Trypanosomatid Diseases (Molecular Routes to Drug Discovery) || Trypanothione-Based Redox Metabolism of Trypanosomatids

9Trypanothione-Based Redox Metabolism of TrypanosomatidsMarcelo A. Comini and Leopold Flohe

AbstractThe intracellular redox balance largely depends on thiol-dependent reactions, and ispivotal to survival and proliferation of all living organisms. Most eukaryotes andprokaryotes use glutathione and thioredoxin complemented with pertinent reduc-tases to maintain their reducing capacity. Strikingly, trypanosomatids lack glutathi-one reductase and thioredoxin reductase, and have developed a unique thiol-basedredox metabolism that differs substantially in some of its molecular entities. Thesystem that controls the thiol redox homeostasis in these parasites comprisesN1,N8-bis(glutathionyl)spermidine called trypanothione, the NADPH-dependent trypano-thione reductase, multipurpose oxidoreductases called tryparedoxins, other redoxins,and tryparedoxin-dependent peroxidases of different classes. Moreover, trypanothioneitself, or assisted by enzymes such as tryparedoxins, glutaredoxins, glyoxalases, andmethionine sulfoxide reductase, acts as reductant, cofactor or ligand in a plethora of keybiological reactions encompassing DNA replication, antioxidant defense, assembly ofironsulfur clusters, and detoxication of ketoaldehydes, xenobiotics, and heavymetals. In consequence, several components of the trypanothione system proved tobe critical for the viability and virulence of Trypanosoma and Leishmania species. Theabsence of trypanothione-independent backup systems in the parasites and lackingconservation of trypanothione and related reactions in the mammalian hosts renderseveral system components promising drug targets. This chapter updates functionalaspects of the trypanothione metabolism and ranks drug target candidates for thedevelopment of novel therapies against trypanosomiasis and leishmaniasis.

ThiolRedoxMetabolismofTrypanosomatids:ABriefHistoricalOverview

Trypanosomatids are exposed to reactive oxygen and nitrogen species, which aremetabolic byproducts or stem from the hosts defense mechanisms. An uncontrolledproduction or insufcient detoxication of these compounds alters the cellular redoxbalance, which may cause a wide range of damage to macromolecules, ultimatelyleading to cell death.Redox-active thiol groupspresent inproteinsor low-molecular-mass

Corresponding Author

Trypanosomatid Diseases: Molecular Routes to Drug Discovery, First edition. Edited by T. Jger, O. Koch, and L. Floh.# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

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compounds play a key role in reducing these oxidants or acting as redox sensors toregulate diverse cellular functions [1]. Long before the components of the thiol redoxsystem of trypanosomatids were identied, the relevance of this metabolism forpathogenic trypanosomatids was recognized by chemotherapeutic approaches employ-ing compounds known to directly (e.g., buthionine sulfoximine, nifurtimox, melarso-prol, and benznidazole) [28] or indirectly (e.g., antimonials) [9,10] interferewith cellularredox balance. Historically, the elucidation of the thiol-dependent redox system oftrypanosomatids resembles apuzzle game.Someof the componentshadbeen identiedlong ago, but their interconnection remained obscure for decades. Indeed, soon after thepresence of an unusual thiol-polyamine conjugate (i.e., mono(glutathionyl)spermidine(Gsp)) had been reported in Escherichia coli [11], Fairlamb and Cerami [12] laid thefoundation stone in trypanosomatid redoxmetabolismby the discovery of trypanothione(T(SH)2), the bis(glutathionyl)-conjugate of spermidine (Spd). T(SH)2 is almost exclu-sively found in the order Kinetoplastida and there represents the main low-molecular-mass thiol [13,14]. One year after the discovery of T(SH)2, its biosynthesis from Spd andGSH [15] and regeneration from its oxidized cyclic disulde form [16] was claried inprinciple, and in the following year the enzyme responsible for T(SH)2 regeneration,trypanothione reductase (TR), was in-depth characterized and crystallized [17]. Incontrast, it took more than a decade to identify the enzymatic entities responsible forthe removal of hydroperoxides, the oxidoreductase tryparedoxin (TXN) [18,19] and twotypes of peroxidases (see next sections) and, more importantly, to recognize theirdependence on T(SH)2 [18,20]. Furthermore, earlier experimental evidence supportingthe lack in trypanosomatids of the major reductases that control the thiol redoxhomeostasis in most eukaryotes and prokaryotes (i.e., glutathione reductase andthioredoxin reductase) was conrmed by sequencing the parasites genomes [2123].Thenal elucidation of T(SH)2 biosynthesiswas only achieved in the last decade [2427].Not surprisingly, the trypanothione system did not remain as simple as seeminglyclaried towards the end of the last century, but became enriched with multipleisoenzymes of mostly unknown specialization [2831] and with unexpected rami-cations, such as ribonucleotide reduction [32], ironsulfur cluster (ISC) metabo-lism [3335], and methionine sulfoxide reduction [36] (Figure 9.1). Finally, thedevelopment of fast and reliable genetic tools enabling the functional characteri-zation of different components of this system and, thus, validating their putativeessentiality for parasites survival and/or virulence was lagging behind and stillremains a challenge [3739].These and other deciencies in basic knowledge and experimental tools

represented major obstacles in the exploitation of the trypanothione system forthe development of novel drugs. Progress in recent years, however, has substan-tially enriched related knowledge: (i) by RNA interference (RNAi) and othertechniques of inverse genetics, most of the system components could be demon-strated to be of vital importance, mostly for T. brucei; (ii) subcellular location andmetabolic context has been claried to a large extent; (iii) structures wereelucidated for at least one representative of each system component; and (iv)reliable molecular models could be generated for the orthologs of other species.Collectively, available data (Table 9.1 and references therein) now promises a safe

168j 9 Trypanothione-Based Redox Metabolism of Trypanosomatids

Figure 9.1 Trypanothione metabolism.Scheme summarizing metabolic pathwaysinvolved in synthesis, reduction, and utilizationof trypanothione. Metabolites and enzymesinvolved in: (i) glutathione (GSH) uptake andbiosynthesis are depicted in yellow with GSHa,c-glutamylcysteine synthetase; GSHb,glutathione synthetase; (ii) polyamine uptakeand biosynthesis are shown in blue with ARG,arginase; ODC, ornithine decarboxylase;AdoMetDC, S-adenosinylmethioninedecarboxylase; SpdS, spermidine synthase; (iii)trypanothione synthesis is shown in light greenwith GspS, mono(glutathionyl)spermidinesynthetase; TryS, trypanothione synthetase; (iv)trypanothione reduction is highlighted in greenolive with TR, trypanothione reductase; TS2,trypanothione disulfide; G6PDH, glucose-6-phosphate dehydrogenase; G6P, glucose-6-phosphate; and other metabolites (6PG,6-phosphogluconate; R5P, ribose-5-phosphate)of the pentose phosphate pathway (box); (v)trypanothione-dependent pathways with

trypanothione as cofactor (e.g., removal ofmethylglyoxal by glyoxalase system and thiolligand of iron-sulfur clusters by glutaredoxins;1-C-Grxs, monothiol glutaredoxins) ornucleophile (e.g., addition to xenobiotics suchas electrophile drugs in reactions catalyzed ornot by trypanothione S-transferase, TST). Thetrypanothione-dependent redoxin-mediated(shown in magenta) pathways involve thosecatalyzed by the main oxidoreductase oftrypanosomatids, tryparedoxin (TXN) (e.g.,reduction of distinct types of peroxidases (Prx)that decompose different hydroperoxides(ROOH) to the corresponding alcohol (ROH)and water, reduction of methionine sulfoxidereductase (MSR) that regenerates methionine(Met) from its sulfoxide form (Met-SO), andreduction of ribonucleotide reductase (RnR),which produces desoxinucleotides (dNTPs)from nucleotides (NTPs), and dithiolglutaredoxins (2-C-Grxs) (e.g., reduction ofglutathione disulfide (GSSG) and protein/GSH-mixed disulfides (Prot-S-SG)).

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prediction how to most effectively hit the system without affecting vital targets in thehost, thus paving the way to create more efcacious and safer trypanocidal drugs.

Trypanothione Biosynthesis

The biosynthesis of T(SH)2 demands the supply of GSH and Spd, metabolites that,depending on the trypanosomatid species, are synthesized de novo as inmammals ortaken up from the extracellular medium. The nal step involves the addition of twoGSH molecules to Spd a reaction catalyzed by one or, in certain trypanosomatidsperhaps, by two related enzymes (Figure 9.1). This link between the polyamine andGSH metabolism is unique to trypanosomatids and, a priori therefore, offersexceptional chances for drug development.

Glutathione Synthesis

The rst and rate-limiting step in GSH biosynthesis is catalyzed by c-glutamylcysteinesynthetase (GshA), which ligates L-glutamate and L-cysteine to produce c-glutamylcys-teine. Glutathione synthetase (GshB) then catalyzes the reaction between c-glutamyl-cysteine and L-glycine to yield GSH. Both reactions are ATP-dependent. Chemicalinhibition of GshA with the irreversible inhibitor L-buthionine-S,R-sulfoximine (BSO)provided the rst evidence for the indispensability of the thiol metabolism in Africantrypanosomes [6]. Subsequent studies conducted in different trypanosomatid speciesand using BSO as model drug conrmed the chemotherapeutic potential of GSHsynthesis and its contribution to drug sensitivity (see sections below) [4244,103]. Theimportance of this pathway was further validated by genetic approaches. RNAi-mediated downregulation of GshA in procyclic T. brucei depleted the cellular poolsof GSH and T(SH)2 with subsequent parasite death [40]. L. infantum mutants withimpaired levels of GshA were more susceptible to oxidative and xenobiotic stress, andshowed decreased survival inside activated macrophages [45]. Kinetic characterizationof recombinant T. brucei GshA suggested the existence of important moleculardifferences with respect to the mammalian enzyme [104,105]. However, analysis ofthe TbGshA crystal structure disclosed a high similarity with its human counterpart,shedding doubt on TbGshA being a useful target for specic inhibitors [41]. Moreover,the ability of T. brucei to incorporate GSH from the extracellular medium implies thatan effective therapy should also target the uptake mechanism (transporter), as, forinstance, BSO does [40]. Although the TriTryp genomes contain sequences coding forputative GshB [2123], the gene products await characterization, before their potentialas targets for selective inhibition can be forecasted.

Polyamine Synthesis, Salvage, and Uptake

The acquisition of Spd varies among different trypanosomatids (Figure 9.1).African trypanosomes depend entirely on de novo synthesis of Spd [68], due to

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their negligible polyamine uptake capability, which probably remainedundeveloped due to the scarce polyamine availability in the hosts bloodstream.In contrast, T. cruzi is auxotrophic for polyamines, and relies on high-afnitytransporters to scavenge putrescine and cadaverine from the host [106108].Leishmania parasites are more versatile being capable of both synthesis andtransport-mediated uptake of polyamines from the extracellular environment[109111].The biosynthetic pathway for polyamine production starts from arginine and

involves its decarboxylation. The genomes of TriTryps harbor sequences encod-ing putative arginases, but none of them has yet been functionally characterized.Nevertheless, a role in polyamine synthesis can be envisaged for arginase fromLeishmania species and T. brucei in the light of their metabolic output (ureaexcretion) or needs (extreme dependence on de novo polyamine synthesis),respectively. The occurrence of an arginase gene in T. cruzi is probably irrelevantfor polyamine metabolism, since this parasite lacks ornithine decarboxylase(ODC) [22,112,113]. In contrast, ODC is present and indispensable for Leish-mania and T. brucei grown in polyamine-decient media [48,52,54]; reviewed in[114,115]. Recent phylogenetic studies propose that parasites from the T. bruceiclade replaced, probably by horizontal gene transfer, their protozoan ODC geneby one of vertebrate origin [116]. Despite the high similarity of sequence (around60% identity) and structure between mammalian and TbODC [27,49,51], thetherapeutic approach based on an irreversible inhibitor (diuoromethylorni-thine (DFMO)) successfully survived laboratory and clinical validations [46,47]and, for two decades already, has become a therapeutic routine to treat bothearly- and late-stage sleeping sickness caused by T. b. gambiense. The effective-ness of DFMO towards T. b. gambiense infections is, in part, explained by thelonger half-life of the trypanosomal ODC as compared to the human ortholog(18 h versus 830min, respectively), and the resulting shortage in polyamines inthe parasite that causes (i) cell growth arrest, (ii) decreased synthesis of macro-molecules (e.g., components of the glycoprotein coat that allows the parasite toescape from host immune recognition), (iii) aberrant methylation due toaccumulation of decarboxylated S-adenosylmethionine (dcAdoMet), and (iv)limited antioxidant capacity due to trypanothione depletion. The failure ofDFMO to act on the more aggressive T. b. rhodesiense has been ascribed tothe higher activity (3-fold) and shorter half-life (around 4 h) of its ODC ascompared to its counterpart in T. b. gambiense [117], and, probably, to a reduceddrug uptake [118]. Recent studies have identied the amino acid transporterTbAAT6 as the solely responsible for laboratory-induced DFMO resistance intrypanosomes [119]. This nding together with the increasing failures reportedfor DFMO treatment of human African trypanosomiasis [119] raises concernsabout the mid-term success of a currently implemented melarsoprolDFMOcombined therapy. Nevertheless, this situation might be reverted if some of therecently discovered potent and selective T. brucei ODC inhibitors overcomesuccessfully clinical trials [53].


Also the ODC of L. donovani exhibits a long-half life (greater than 20 h) [120,121].However, Leishmania species are mostly tolerant to DFMO, which in part has beenascribed to increased ODC activity in DFMO-resistant strains [122], but also to theintracellular life style of Leishmania, which favors polyamine uptake from the hostcell [110,123]. On this background, the development of polyamine-targeting inhibi-tors to treat leishmaniasis remains a challenge, since a successful strategy wouldlikely have to target both transport-mediated uptake and endogenous synthesis.AdoMetDC is expressed in all three trypanosomatids, shares a low sequence

identity with the mammalian enzymes (around 30%), and has been shown to beessential for L. donovani promastigotes and T. brucei species by means of genetic orchemical approaches [5559,61,62]. Themechanism of cell death in parasites treatedwith AdoMetDC inhibitors appears rather to be associated with aberrant methyla-tion due to AdoMet accumulation than with polyamine depletion [57,58]. Earlypromising therapeutic data on AdoMetDC inhibitors in experimental T. b. rhode-siense infection [56,60] has recently been corroborated by novel more potent drugcandidates [63,64]. Resolution of the three-dimensional structure of KinetoplastidaAdoMetDC and a thorough comparison with the human ortholog [124] mightreanimate related drug design efforts.Spermidine synthase (SpdS) has been biochemically characterized, and shown to

be indispensable for the growth of T. brucei, L. donovani, and T. cruzi in the presenceand the absence of exogenous Spd, respectively [52,6569]. Inhibition of SpdS maybe a suitable monotherapeutic option for T. brucei, because of its dependency on denovo polyamine synthesis, while in trypanosomatids that rely on polyamine uptakethe transporters have to be targeted simultaneously. Unfortunately, potent andspecic inhibitors have not yet been identied for this enzyme [65], and thesequence similarities withmammalian SpdS (4050% identities) are not particularlyencouraging.Many eukaryotes also use an alternative pathway to synthesize putrescine that

depends on the concerted action of arginine decarboxylase (ADC), which producesagmatine from arginine, followed by agmatine hydrolysis by agmatinase to nallyyield putrescine and urea. Agmatinase-like sequences are present in the genome ofthe trypanosomatids [2123]. However, the corresponding agmatinase activitieshave not yet been detected [125], and the submicromolar concentration of agmatinein mammalian tissues and uids [126] would require highly efcient transporters tofulll the parasites needs for polyamine synthesis in the absence of ADC.

Trypanothione Synthesis

The rst step in the biosynthesis of T(SH)2 consists in the ATP-dependent additionof GSH to one of the amino groups of Spd, preferentially to the N1, to form Gsp.Depending on the genetic background of the trypanosomatid this reaction can becatalyzed by either glutationylspermidine synthetase (GspS) or trypanothionesynthetase (TryS). The addition of a second GSH molecule to Gsp is a reaction

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exclusively catalyzed by TryS. These proteins have no counterpart in mammals, theirclosest and ancestral relative being a GspS from c -proteobacteria [127]. Although thegenomes of T. cruzi and L. brasiliensis contain putative genes for GspS (www.tritrypdb.org), GspS activity has not yet been reported for any of these species. Therole of a GspS in pathogenic trypanosomatids remains uncertain, since TryS fromseveral species have been shown to produce T(SH)2 from GSH and Spd. In fact,T. brucei [70,128], L. major [27], and probably L. infantum (Castro and Tomas,unpublished observations) rely entirely on TryS for T(SH)2 biosynthesis irrespectiveof the presence of a GspS. The gene encoding GspS, thus, may be on the way to belost, if it does not meet specic requirement other than T(SH)2 synthesis (seebelow).GspS and TryS are composed of two functional domains harboring opposite

catalytic activities. The N-terminal domain presents a typical Cys-protease motif thatconfers hydrolytic activity to convert Gsp and T(SH)2 back to the building blocks,GSH and Spd. The C-terminal synthetase domain belongs to the ATP-grasp enzymefamily (i.e., GSHb, AlaAla synthetase, and other ATP-dependent CN ligases) [129].The biological role of the amidase activity of these enzymes remains obscure. Ininfective T. brucei, the amidase activity of TryS does not appear to confer anyadvantage in respect to in vivo survival [72]. However, the strict conservation ofcatalytic residues in the amidase domain of GspS and TryS from different trypa-nosomatids is suggestive of a peculiar biological role. Otherwise, this activity wouldsimply maintain a costly futile cycle of T(SH)2 synthesis and degradation. Theamidase activity may, for instance, contribute to Spd salvage in Leishmania parasites,since ODC-null mutants have decreased levels of putrescine and T(SH)2, but not ofSpd [54], suggesting that either Spd transport or back conversion from Gsp orT(SH)2 may sufce to maintain Spd homeostasis. In contrast, the synthetase activityof TryS is considered to be essential. TryS downregulation by RNAi or conditionalknockout in T. brucei resulted in depletion of Gsp and T(SH)2 and accumulation ofGSH and, at the biological level, in growth arrest, impaired antioxidant capacity, andinfectivity, and ultimately in death. Although the essentiality of TryS has so far beendemonstrated for African trypanosomes only [70,72,128], the enzyme may beconsidered to be equally indispensable for species that lack a GspS. However,the phenotype of TryS depletion or inhibition may substantially differ in trypano-somatids expressing a GspS, since at least in vitro Gsp has been shown to be areasonable substrate and reducing agent for TR and TXN, respectively [130,131]. InL. major, T. cruzi, and T. brucei TryS presents a non-uniform cytosolic distribution([(27)] Medeiros and Comini, unpublished observations). In this respect, the T(SH)2requirement of specic organelles such as the mitochondrion still poses intriguingquestions [132,133].The crystal structure of L. major TryS has been solved at 2.8 A

[71]. Modeling of

substrates and analogs to the presumed active site revealed well-structured bindingsites for ATP and GSH, while the one for Spd appears less characteristic. Inagreement with functional data [26], the substrate orientations comply with amechanism by which GSH is rst activated by phosphorylation at its glycinecarboxylate for conjugation to N1 of Spd. Interestingly, the Spd binding site extends


into a second binding site for GSH, which suggests that theN1-glutathionylated Spdformed in the initial step of T(SH)2 synthesis ips around and is then re-bound in away that its still free N8 can also be glutathionylated (Koch and Flohe, unpublished).The detailed information on mechanism and substrate binding sites predict anexcellent druggability of TryS and certainly will prove to be of outstanding impor-tance for rational drug design. Before these structural and mechanistic insights, thedesign of TryS inhibitors was only possible on the basis of substrate similarity [134136]. Inhibitors thus obtained showed indeed convincing trypanocidal activities withcultured parasites, but their peptidic character remained an obstacle for furtherdevelopment. More recently, novel drug-like TryS inhibitors have been identied bymeans of high-throughput drug screening [73,74], which conrms the druggabilityof TryS.Collectively, therefore, TryS proved to be a most attractive drug target by several

criteria: (i) uniqueness of sequence, (ii) low abundance, (iii) genetic proof of essen-tiality, and (iv) demonstrated druggability by chemical validation [26,31,70,73,74]. Theonly disadvantage of this target that emerges from ongoing drug design efforts is itsspeciesspecic susceptibility for inhibition. In fact, differences in IC50 of more thanthree orders of magnitude have been observed for the very same inhibitor for TryS ofdifferent trypanosomatid species [137,138].

Trypanothione Recycling

The maintenance of steady-state levels of dihydrotrypanothione (reduced form) iswarranted in trypanosomatids by the presence of an NADPH-dependent avoen-zyme named TR. Although sharing homology with mammalian glutathione reduc-tase (around 40% protein identity), TR has a remarkable specicity for the disuldeform of trypanothione and Gsp [130]. The protein has been shown to be essential forinfective T. brucei [80], and to be important for the proliferation of L. donovani andL. major inside activated macrophages [77,78]. The crystal structure has been solvedfor TR from T. cruzi [17,75,76,79,139], L. infantum [83], and T. brucei [81,82], also incomplex with ligands and inhibitors (for review, see [140] and references therein). Arecent thorough comparative study reveals that the T. brucei and T. cruzi homologsare almost indistinguishable in terms of kinetics and active site structure, whichfacilitates the design of a promiscuous compound targeting the enzymes of bothspecies [141]. Since its identication in the early 1990s, TR has been investigated asthe preferred drug target candidate of trypanothione-dependent metabolism and,despite structural similarity with several mammalian avoproteins, numerousspecic TR inhibitors with good in vitro activities have been synthesized (reviewedin [140,141]). Nevertheless, none of these inhibitors was potent enough in vivo towarrant drug development. A likely explanation of this disappointing situation isoffered by the conditional knockout of TR in T. brucei [80]. Although this geneticapproach convincingly demonstrated the essentiality of TR, it simultaneouslyrevealed that a more than 95% inhibition of the enzyme is required to criticallyaffect the parasites redox homeostasis. Evidently, such degree of enzyme inhibition

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is not easily sustained long enough by any kind of reversible inhibitor to achieve anefcient trypanocidal action in vivo.The alternate option to interfere with T(SH)2 regeneration, inhibition of the

oxidative part of the pentose phosphate pathway, has not yet been intensivelyexplored. The NADPH required for regeneration of T(SH)2 is considered to belargely derived from glucose-6-phosphate dehydrogenase and 6-phosphoglunoco-lactone dehydrogenase of the pentose phosphate shunt. The role of carbohydratemetabolism in trypanosomatids and the potential use of related enzymes as drugtargets are discussed in Chapters 7 and 16 of this volume.

Trypanothione Utilization

Redoxin-Independent Functions of Trypanothione

Trypanothione has been shown to be largely more efcient than GSH in: (i) reducingsmall molecules such as dehydroascorbate, hydrogen peroxide, and peroxynitrite[142144], (ii) reducing protein disuldes such as those of Trx, TXN, RnR,and glutaredoxins [18,19,32,33,35,145,146], (iii) scavenging radiation-induced radi-cals [147,148], (iv) acting as cofactor of the trypanosomatid glyoxylase system[149,150], and ligand of ISCs bound to T. brucei 1-C-Grx1 and 2-C-Grx1[3335] and of nitrosyl-iron complexes [151], and (v) reacting with electrophilicxenobiotics [9]. Some of these functions will be shortly discussed here.

Trypanothione-Dependent Ligation of IronNO Complexes and ISCs

Nitric oxide (NO) is a highly reactive gas that can be produced by endothelial andimmune cells. If not neutralized rapidly, NO can modify proteins (nitrosylation)[152] or, in the presence of O2 , can originate peroxynitrite a highly reactive andcytotoxic oxidant that mediates intracellular killing of T. cruzi [153,154]. T(SH)2 hasbeen shown to form a stable dinitrosyl-iron complex (DNIC) with a 600-fold higherefciency than GSH. Interestingly, the complex formed with T(SH)2, even atmillimolar concentrations, did not inactivate TR in comparison to the potentinhibitory effect exerted by micromolar concentrations of dinitrosyl-digluta-thionyl-iron complex on human glutathione reductase [155]. Formation of theDNICT(SH)2 complex was demonstrated in T. brucei and L. infantum exposedto NO, which led to the proposal for a protective role of T(SH)2 in defense againstmacrophage-derived reactive nitrogen species [151].As for other cells, ISCs are important structural elements and redox-active

cofactors of ironsulfur proteins of trypanosomatids [156,157]. A role in ironhomeostasis has recently been recognized for certain dithiol and most monothiolglutaredoxins from distantly related organisms [158], which was, at least in part,linked to their capacity to assemble an ISC at the expense of their active-site cysteineand GSH as additional low-molecular-mass thiol ligand. Analogous proteinISC


holocomplexes have been reported for T. brucei 2-C-Grx1 and 1-C-Grx1 withthe novelty that in these proteins T(SH)2 efciently replaced GSH as thiol ligand[3335,102]. The ISC assembled by 2-C-Grx1 is supposed to play a regulatory role inenzyme activity with the holo-form of the protein being redox inactive [33]. 1-C-Grx1fullls an indispensable role in the mitochondrion of T. brucei [34], probablyparticipating in the nal step of ironsulfur protein biogenesis that involves thetransfer of the bound ISC to apo-proteins [102]. Recent structural characterization ofT. brucei 1-C-Grx1 has pinpointed important differences with its human counterpart[34]. Moreover, these studies revealed that even in the absence of scaffold proteins,T(SH)2 alone can coordinate and transfer an ISC to 2-C-Grx1 [33,34] raising thepossibility for a new physiological role for the low-molecular-mass dithiol as aputative ISC carrier or ligand in trypanosomes [35].

Trypanothione-Dependent Detoxification Reactions and Drug Resistance

Methylglyoxal is a highly reactive and mutagenic byproduct of glycolysis that demandsdedicated enzymes for its efcient detoxication. Trypanosomatids metabolize thisoxoaldehyde through different pathways. T. cruzi, L. major, and L. donovani have a fullyactive glyoxalase system that uses T(SH)2 as a reaction cofactor [98,100,101,150,159161], while T. brucei lacks a complete glyoxalase system; the glyoxalase I gene ismissingand glyoxalase II appears to be dispensable [99,100]. An alternative pathway tometabolize methylglyoxal relies on the concerted action of a NADPH-dependentmethylglyoxal reductase that produces lactaldehyde, which is further converted intoL-lactate by a NAD(P)H-dependent lactaldehyde dehydrogenase. Both activities weredetected in cell extracts of trypanosomatids [100,160,162], but the enzymatic entitieshave not yet been isolated. The problem of oxoaldehyde detoxication is the subject ofChapter 8 in this volume. Based on metabolic modeling, the authors conclude that thetrypanosomatid oxoaldehydemetabolism is likely too robust to be critically inhibited bytargeting a single enzyme [163].The role of T(SH)2 in the detoxication of the trivalent arsenic compound

melarsoprol, the trivalent antimonial triostam, and the nitroheterocyclic compoundsnifurtimox, megazol, and benznidazole, has been studied intensively, and the hintstoward T(SH)2-dependent drug resistance are numerous [8,103,128,164169]. Themechanisms by which T(SH)2 interferes with different drugs may comprise (i)direct binding of trivalent arsenicals and likely antimonials, (ii) spontaneous or (iii)enzyme-catalyzed conjugation with electrophiles, but (iv) also the TXN/TXN-dependent Prx (TXNPx)-mediated removal of hydroperoxides generated fromredox-cycling drugs, as outlined in the next section. To what extent drug resistanceis due to direct or enzyme-mediated actions of T(SH)2 appears to be far from clear.Certainly, electrophilic drugs or metabolites may be conjugated by T(SH)2 directly,but more likely this detoxication is accelerated by a trypanothione-S-transferase, anenzyme found in different trypanosomatids and showing similarity with theeukaryotic elongation factor 1B (eEF1B) [170,171]. Also genes encoding aneEF1B homolog and a putative glutathione-S-transferase-related glutaredoxin are

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present in the T. brucei genome [21], but none of these enzymes have beencharacterized sufciently to justify an attempt to overcome drug resistance by atargeted inhibition.Also the seemingly straightforward binding of arsenicals and antimonials by

T(SH)2 turns out to be more complex than anticipated. For sure, trivalent arsenicalsand antimonials can covalently bind to the dithiol T(SH)2, but it does not imply thatsuch mode of action is the only or the therapeutically relevant one. They couldequally attack any of the exposed thiols in TR, as discussed [164], or the thiols of thevarious thiol peroxidases or of redoxins, which are the prototype of proteinspreferentially labeled with arsenicals [172,173]. In line with this reasoning, resist-ance to arsenicals, benznidazole, and antimonials was found to be associated withnatural or experimental elevation of TXNPx in Leishmania species [91,174177].However, recent studies on Leishmania spp. have revealed a more complex scenariofor the mechanism of drug resistance generated in laboratories versus thoseoccurring in the eld [178,179].

Redoxin-Dependent Functions of Trypanothione

Without the assistance of thiol/disulde oxidoreductases, the ow of reducingequivalents from T(SH)2 to its different protein targets would likely be too slow tocope with cellular demands. Accordingly, trypanosomatids encode three types ofdithiol oxidoreductases that are characterized by a CxxC active-site motif, belong tothe thioredoxin (Trx) superfamily and are now commonly called redoxins:(i) typical Trx(s) with an active site WCGPCK motif, (ii) glutaredoxin(s) with aCPYC (exceptionally CQFC) motif [33], and (iii) tryparedoxin(s) characterized by aWCPPCR motif. The latter have so far only been detected in trypanosomatids. Atypical Trx was found in T. brucei and T. cruzi, but proved to be functionallydispensable in T. brucei by inverse genetics [89,90,180,181]. Moreover, the TriTrypgenomes completely lack sequences encoding for a thioredoxin reductase homologgene, suggesting that Trx is a phylogenetic relict in trypanosomatids. In fact, allfunctions known of Trx in other organisms appear to be exerted by the trypano-somatid-specic TXN.Twodithiolglutaredoxins (2-C-Grxs)were identied inT. cruzi [182]andT.brucei [33].

Although displaying different efciencies and specicities, cytosolic Grx1 andmitochondrial Grx2 from T. brucei were able to not only reduce GSSG, but alsoproteinGSHmixeddisuldes (deglutathionylation) aswell as inter- or intramoleculardisuldes [33]. Not surprisingly for organisms that lack glutathione reductaseand strictly depend on T(SH)2 to maintain the intracellular thiol/disuldemilieu, the trypanosomatid 2-C-Grxs evolved as catalysts for the reduction of GSSGby T(SH)2 [33]. Although 2-C-Grxs cannot compete with TXN in the reduction ofprotein disuldes, except the former would have specic protein targets, they largelysurpass TXN in their capacity to deglutathionylate proteins [33]. Protein glutathiony-lation is discussed as a reversible modication of thiols that protects proteins againstloss of function associated with overoxidation of cysteines [152,183]. Cysteine-specic


glutathionylation of recombinant forms of different trypanosomatid proteins hasrecently been reported [184]. However, and regardless of the physiological role of 2-C-Grxs in trypanosomes (e.g., kinetic control of GSSG reduction or protein deglutathio-nylation), none of theGrx activities appear critical for the viability of infective forms ofT. brucei cultured in vitro [33].In the parasite cytosol, TXN is the most abundant redoxin [18,26]. Despite

belonging to the Trx superfamily, several functional and structural studiescorroborate the preference of the enzyme for T(SH)2 and Gsp, and the negligiblecapacity of GSH to act as reducing substrate [19,20,131,185187]. In contrast toTrxs and 2-C-Grxs, downregulation of cytosolic TXN was detrimental for theproliferation of bloodstream T. brucei grown under optimal conditions [85,86].TXN was equally indispensable for L. infantum where knockout of both chromo-somal TXN1 alleles was only possible upon complementation with an episomalcopy of the gene [87]. TXN has been shown to mediate important cellularprocesses such as the synthesis of deoxyribonucleotides [32], the decompositionof hydroperoxides [18,85,86,94,131,188,189], and the repair of (methionine)oxidized proteins [36]. By these and other suggested roles, TXN supports parasitevirulence [13,87,190]. Altogether, this highlights the dominant role of the parasite-specic redoxin TXN in mastering the trypanothione-dependent intracellularredox homeostasis.

Hydroperoxide Metabolism

In trypanosomatids, TXNs as well as peroxiredoxin-type peroxidases (Prx) andcysteine-homologs of the classical selenocysteine-containing glutathione peroxi-dase-type proteins (GPx), complemented in certain species with a heme-typeascorbate peroxidase [191,192], form a most complex antioxidant defense system(for extensive reviews see [28,29,193195], and Chapters 10 and 11 of this volume).TXNs, Prxs, and GPxs occur in multiple isoforms that display discrete differences insubstrate specicities and are localized in different subcellular compartments. Thelink between TXN and peroxidases was rst disclosed in Crithidia fasciculata [18],and then found to be common to all trypanosomatids [131,196198]. The novelredoxin, thereby, emerged as the missing factor responsible for the reduction ofhydroperoxides by the vaguely dened trypanothione-dependent peroxidase activityof trypanosomatids [18], which were later recognized to belong to the Prx type. Theincreased sensitivity towards hydrogen peroxide or the organic tert-butyl hydro-peroxide exhibited by bloodstream T. brucei with a low content of TryS and TXNfurther conrmed the exclusive dependence of the hydroperoxide metabolism oftrypanosomatids on the T(SH)2/TXN couple [70,86].Noticeably, trypanosomatids are endowed with Prxs only from the typical 2-Cys

class [199] that are characterized by the formation of an intermolecular disuldeupon oxidation by hydroperoxide and by assembling into large quaternary structures(decamers and stacks of decamers) [92,200]. Different isoforms of TXNPx have beenshown to be important for parasite survival and virulence [85,93,201203]. Their


biochemical, kinetic, and biological features are treated elsewhere in this volume(Chapters 10 and 11).TXN also substitutes for Trx in the reduction of GPxs. The unexpected redoxin

specicity of GPx-type proteins was rst detected for a glutathione peroxidasefrom Plasmodium falciparum [204], and soon after in various plants, yeasts, insects,bacteria, and nally by Wilkinson et al. [205] and Hillebrand et al. [198] inkinetoplasts. It is likely more common to this type of proteins than the name-giving GPx activity of the mammalian prototype GPx1 [206]. The redoxin-specicGPxs are characterized as monomeric non-selenium GPxs having a second cysteinelocated in a exible loop that forms an intramolecular disulde bridge with theactive-site (or peroxidatic) cysteine upon oxidation by a hydroperoxide [9597,207],thus presenting a typical substrate structure for a redoxin-type disulde reductase[208]. Similarly to GPx from other organisms, the parasite proteins showedspecicity for reducing lipid hydroperoxides [94,188,195]. RNAi-mediated genesilencing depleted transcripts from all three gene copies present in the genomeof T. brucei, and revealed the indispensability of GPx-type peroxidases for parasitegrowth [188]. Recently, targeted gene replacement (knockout) of GPx-type peroxi-dases demonstrated that the cytosolic isoforms of these proteins were majordeterminants for cell viability [94]. Interestingly, the detrimental effect caused bythe lack of cytosolic GPx activity could be reverted by cultivating the GPx-decientparasites in the presence of Trolox, a water-soluble version of a-tocopherol and well-known hydrophobic antioxidant, thus conrming that the main function of theseproteins is to provide protection against lipid peroxidation and membrane damage.Knockout of the third GPx-type isoform localized in the parasite mitochondrionrevealed an increase in cardiolipin oxidation, but only a transitory growth arrest [94].

Redoxin and DNA Synthesis

Ribonucleotide reductase (RnR) is the enzyme that catalyzes the reduction of the20-hydroxyl group of ribonucleotides to the corresponding deoxyribonucleotides andthus is indispensible for DNA synthesis [209]. RnRs require Trx or Grx as reducingcosubstrate in practically all organisms [210,211]. Trypanosomatid RnR had for longbeen measured with non-physiological thiols or with T(SH)2. Finally, cytosolicTbTXN was shown to be a much more efcient reductant of RnR than any otherthiol previously applied and, thus, is likely the physiological RnR reductant [32].Later attempts to replace TXN as RnR substrate by TbTrx [212] or 2-C-Grxs [33] didnot challenge this conclusion, since they were less active. In line with the assump-tion that TXN is the relevant substrate of RnR, T. brucei depleted in TXN ortrypanothione displayed a decreased proliferation rate under normal culture condi-tions [70,86], whereas knockout of Trx and RNAi of cytosolic Tb 2-C-Grx1 remainedwithout any obvious phenotype [33,89]. It is, however, hard to decide to what extentgrowth retardation in TXN- or T(SH)2-decient T. brucei results from deoxynucleo-tide shortage or a disturbed redoxmetabolism, since the RnR is under redox control,being inhibited by oxidized trypanothione [32]. RnR remains as an attractive drug


target candidate out of the redoxin-dependent enzymes. Orthologs of this enzyme inother species have attracted considerable interest, particularly in the context of anti-cancer drug research, but so far not for the design of trypanocidal drugs.

New Putative Functions for TXN and Peroxidases

Methionine sulfoxide reductase (MSR) is a ubiquitous enzyme that catalyzes thereduction of methionine sulfoxide to methionine. MSR is considered to be part ofthe antioxidant armamentarium of cells by participating in the second line ofdefense that includes the repair of oxidized proteins [213]. The reaction mechanismis mediated by redox active thiols: subtraction of a single oxygen atom frommethionine sulfoxide by a nucleophilic cysteine of MSR leads to formation of asulfenic acid on the thiol and reduced methionine; a second cysteine from the sameMSR subunit attacks the sulfenic acid with concomitant formation of an intra-molecular disulde, which is typically reduced by Trx(s). Recent work has reportedon the characterization of genes encoding for putative MSR type A in T. cruzi(TcMSR10 and TcMSR180) and T. brucei (TbMSR) [36]. The recombinant proteinsfrom all three isoforms displayed specicity for L-Met(S) sulfoxide and cytosolic T.cruzi TXN I as substrate. Active expression of the proteins was detected only in thereplicative forms of T. cruzi (e.g., epimastigotes and amastigotes) and in both lifestages of T. brucei. A 15- to 20-fold overexpression of TcMSR10 in non-infectiveepimastigotes conferred a 2-fold resistance against H2O2, supporting a role of theprotein in antioxidant protection. Although the biological relevance of MSR forparasite survival remains to be elucidated this work adds a new target to the list ofT(SH)2/TXN-dependent functions in trypanosomatids.Admittedly, the complexity of the trypanothione-dependent hydroperoxide metab-

olism may be underestimated when only regarded as a defense system that protectsthe parasites against the hostile environment created by the hosts innate immuneresponse. The substrates of TXNPxs and GPxs such as H2O2, lipid hydroperoxides,or peroxynitrite (for reviews, see [214,215]) are no longer considered just as toxiccompounds responsible for oxidative cell damage, but are increasingly recognized assignaling molecules [1,183,216,217]. Like the GPx-type ORP1 of Saccharomycescerevisiae [218] and the Prx-type TPx1 of Schizosaccharomyces pombe [219], a TXNPxof C. fasciculata was also reported to act as sensor for H2O2 and, as oxidizedperoxidase, to oxidize a transcription factor, here the universal mini circle sequencebinding protein implicated in replication of mitochondrial DNA [220]. The role ofTXN in this context would be to prevent signaling by reducing the oxidized peroxidaseand to reverse the signaling event by regenerating the reduced transcription factor.So far, however, the in vivo relevance of this regulatory concept could not bedemonstrated, since knockdown of the mitochondrial TXNPx in T. brucei [85], aswell as knockout inL. infantum [88], did not affect the proliferation rate of theparasites.The reaction mechanism common to redoxins attack of a (protein) disulde by

means of their exposed active-site cysteine residue followed by further thiol disuldeexchange can be used to identify TXN targets in the form of disulde-linked


heterodimers, if the redoxins coreacting (resolving) cysteine is mutated to serine[207,221]. By means of this technology, several putative reaction partners ofTcTXN1C43S could be trapped from T. cruzi [190] and T. brucei ([13] and Cominiunpublished), inter alia proteins involved in cysteine biosynthesis (cystathionase),methionine/adenine salvage (methylthioadenosine phosphorylase), and proteinsynthesis/degradation (eIF4AI, a subunit of the eukaryotic translation initiationfactor eIFaF and ubiquitin-activating enzyme e1). Although further validations ofthese interactions are required, these results open up novel perspectives for furthercellular functions of TXN and ultimately of T(SH)2.

Redoxins and Peroxidases as Drug Targets?

In general, redoxins are considered to bepleiotropic, whichmeans they are designed bynature to interact with multiple partners without being entirely unspecic. The mostprominent example of pleiotropism is represented by Trx, which, apart from being acosubstrate of RnR and peroxidases, is implicated as a transducer or terminator insignaling cascades, as a universal disulde reductant and, extracellularly, as a kind ofcytokine [210,217,222,223]. Its trypanosomal counterpart, TXN, appears not tomake anexception to this rule: as outlined above, it can interact covalently with RnR, structurallydistinct peroxidases, new protein partners engaged in different metabolic processes aand with its peptidic substrate T(SH)2. This functional diversication is hard tounderstand on the basis of a highly specic enzyme substrate interaction accordingto the lock and keymodel and, indeed, TXNstructures donot reveal any characteristicsubstrate-binding pockets. Instead, substrate interaction studies suggest that substraterecognition is essentially basedonelectrostatic interactions [185,187,199,224]. Recently,however, high-throughput screening against the components of the hydroperoxide-detoxifying cascade of African trypanosomes (i.e., TR, TXN, GPx and T(SH)2) witharound80000 chemicals identied a few systeminhibitors [225]. Surprisingly, themostactive compoundswere time-dependent inhibitors ofTXN that reacted irreversiblywithits exposed N-terminal active-site cysteine. Although the molecules (e.g., thienopyr-imidine-4-ones and purine-2,6-diones) interacted also with recombinant human Trxthey displayed an at least greater than 10-fold cytotoxic potency against infectiveT. bruceithan towards mammalian HeLa cells. These results may again place TXN into thepreviously discussed rank of useful targets [26].Also, GPx-type proteins do not have any characteristic binding pocket for their

reducing substrates and, with the exception of mammalian GPx1, are not particu-larly specic enzymes. As far as investigated, their interaction with substrates is alsodominated by weak electrostatic attraction [208]. In vitro studies with knockout celllines of T. brucei rule out a moonlighting function for the trypanosomatid GPxs, aswas observed with mammalian and yeast orthologs [216,218,226228]. In contrast,their crucial role appears to be restricted to the detoxication of lipid hydroperoxidesand, hence, in protecting parasites against membrane damage [94]. The druggabilityof trypanosomatid non-selenium GPx may, indeed, be questioned based on thenegative results obtained in a recent high-throughput screening approach [225].


Pleiotropism is also a hallmark of Prxs. Long before the discovery of theirperoxidase nature, they were identied in different biological contexts [229,230],and moonlighting, here between peroxidase and chaperone function, appears tobe a common feature [88]. Thus far, TXNPx inhibitors have not been reported andthe design of specic ones appears to be a highly challenging task. Nevertheless,synthetic (conoidin A) and natural (adenanthin) compounds have recently beenreported to inhibit PrxII of Toxoplasma gondii and/or mammalian PrxI and PrxII,respectively [231,232]. These compounds might therefore be considered as rststructural scaffolds for developing specic Prxs inhibitors.In respect of target selection, it can be stated that cTXN, cTXNPx, and the GPx-

type TXNPx were shown to be essential by inverse genetics for survival ofTrypanosoma and/or Leishmania species (Table 9.1 and references therein), andknockout of the mTXNPx in L. infantum at least impaired virulence in an animalinfection model [88]. However, all these proteins share a more or less pronouncedpleiotropism. In line with this functional versatility, they are devoid of any well-structured substrate-binding pocket, where a high-afnity inhibitor could beaccommodated with a specicity that reliably excludes cross-reaction with mamma-lian homologs. In short, Prxs and GPx-type peroxidases are not easily druggable.

Conclusions

The trypanothione system is indeed a unique feature of trypanosomatids, and it is ofoutstanding importance for the parasites viability and virulence. Accordingly, itsexploitation for therapeutic intervention merits consideration. The uniqueness ofthe system, however, does not imply that all of its components are unique nor are itsindividual components equally essential. If target druggability is also taken intoaccount as an additional criterion for target selection, the following conclusionsappear justied:

Trypanosomatid glyoxalases and S-transferases should be disregarded as drugtarget because of lacking evidence of essentiality.

Redoxins and peroxidases may be disregarded as preferred drug targets becauseof lack of essentiality, uniqueness, and/or poor druggability.

Enzymes of polyamine synthesis and RnR deserve more attention, but selectivityof inhibition will remain a challenge.

TR as target for reversible inhibitors disappointed, but irreversible inhibitionremains a realistic option, if adequate selectivity can be achieved.

To consider GspS as a drug target appears premature with regard to its stillobscure biological role.

TryS is the target of choice because of the uniqueness of its sequence andstructure, low abundance, genetic support of essentiality, and structural evidenceas well as chemical proof of druggability. Moreover, TryS shares with TR theimportant target criterion of controlling the metabolic ux through the entirepathway [163,233].

Conclusions j185

Acknowledgments

M.A.C. acknowledges Agencia Nacional de Investigacion e Innovacion (grantInnova Uruguay, agreement DCI-ALA/2007/19.040 between Uruguay and theEuropean Commission) for nancial support.

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Trypanosomatid Diseases (Molecular Routes to Drug Discovery) || Trypanothione-Based Redox Metabolism of Trypanosomatids