Trypanothione reductase from Trypanosoma cruzi

Abstract

Trypanothione reductase of Trypanosoma cruzi is a key enzyme in the antioxidant metabolism of the parasite. Here we report on the enzymic and pharmacological properties of trypanothione reductase using glutathionylspermidine disulfide as a substrate.1. Both pH optimum (7.5) and the ionic strength optimum (at 30 mM) are unusually narrow for this enzyme. 40 mM Hepes, 1 mM EDTA, pH 7.5 was chosen as a standard assay buffer because in this system the k,,,/K, ratio had the highest values for both natural substrates, glutathionylspermidine disulfide (2.65 x lo6 M-' s-' 1 and trypanothione disulfide (4.63 x lo6 M-' s f l 1.2. Using the standardized assay, trypanothione reductase and the phylogenetically related host enzyme, human glutathione reductase, were studied as targets of inhibitors. Both enzymes, in their NADPH-reduced forms, were irreversibly modified by the cytostatic agent, 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU). Nifurtimox, the drug used in the treatment of Chagas' disease, is a stronger inhibitor of glutathione reductase (Ki = 40 pM) than of trypanothione reductase (ICso = 200 pM).3. Of the newly synthesized trypanocidal compounds [Henderson, G. B., Ulrich, P., Fairlamb, A. H., Rosenberg, I., Pereira, M., Sela, M. & Cerami, A. (1988) Proc. Natl Acad. Sci., 85, 5374-53781 a nitrofuran derivative, 2-(5 -nitro -2-furanylmethy1idene)-N,W-[1,4-piperazinediylbis (1,3 -propanediyl)]bishydrazinecarboximidamide tetrahydrobromide, was found to be a better inhibitor for trypanothione reductase (Ki = 0.5 pM) than for glutathione reductase (ICso = 10 pM). A naphthoquinone derivative, 2,3-bis[3-(2-amidinohydrazono)-butyl]-l,4-naphthoquinone dihydrochloride, turned out to be both an inhibitor = 1 pM) and an NADPHoxidation-inducing substrate (K, = 14 pM). This effect was not observed with human glutathione reductase. Such compounds which lead to oxidative stress by more than one mechanism in the parasite are promising starting points for drug design based on the three-dimensional structures of glutathione and trypanothione reductases. Enzymes. Trypanothione reductase (EC 1.6.4.-); glutathione reductase (EC 1.6.4.2). donovani) and oriental sore (Leishmania tropica) are caused by these protozoa.In contrast to all other known eukaryotes [l, 21 Trypanosomatidae do not possess the flavoenzyme glutathione reductase that is responsible for a high intracellular glutathione/ glutathione disulfide ratio. The thiol metabolism of trypanosomes and leishmanias is based on conjugates between glutathione and spermidine, namely trypanothione [3] and glutathionylspermidine [4]. These thiols are kept in the reduced state by trypanothione reductase which catalyzes the following reactions: trypanothione disulfideTrypanothione reductase is an FAD-cystine oxidoreductase which has been purified from the insect parasite Crithidia fasciculata [5] and, in crystalline form, from T . cruzi [6]. The DNA sequence is known for the enzyme from T. congolense [7].In vitro studies on trypanothione reductase are hampered by the fact that the natural dis...

Role of Δ1-Pyrroline-5-Carboxylate Dehydrogenase Supports Mitochondrial Metabolism and Host-Cell Invasion of Trypanosoma cruzi

“…For example, NADH might promote oxidative phosphorylation, generating the proton-driven force in the form of an electrochemical potential that can drive the synthesis of ATP (101). Moreover, NADPH serves as a cofactor for trypanothione reductase (102). Indeed, we have observed that P5C can serve as a viable energy source in the ATP production, which was supported by our data showing that antimycin A inhibits this process.…”

Pharmacokinetics of Hydroxymethylnitrofurazone, a Promising New Prodrug for Chagas' Disease Treatment

“…The literature showed that the inhibition of the trypanothione reductase was a possible mechanism of action of nitrofurazone (22,23,24); Trossini and coworkers (16) showed cruzipain inhibition and an increase of 30% in affinity for the enzyme. In addition, the most interesting and important point is the simplicity of the NFOH synthesis and its high yield (10), suggesting a very cheap compound for chemical scale-up.…”

8-Methoxy-naphtho[2,3-b]thiophen-4,9-quinone, a non-competitive inhibitor of trypanothione reductase

“…After the initial work of Henderson and co-workers (1988) naphthoquinone derivatives were further investigated for their action on TR (Jockers-Scherubl et al 1989, Salmon-Chemin et al 2000. In a previous work we reported the in vitro effect of several naphthothiophenquinone derivatives against epimastigote and trypomastigote forms of T. cruzi and the inhibition of the recombinant enzyme trypanothione reductase (Zani et al 1997).…”

“…Using either rational or empiric approaches, different classes of compounds, including peptides and peptoid, substituted polyamines, quinacrine and acridine analogues, 2-aminodiphenylsulfides, phenothiazine derivatives, substituted piperazines and nitrofuran derivatives, were studied for their inhibitory potential (see reviews by Werbovetz 2000, Augustyns et al 2001, Schmidt & KrauthSiegel 2002. Investigation of natural products, although not so extensive, disclosed interesting inhibitors such as ajoene from garlic (Gallwitz et al 1999), bisbenzylisoquinoline alkaloids (Fournet et al 1998(Fournet et al , 2000, and polyamines such as lunarine (Bond et al 1999) and kukoamine (Ponasik et al 1995).After the initial work of Henderson and co-workers (1988) naphthoquinone derivatives were further investigated for their action on TR (Jockers-Scherubl et al 1989, Salmon-Chemin et al 2000. In a previous work we reported the in vitro effect of several naphthothiophenquinone derivatives against epimastigote and trypomastigote forms of T. cruzi and the inhibition of the recombinant enzyme trypanothione reductase (Zani et al 1997).…”