Drug target validation of the trypanothione pathway enzymes through metabolic modelling

Abstract

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

Deciphering the interplay between cysteine synthase and thiol cascade proteins in modulating Amphotericin B resistance and survival of Leishmania donovani under oxidative stress

“…In addition, a previous report describing kinetics of T(SH) 2 biosynthesis in Trypanosoma cruzi [80] also supports the role of γ -GCS and TryS proteins in counteracting oxidative stress conditions. This study suggested that main control flux is regulated by γ -GCS and TryS proteins, followed by spermidine transporter, TryR and T(SH) 2 -dependent thiol pathway enzymes [80]. CS may also play a crucial role in regulating the T(SH) 2 biosynthesis pathway because it provides cysteine for the biosynthesis of γ -glutamylcysteine and glutathione by γ -GCS and glutathione synthetase enzymes, respectively [6].…”

“…This suggests that either these isolates are different or canine isolated parasites differ from human isolates because antimony resistant isolates reported so far showed up-regulation of cytosolic isoforms of TXN and TXNPx [77], [78], [79]. In addition, a previous report describing kinetics of T(SH) 2 biosynthesis in Trypanosoma cruzi [80] also supports the role of γ -GCS and TryS proteins in counteracting oxidative stress conditions. This study suggested that main control flux is regulated by γ -GCS and TryS proteins, followed by spermidine transporter, TryR and T(SH) 2 -dependent thiol pathway enzymes [80].…”

Mathematical Modelling of Polyamine Metabolism in Bloodstream-Form Trypanosoma brucei: An Application to Drug Target Identification

“…Our polyamine model complements a recent attempt at modelling trypanothione () metabolism in the related parasite T. cruzi [6]. The T. cruzi model focuses on the glutathione synthesis branch and the redox cycle of .…”

“…These features have ensured that the polyamine pathway in T. brucei has been subject to investigation and details are available for enough of the enzymes to allow a mathematical model to be constructed. A recent attempt to model trypanothione () metabolism in Trypanosoma cruzi ( T. cruzi ) [6] also points to the value in modelling of this branch of metabolism in trypanosomatids.…”

Genetic Validation of Trypanosoma brucei Glutathione Synthetase as an Essential Enzyme

“…Polyamines are positively charged, flexible hydrocarbons that are essential for growth and proliferation of eukaryotic cells, with roles in transcription, translation, chromatin structure, and ion channel function (7)(8)(9)(10). Trypanosomes have evolved to use the polyamine spermidine in an additional role through conjugation with two molecules of glutathione to form the essential redox cofactor trypanothione [N1,N8-bis(glutathionyl)-spermidine] (11).…”