Drug target validation of the trypanothione pathway enzymes through metabolic modelling

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 γ-glutamylcysteine synthetase (γECS) 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, γECS 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 γECS 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 γECS 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.