Expression, purification, and characterization of Leishmania donovani trypanothione reductase in Escherichia coli

Trypanothione reductase (TR) is an NADPH-dependent flavoprotein oxidoreductase central to thiol metabolism in all the trypanosomatids including Leishmania. The unique presence of this enzyme in trypanosomatids and absence in mammalian host make this enzyme an attractive target for the development of the antileishmanials. Complete open reading frame encoding trypanothione reductase from Leishmania donovani (Dd8 strain, causative agent of Indian visceral leishmaniasis) was cloned, sequenced, and expressed in Escherichia coli strain BL21 (DE3) as glutathione S-transferase fusion protein. The conditions were developed for overexpression of fusion protein in soluble form and purification of the recombinant protein to homogeneity. The recombinant LdTR was 54.68 kDa in size, dimeric in nature, and reduces oxidized trypanothione to reduced form. The kinetic parameters for trypanothione disulfide are K(m), 50 microM; k(cat), 18,181 min(-1); and k(cat)/K(m), 6.06x10(6) M(-1) s(-1). The yield of recombinant LdTR was approximately 16 mg/L bacterial culture and accounted for 6% of the total soluble proteins. The expressed protein was inhibited by known TR inhibitors as well as by SbIII, the known antileishmanial compound. This is the first report of large-scale production of any leishmanial TR in E. coli.     

Genetic and chemical analyses reveal that trypanothione synthetase but not glutathionylspermidine synthetase is essential for Leishmania infantum

Trypanothione is a unique and essential redox metabolite of trypanosomatid parasites, the biosynthetic pathway of which is regarded as a promising target for antiparasitic drugs. Synthesis of trypanothione occurs by the consecutive conjugation of two glutathione molecules to spermidine. Both reaction steps are catalyzed by trypanothione synthetase (TRYS), a molecule known to be essential in Trypanosoma brucei. However, other trypanosomatids (including some Leishmania species and Trypanosoma cruzi) potentially express one additional enzyme, glutathionylspermidine synthetase (GSPS), capable of driving the first step of trypanothione synthesis yielding glutathionylspermidine. Because this monothiol can substitute for trypanothione in some reactions, the possibility existed that TRYS was redundant in parasites harboring GSPS. To clarify this issue, the functional relevance of both GSPS and TRYS was investigated in Leishmania infantum (Li). Employing a gene-targeting approach, we generated a gsps^−/− knockout line, which was viable and capable of replicating in both life cycle stages of the parasite, thus demonstrating the superfluous role of LiGSPS. In contrast, elimination of both LiTRYS alleles was not possible unless parasites were previously complemented with an episomal copy of the gene. Retention of extrachromosomal LiTRYS in the trys^−/−/+TRYS line after several passages in culture further supported the essentiality of this gene for survival of L. infantum (including its clinically relevant stage), hence ruling out the hypothesis of functional complementation by LiGSPS. Chemical targeting of LiTRYS with a drug-like compound was shown to also lead to parasite death. Overall, this study disqualifies GSPS as a target for drug development campaigns and, by genetic and chemical evidence, validates TRYS as a chemotherapeutic target in a parasite endowed with GSPS and, thus, probably along the entire trypanosomatid lineage.     

Preparative enzymatic synthesis of trypanothione and trypanothione analogues

Trypanosomatids, the causative agents of several tropical diseases, have a unique thiol metabolism based on trypanothione [bis(glutathionyl)spermidine]. Enzymes of the pathway are attractive drug target molecules but the availability of trypanothione remains an obstacle. Here, we present a convenient method for the production of trypanothione and trypanothione disulfide in >200 mg quantities using a mutant of Crithidia fasciculata trypanothione synthetase in which Cys59 has been replaced by an alanine residue. The reagent costs less than 1% of the commercial price of trypanothione disulfide. The protocol also allows the synthesis of related glutathione conjugates. It will greatly facilitate the thorough analysis of this parasite’s metabolism and drug screening approaches against trypanothione-dependent enzymes.     

Trypanothione: A unique bis-glutathionyl derivative in trypanosomatids

Background

Trypanosomatids are early-diverging eukaryotes devoid of the major disulfide reductases – glutathione reductase and thioredoxin reductase – that control thiol-redox homeostasis in most organisms. These protozoans have evolved a unique thiol-redox system centered on trypanothione, a bis-glutathionyl conjugate of spermidine. Notably, the trypanothione system is capable to sustain several cellular functions mediated by thiol-dependent (redox) processes.

Scope of review

This review provides a summary of some historical and evolutionary aspects related to the discovery and appearance of trypanothione in trypanosomatids. It also addresses trypanothione's biosynthesis, physicochemical properties and reactivity towards biologically-relevant oxidants as well as its participation as a cofactor for metal binding. In addition, the role of the second most abundant thiol of trypanosomatids, glutathione, is revisited in light of the putative glutathione-dependent activities identified in these organisms.

Major conclusions

Based on biochemical and genome data, the occurrence of a thiol-redox system that is strictly dependent on trypanothione appears to be a feature unique to the order Kinetoplastida. The properties of trypanothione, a dithiol, are the basis for its unique reactivity towards a wide diversity of oxidized and/or electrophilic moieties in proteins and low molecular weight compounds from endogenous or exogenous sources. Novel functions have emerged for trypanothione as a potential cofactor in iron metabolism.

General significance

The minimalist thiol-redox system, developed by trypanosomatids, is an example of metabolic fitness driven by the remarkable physicochemical properties of a glutathione derivative. From a pharmacological point of view, such specialization is the Achilles' heel of these ancient and deadly parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Leishmania amazonensis trypanothione reductase: evaluation of the effect of glutathione analogs on parasite growth, infectivity and enzyme activity

Trypanothione reductase (TR) is a major enzyme in trypanosomatids. Its substrate, trypanothione is a molecule containing a tripeptide (L-glutamic acid-cysteine-glycine) coupled to a polyamine, spermidine. This redox system (TR/Trypanothione) is vital for parasite survival within the host cell and has been described as a good target for chemotherapy anti-Leishmania. The use of tripeptides analogs of glutathione would result in a decrease in trypanothione synthesis and as a consequence in TR activity. In this work, besides the enzyme potential inhibition, it also evaluated the influence of those analogs on parasite growth and on its infective capacity. The results showed a significant effect on parasite growth and infectivity and in addition TR activity was highly inhibited. These results are very promising, suggesting a potential use of those analogs as therapeutic drugs against experimental diseases caused by trypanosomatids.     

Leishmania Subtilisin Is a Maturase for the Trypanothione Reductase System and Contributes to Disease Pathology

Proteases are a ubiquitous group of enzymes that play key roles in the life cycle of parasites, in the host-parasite relationship, and in the pathogenesis of parasitic diseases. Furthermore, proteases are druggable targets for the development of new anti-parasitic therapy. The subtilisin protease (SUB; Clan SB, family S8) of Leishmania donovani was cloned and found to possess a unique catalytic triad. This gene was then deleted by gene knock-out, which resulted in reduced ability by the parasite to undergo promastigote to amastigote differentiation in vitro. Electron microscopy of SUB knock-out amastigotes revealed abnormal membrane structures, retained flagella, and increased binucleation. SUB-deficient Leishmania displayed reduced virulence in both hamster and murine infection models. Histology of spleens from SUB knock-out-infected hamsters revealed the absence of psammoma body calcifications indicative of the granulomatous lesions that occur during Leishmania infection. To delineate the specific role of SUB in parasite physiology, two-dimensional gel electrophoresis was carried out on SUB^−/− versus wild-type parasites. SUB knock-out parasites showed altered regulation of the terminal peroxidases of the trypanothione reductase system. Leishmania and other trypanosomatids lack glutathione reductase, and therefore rely on the novel trypanothione reductase system to detoxify reactive oxygen intermediates and to maintain redox homeostasis. The predominant tryparedoxin peroxidases were decreased in SUB^−/− parasites, and higher molecular weight isoforms were present, indicating altered processing. In addition, knock-out parasites showed increased sensitivity to hydroperoxide. These data suggest that subtilisin is the maturase for tryparedoxin peroxidases and is necessary for full virulence.     

The parasite-specific trypanothione metabolism of trypanosoma and leishmania

The bis(glutathionyl)spermidine trypanothione exclusively occurs in parasitic protozoa of the order Kinetoplastida, such as trypanosomes and leishmania, some of which are the causative agents of several tropical diseases. The dithiol is kept reduced by the flavoenzyme trypanothione reductase and the trypanothione system replaces in these parasites the nearly ubiquitous glutathione/glutathione reductase couple. Trypanothione is a reductant of thioredoxin and tryparedoxin, small dithiol proteins, which in turn deliver reducing equivalents for the synthesis of deoxyribonucleotides as well as for the detoxification of hydroperoxides by different peroxidases. Depending on the individual organism and the developmental state, the parasites also contain significant amounts of glutathione, mono-glutathionylspermidine and ovothiol, whereby all four low molecular mass thiols are directly (trypanothione and mono-glutathionylspermidine) or indirectly (glutathione and ovothiol) maintained in the reduced state by trypanothione reductase. Thus the trypanothione system is central for any thiol regeneration and trypanothione reductase has been shown to be an essential enzyme in these parasites. The absence of this pathway from the mammalian host and the sensitivity of trypanosomatids toward oxidative stress render the enzymes of the trypanothione metabolism attractive target molecules for the rational development of new drugs against African sleeping sickness, Chagas' disease and the different forms of leishmaniasis.     

Cytoplasmic localization of the trypanothione peroxidase system in Crithidia fasciculata

Tryparedoxin I (TXNI) and tryparedoxin peroxidase (TXNPx), novel proteins isolated from Crithidia fasciculata, have been reported to reconstitute a trypanothione peroxidase activity in vitro (Nogoceke, E.; Gommel, D. U.; Kiess, M.; Kalisz, H. M.; Flohé, L. Biol. Chem. 378:827-836; 1997). Combined with trypanothione reductase, they may form an NADPH-fueled trypanothione-mediated defense system against hydroperoxides in the trypanosomatids. In situ confocal microscopy of antibody-stained TXNI and TXNPx and electron microscopy of the immunogold labeled proteins revealed their colocalization in the cytosol. Insignificant amounts of the enzymes were detected in the nucleus and vesicular structures, whereas the kinetoplast and the mitochondrion are virtually free of any label. Comparison of the PCR product sequences obtained with genomic and cDNA templates rules out any editing typical of kinetoplast mRNA. Sequence similarities with any of the established maxicircle genes of trypanosomatids were not detectable. It is concluded that both, TXNI as well as TXNPx are encoded by nuclear DNA and predominantly, if not exclusively localized in the cytosol. Working in concert with trypanothione reductase, they can function as an enzymatic system that reduces hydroperoxides at the expense of NADPH without any impairment of the flux of reduction equivalents by cellular compartmentation.     

Thiol-based redox metabolism of protozoan parasites

The review considers redox enzymes of Plasmodium spp., Trypanosomatida, Trichomonas, Entamoeba and Giardia, with special emphasis on their potential use as targets for drug development. Thiol-based redox systems play pivotal roles in the success and survival of these parasitic protozoa. The synthesis of cysteine, the key molecule of any thiol metabolism, has been elucidated in trypanosomatids and anaerobes. In trypanosomatids, trypanothione replaces the more common glutathione system. The enzymes of trypanothione synthesis have recently been identified. The role of trypanothione in the detoxification of reactive oxygen species is reflected in the multiplicity of trypanothione-dependent peroxidases. In Plasmodium falciparum, the crystal structures of glutathione reductase and glutamate dehydrogenase are now available; another drug target, thioredoxin reductase, has been demonstrated to be essential for the malarial parasite.     

Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems

This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.     

The Dithiol Glutaredoxins of African Trypanosomes Have Distinct Roles and Are Closely Linked to the Unique Trypanothione Metabolism

Trypanosoma brucei, the causative agent of African sleeping sickness, possesses two dithiol glutaredoxins (Grx1 and Grx2). Grx1 occurs in the cytosol and catalyzes protein deglutathionylations with k_cat/K_m-values of up to 2 × 10⁵ m⁻¹ s⁻¹. It accelerates the reduction of ribonucleotide reductase by trypanothione although less efficiently than the parasite tryparedoxin and has low insulin disulfide reductase activity. Despite its classical CPYC active site, Grx1 forms dimeric iron-sulfur complexes with GSH, glutathionylspermidine, or trypanothione as non-protein ligands. Thus, contrary to the generally accepted assumption, replacement of the Pro is not a prerequisite for cluster formation. T. brucei Grx2 shows an unusual CQFC active site, and orthologues occur exclusively in trypanosomatids. Grx2 is enriched in mitoplasts, and fractionated digitonin lysis resulted in a co-elution with cytochrome c, suggesting localization in the mitochondrial intermembrane space. Grx2 catalyzes the reduction of insulin disulfide but not of ribonucleotide reductase and exerts deglutathionylation activity 10-fold lower than that of Grx1. RNA interference against Grx2 caused a growth retardation of procyclic cells consistent with an essential role. Grx1 and Grx2 are constitutively expressed with cellular concentrations of about 2 μm and 200 nm, respectively, in both the mammalian bloodstream and insect procyclic forms. Trypanothione reduces the disulfide form of both proteins with apparent rate constants that are 3 orders of magnitude higher than those with glutathione. Grx1 and, less efficiently, also Grx2 catalyze the reduction of GSSG by trypanothione. Thus, the Grxs play exclusive roles in the trypanothione-based thiol redox metabolism of African trypanosomes.     

Molecular characterization of the trypanothione reductase gene from Crithidia fasciculata and Trypanosoma brucei: comparison with other flavoprotein disulphide oxidoreductases with respect to substrate specificity and catalytic machanism

Trypanothione reductase belongs to the family of flavoprotein disulphide oxidoreductases that include glutathione reductases, dihydrolipoamide dehydrogenases and mercuric reductases. Trypanothione reductase and its substrate, trypanothione disulphide, are unique to parasitic trypanosomatids responsible for several tropical diseases. The crystal structure of the enzyme from Crithidia fasciculata is currently under investigation as an aid in the design of selective inhibitors with a view to producing new drugs. We report here the cloning and sequencing of the genes for trypanothione reductase from C. fasciculata and Trypanosoma brucei. Alignment of the deduced amino acid sequences with 21 other members of this family provides insight into the role of certain amino acid residues with respect to substrate specificity and catalytic mechanism as well as conservation of certain elements of secondary structure.     

Redox enzyme engineering: conversion of human glutathione reductase into a trypanothione reductase

The substrate specificity of the human enzyme glutathione reductase was changed from its natural substrate glutathione to trypanothione [N1,N8-bis(glutathionyl)spermidine] by site-directed mutagenesis of two residues. The glutathione analogue, trypanothione, is the natural substrate for trypanothione reductase, an enzyme found in trypanosomatids and leishmanias, the causative agents of diseases such as African sleeping sickness, Chagas disease, and Oriental sore. The rational bases for our mutational experiments were the availability of a high-resolution X-ray structure for human glutathione reductase with bound substrates, the active site sequence comparisons of human glutathione reductase and the trypanothione reductases from Trypanosoma congolense and Trypanosoma cruzi, a complementary set of mutants in T. congolense trypanothione reductase, and the properties of substrate analogues of trypanothione. Mutation of two residues, A34----E34 and R37----W37, in the glutathione-binding site of human glutathione reductase switches human glutathione reductase into a trypanothione reductase with a preference for trypanothione over glutathione by a factor of 700 using kcat/Km as a criterion.     

The thiol-based redox networks of pathogens: unexploited targets in the search for new drugs

Hydroperoxide metabolism in diverse pathogens is reviewed under consideration of involved enzymes as potential drug targets. The common denominator of the peroxidase systems of Trypanosoma, Leishmania, Plasmodium, and Mycobacterium species is the use of NAD(P)H to reduce hydroperoxides including peroxynitrite via a flavin-containing disulfide reductase, a thioredoxin (Trx)-related protein and a peroxidase that operates with thiol catalysis. In Plasmodium falciparum, thioredoxin- and glutathione dependent systems appear to be linked via glutaredoxin and plasmoredoxin to terminal thioredoxin peroxidases belonging to both, the peroxiredoxin (Prx) and glutathione peroxidase (GPx) family. In Mycobacterium tuberculosis, a catalase-type peroxidase is complemented by the typical 2-C-Prx AhpC that, in contrast to most bacteria, is reduced by TrxC, and an atypical 2-C-Prx reduced by TrxB or C. A most complex variation of the scheme is found in trypanosomatids, where the unique redox metabolite trypanothione reduces the thioredoxin-related tryparedoxin, which fuels Prx- and GPx-type peroxidases as well as ribonucleotide reductase. In Trypanosoma brucei and Leishmania donovani the system has been shown to be essential for viability and virulence by inversed genetics. It is concluded that optimum efficacy can be expected from inhibitors of the most upstream components of the redox cascades. For trypanosomatids attractive validated drug targets are trypanothione reductase and trypanothione synthetase; for mycobacteria thioredoxin reductase appears most appealing, while in Plasmodium simultaneous inhibition of both the thioredoxin and the glutathione pathway appears advisable to avoid mutual substitution in co-substrate supply to the peroxidases. Financial and organisational needs to translate the scientific progress into applicable drugs are discussed under consideration of the socio-economic impact of the addressed diseases.     

Rational design of peptide-based inhibitors of trypanothione reductase as potential antitrypanosomal drugs

Summary The rational design of ligands for the substrate-binding site of a homology-modelled trypanothione reductase (TR) was performed. Peptides were designed to be selective for TR over human glutathione reductase (GR). The design process capitalized on the proposed differences between the activesites of TR and human GR, subsequently confirmed by the TR crystal structure. Enzyme kinetics confirmed that forT. cruzi TR benzoyl-Leu-Arg-Arg-ß-naphthylamide was an inhibitor (K_i 13.8µM) linearly competitive with the native substrate, trypanothione disulphide, and did not inhibit glutathione reductase.     

Glyoxalase II of African trypanosomes is trypanothione-dependent

The glyoxalase system is a ubiquitous pathway catalyzing the glutathione-dependent detoxication of ketoaldehydes such as methylglyoxal, which is mainly formed as a by-product of glycolysis. The gene encoding a glyoxalase II has been cloned from Trypanosoma brucei, the causative agent of African sleeping sickness. The deduced protein sequence contains the highly conserved metal binding motif THXHXDH but lacks three basic residues shown to fix the glutathione-thioester substrate in the crystal structure of human glyoxalase II. Recombinant T. brucei glyoxalase II hydrolyzes lactoylglutathione, but does not show saturation kinetics up to 5 mm with the classical substrate of glyoxalases II. Instead, the parasite enzyme strongly prefers thioesters of trypanothione (bis(glutathionyl)spermidine), which were prepared from methylglyoxal and trypanothione and analyzed by high performance liquid chromatography and mass spectrometry. Mono-(lactoyl)trypanothione and bis-(lactoyl)trypanothione are hydrolyzed by T. brucei glyoxalase II with k(cat)/K(m) values of 5 x 10(5) m(-1) s(-1) and 7 x 10(5) m(-1) s(-1), respectively, yielding d-lactate and regenerating trypanothione. Glyoxalase II occurs in the mammalian bloodstream and insect procyclic form of T. brucei and is the first glyoxalase II of the order of Kinetoplastida characterized so far. Our results show that the glyoxalase system is another pathway in which the nearly ubiquitous glutathione is replaced by the unique trypanothione in trypanosomatids.     

Substrate specificity of the flavoprotein trypanothione disulfide reductase from Crithidia fasciculata

The substrate specificity of the trypanosomatid enzyme trypanothione reductase has been studied by measuring the ability of the enzyme to reduce a series of chemically synthesized cyclic and acyclic derivatives of N1,N8-bis(glutathionyl)spermidine disulfide (trypanothione). Kinetic analysis of the enzymatic reduction of these synthetic substrates indicates that the mutually exclusive substrate specificity observed by the NADPH-dependent trypanothione disulfide reductase and the related flavoprotein glutathione disulfide reductase is due to the presence of a spermidine binding site in the substrate binding domain of trypanothione reductase. Trypanothione reductase will reduce the disulfide form of N1-monoglutathionylspermidine and also the mixed disulfide of N1-monoglutathionylspermidine and glutathione. The Michaelis constants for these reactions are 149 microM and 379 microM, respectively. Since the disulfide form of N1-monoglutathionylspermidine and the mixed disulfide of N1-monoglutathionylspermidine and glutathione could be formed in trypanosomatids, the binding constants and turnover numbers for the enzymatic reduction of these acyclic disulfides are consistent with these being potential alternative substrates for trypanothione reductase in vivo.     

Cynaropicrin targets the trypanothione redox system in Trypanosoma brucei

In mice cynaropicrin (CYN) potently inhibits the proliferation of Trypanosoma brucei—the causative agent of Human African Trypanosomiasis—by a so far unknown mechanism. We hypothesized that CYNs α,β-unsaturated methylene moieties act as Michael acceptors for glutathione (GSH) and trypanothione (T(SH)₂), the main low molecular mass thiols essential for unique redox metabolism of these parasites. The analysis of this putative mechanism and the effects of CYN on enzymes of the T(SH)₂ redox metabolism including trypanothione reductase, trypanothione synthetase, glutathione-S-transferase, and ornithine decarboxylase are shown. A two step extraction protocol with subsequent UPLC–MS/MS analysis was established to quantify intra-cellular CYN, T(SH)₂, GSH, as well as GS-CYN and T(S-CYN)₂ adducts in intact T. b. rhodesiense cells. Within minutes of exposure to CYN, the cellular GSH and T(SH)₂ pools were entirely depleted, and the parasites entered an apoptotic stage and died. CYN also showed inhibition of the ornithine decarboxylase similar to the positive control eflornithine. Significant interactions with the other enzymes involved in the T(SH)₂ redox metabolism were not observed. Alongside many other biological activities sesquiterpene lactones including CYN have shown antitrypanosomal effects, which have been postulated to be linked to formation of Michael adducts with cellular nucleophiles. Here the interaction of CYN with biological thiols in a cellular system in general, and with trypanosomal T(SH)₂ redox metabolism in particular, thus offering a molecular explanation for the antitrypanosomal activity is demonstrated. At the same time, the study provides a novel extraction and analysis protocol for components of the trypanosomal thiol metabolism.     

The effect of buthionine sulfoximine on the growth of Leishmania donovani in culture

Changes in composition of the principal low molecular mass thiols of Leishmania donovani were monitored during the transformation of promastigotes, first to stationary phase metacyclic forms and then to amastigotes. No consistent variation in the thiol composition of the parasite which could account for the known increase in resistance of metacyclic and amastigote lifecycle forms to oxidant stress could be established. Amastigotes cultivated at 37 degrees C also produced ovothiol A, as judged by incorporation of radiolabel from [3-methyl]methionine and [14C]histidine, and the incorporation of radiolabel from [35S]cysteine into ovothiol A represented about 10-15% of the total label recovered in ovothiol A, glutathione and trypanothione. Amastigotes were less susceptible than promastigotes to the effects of the redox cyclers paraquat and menadione and grew in culture in the presence of up to 20 mM buthionine sulfoximine, which completely blocked the synthesis of glutathione and its spermidine conjugates. Glutathione and trypanothione biosynthesis is, therefore, not necessary for the replication of L. donovani amastigotes in culture. Inhibition of the formation of glutathione and trypanothione did not result in an upregulation of ovothiol A production.     

High throughput screening against the peroxidase cascade of African trypanosomes identifies antiparasitic compounds that inactivate tryparedoxin

In African trypanosomes, the detoxification of broad spectrum hydroperoxides relies on a unique cascade composed of trypanothione (T(SH)(2)), trypanothione reductase, tryparedoxin (Tpx), and nonselenium glutathione peroxidase-type enzymes. All three proteins are essential for Trypanosoma brucei. Here, we subjected the complete system to a high throughput screening approach with nearly 80,000 chemicals. Twelve compounds inhibited the peroxidase system. All but one carried chloroalkyl substituents. The detailed kinetic analysis showed that two compounds weakly inhibited trypanothione reductase, but none of them specifically interacted with the peroxidase. They proved to be time-dependent inhibitors of Tpx-modifying Cys-40, the first cysteine of its active site WCPPC motif. Importantly, gel shift assays verified Tpx as a target in the intact parasites. T(SH)(2), present in the in vitro assays and in the cells in high molar excess, did not interfere with Tpx inactivation. The compounds inhibited the proliferation of bloodstream T. brucei with EC(50) values down to <1 μM and exerted up to 83-fold lower toxicity toward HeLa cells. Irreversible inhibitors are traditionally regarded as unfavorable. However, a large number of antimicrobials and anticancer therapeutics acts covalently with their target protein. The compounds identified here also interacted with recombinant human thioredoxin, a distant relative of Tpx. This finding might even be exploited for thioredoxin-based anticancer drug development approaches reported recently. The fact that the T(SH)(2)/Tpx couple occupies a central position within the trypanosomal thiol metabolism and delivers electrons also for the synthesis of DNA precursors renders the parasite-specific oxidoreductase an attractive drug target molecule.