with spermidine or putrescine, increased the trypanothione pool which was in agreement with the flux-control distribution obtained by modeling. In the trypanothione-peroxide detoxification system, tryparedoxin was the main controlling enzyme, which is in agreement with pathway reconstitution data that indicated the tryparedoxin/tryparedoxin-peroxidase redox pair fully controlled the pathway flux, with negligible control exerted by trypanothione reductase.
CONCLUSIONS: The most controlling steps of the trypanothione metabolism in T. cruzi were identified. The results indicated that inhibition of either gamma-glutamycysteine synthetase, trypanothione synthetase or tryparedoxin will have much stronger adverse effects on the parasite antioxidant defense than inhibition of low-controlling enzymes such as trypanothione reductase, a popular protein for drug-target studies.