A kinetic model of trypanothione [T(SH) 2 ] metabolism in Trypanosoma cruzi was constructed based on enzyme kinetic parameters determined under near-physiological conditions (including glutathione synthetase), and the enzyme activities, metabolite concentrations and fluxes determined in the parasite under control and oxidizing conditions. The pathway structure is characterized by a T(SH) 2 synthetic module of low flux and low catalytic capacity, and another more catalytically efficient T(SH) 2 -dependent antioxidant ⁄ regenerating module. The model allowed quantification of the contribution of each enzyme to the control of T(SH) 2 synthesis and concentration (flux control and concentration control coefficients, respectively). The main control of flux was exerted by c-glutamylcysteine synthetase (cECS) and trypanothione synthetase (TryS) (control coefficients of 0.58-0.7 and 0.49-0.58, respectively), followed by spermidine transport (0.24); negligible flux controls by trypantothione reductase (TryR) and the T(SH) 2 -dependent antioxidant machinery were determined. The concentration of reduced T(SH) 2 was controlled by TryR (0.98) and oxidative stress ()0.99); however, cECS and TryS also exerted control on the cellular level of T(SH 2 ) when they were inhibited by more than 70%. The model predicted that in order to diminish the T(SH) 2 synthesis flux by 50%, it is necessary to inhibit cECS or TryS by 58 or 63%, respectively, or both by 50%, whereas more than 98% inhibition was required for TryR. Hence, simultaneous and moderate inhibition of cECS and TryS appears to be a promising multi-target therapeutic strategy. In contrast, use of highly potent and specific inhibitors for TryR and the antioxidant machinery is necessary to affect the antioxidant capabilities of the parasites. DatabaseThe glutathione synthetase gene sequences from the Ninoa and Queretaro strains have been submitted to the GenBank database under accession numbers HQ398240 and HQ398239, respectively Abbreviations

No abstract

Trypanothione (T(SH) 2 ) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi ; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH) 2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH) 2 synthesis by 60–74% and 15–31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH) 2 concentration, whereas TryS overexpression did not render an increase in T(SH) 2 levels as a consequence of high T(SH) 2 degradation. The peroxide reduction flux was controlled for 64–73% by TXN1, 17–20% by TXNPx and 11–16% by TryR. TXN1 and TryR overexpression increased H 2 O 2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH) 2 antioxidant pathway may compromise the parasite's viability and infectivity.

Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH) ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH) metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH) even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.

In the search for therapeutic targets in the intermediary metabolism of trypanosomatids the gene essentiality criterion as determined by using knock-out and knock-down genetic strategies is commonly applied. As most of the evaluated enzymes/transporters have turned out to be essential for parasite survival, additional criteria and approaches are clearly required for suitable drug target prioritization. The fundamentals of Metabolic Control Analysis (MCA; an approach in the study of control and regulation of metabolism) and kinetic modeling of metabolic pathways (a bottom-up systems biology approach) allow quantification of the degree of control that each enzyme exerts on the pathway flux (flux control coefficient) and metabolic intermediate concentrations (concentration control coefficient). MCA studies have demonstrated that metabolic pathways usually have two or three enzymes with the highest control of flux; their inhibition has more negative effects on the pathway function than inhibition of enzymes exerting low flux control. Therefore, the enzymes with the highest pathway control are the most convenient targets for therapeutic intervention. In this review, the fundamentals of MCA as well as experimental strategies to determine the flux control coefficients and metabolic modeling are analyzed. MCA and kinetic modeling have been applied to trypanothione metabolism in Trypanosoma cruzi and the model predictions subsequently validated in vivo. The results showed that three out of ten enzyme reactions analyzed in the T. cruzi anti-oxidant metabolism were the most controlling enzymes. Hence, MCA and metabolic modeling allow a further step in target prioritization for drug development against trypanosomatids and other parasites.