The therapeutic potential of inhibitors of the trypanothione cycle

Review

Ashley Publicationswww.ashley-pub.com

1. Introduction

2. Trypanothione cycle

3. Trypanothione reductase

4. Tryparedoxins

5. Conclusion and expert opinion

Monthly Focus: Anti-infectives

The therapeutic potential of inhibitors of the trypanothione cycleClaudius D’Silva† & Sylvie DaunesDepartment of Chemistry & Materials, The Manchester Metropolitan University,John Dalton Building, Chester Street, Manchester, M1 5GD, UK

There is an urgent need for new drugs in the treatment of human Africantrypanosomiasis, Chagas’ disease and leishmaniasis. This article provides anoverview of current drugs, formulations and their deficiencies. Targets forthe design of new drugs and the rational provided for targeting enzymes ofthe trypanothione cycle are described. Biochemical aspects of the cycle andthe currently investigated target trypanothione reductase are discussed asare the several classes of inhibitors and their in vitro potencies. Evidence isprovided for considering the tryparedoxins as a new target for antiprotozoalchemotherapy and a summary of glutathione-based inhibitors with signifi-cant in vitro activity is reported.

Keywords: chemotherapy, glutathione, leishmaniasis, protozoan diseases, Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, trypanothione, tryparedoxin, inhibitors, trypanothione reductase

Expert Opin. Investig. Drugs (2002) 11(2):217-231

1. Introduction

Blood-sucking insects transmit various forms of fatal parasitic protozoan diseases,both in the Old and New Worlds. Amongst these, the pathogenic parasitesbelonging to the order Kinetoplastida cause three main groups of mammaliandiseases. African trypanosomes (e.g., Trypanosoma brucei) cause sleeping sickness,South American trypanosomes (e.g., Trypanosoma cruzi) cause Chagas’ diseasewhilst Leishmania parasites cause a range of diseases (leishmaniasis). Kala-azar(visceral leishmaniasis) is caused by Leishmania donovani, oriental sore byLeishmania tropica and mucocutaneous leishmaniasis by Leishmania braziliensis.Vaccination is not a solution in the case of African trypanosomes due to antigenicvariations in the VSG coat proteins of these parasites [1], therefore chemotherapyremains the major form of treating and controlling this disease, despite recentsetbacks due to resistance [1-2]. The drugs used today are few in number and manyare strain specific due to differences in metabolism [3-5]. Many of the first line drugsused in the treatment of human African trypanosomiasis (sleeping sickness or HAT)[6] such as pentamidine, suramin, the organoarsenical drugs (melarsoprol [Arsobal®,MelB]) and those used against leishmaniasis, for example pentamidine, theorganoantimonial drugs (sodium stibogluconate [Pentostam®]) and meglumineantimoniate (Glucantime®) were developed decades ago and are now inadequatewith respect to potency, toxicity and safety [7] (Figure 1). In the case of Mel B, whichis used to treat late stage infections of T.b. rhodesiense, the side effects include acutereactive encephalopathy (which can result in paralysis) in 10% of cases, braindamage and death in ≤ 5% of treated patients [6].

African trypanosomes require spermidine for the biosynthesis of trypanothione,the main dithiol defence against oxidants, toxic heavy metals and probably xenobi-otics. Trypanothione biosynthesis [8] involves conjugation of glutathione with sper-

2002 © Ashley Publications Ltd ISSN 1354-3784 217

The therapeutic potential of inhibitors of the trypanothione cycle

Figure 1. Structure of current antiplastic drugs.

NH2

NH2

O

O

NH2

NH2

Pentamidine

+

+

SAs

S

N

N

N

NH

NH2

NH2

OH

Melarsoprol

NH

O

NH

O

NH

O

n

n = 2

Suramin

O

CHOH

O

O

COO-

CHOH

O

CHOH

O

O

COO-

CHOH

Sb

OH

Sb

O3Na+

Sodium stibogluconate

CH2NHCH3

OH

OH

OHH

OHH

CH2OH

Sb

CH2OH

O

HO

HOH

HOH

CH2NHCH3

H+

Meglumine antimoniate

SO3-Na+

SO3-Na+

Na+SO3-

midine by glutathionylspermidine synthetase to form mono-glutathionylspermidine and then with glutathione to formtrypanothione by trypanothione synthetase. The enzymesornithine decarboxylase (ODC), S-adenosylmethionine syn-thetase, S-adenosylmethionine decarboxylase and spermidinesynthetase [8] are collectively involved in spermidine biosyn-thesis. Enzymes of the trypanothione and spermidine biosyn-thetic pathways are therefore potential targets for the designof antiparasitic drugs, depending on turnover and bioavaila-bility.

Drugs and drug formulations developed over the past dec-ade (Figure 2) include eflornithine (DL-α-difluoromethylorni-thine (DFMO), Ornidyl™), a selective irreversible inhibitor(suicide substrate) of ODC and the only drug apart frommelarsoprol licensed for the treatment of late-stage HAT aftermanifesting itself in the central nervous system (CNS) [6].DFMO shows excellent penetration of the CNS and has been

successfully used to treat late-stage T.b. gambiense infectionsbut is ineffective against T.b. rhodesiense, probably due to dif-ferences in metabolism of S-adenosylmethionine [4] as well asthe presence of high concentrations of ODC [5]. As DFMO isa substrate of ODC, it requires high dose regimes of 400 mg/kg per day over 14 days, which is partly responsible for thehigh cost of treatment. Side effects include a 40 - 50% risk ofdeveloping leukopoenia and anaemia.

The treatment of Chagas’ disease is dependent upon theuse of the drug 2-nitroimidazole benznidazole (Radanil ),introduced in 1971, and nifurtimox (Lampit ), the latter’spresent availability a courtesy of Bayer Leverkusen. Benznida-zole is thought to act by covalent reaction of the partiallyreduced nitro group of the drug with macromolecules [7]. Thedrug is effective in the acute phase of the disease, althoughreports of failure have appeared due to the sensitivity of para-sitic populations throughout Central and South America to

218 Expert Opin. Investig. Drugs (2002) 11(2)

D’Silva & Daunes

the drug. Side effects of this drug include anorexia, vomiting,peripheral polyneuropathy and allergic dermopathy.

Leishmania and T. cruzi [9], like fungi, possess membranesrich in ergosterol to which polyene drugs such as amphoter-icin B preferentially bind. Amphotericin B (Figure 2) is anantifungal agent which as a unilamellar liposomal formulationAmbisome exhibits reduced toxicity, this allows its use inhigh dose regime treatments of systemic mycoses and visceralleishmania (VL). Use of this formulation proved effectiveagainst antimony resistant VL in the Mediterranean, Sudanand India [10]. Other commercial formulations of this druginclude Amphocil , an amphotericin B colloidal dispersioneffective in the treatment of VL in Brazil, [11] and Abelcet anamphotericin B lipid complex [12].

The aminoglycoside paromomycin (PM) (Figure 2) alsoknown as aminosidine is an antibiotic with broad antiproto-zoal activity which, as a commercial formulation (15% PMplus 12% methyl benzenethonium chloride), is effectiveagainst cutaneous leishmania (CL) [13] but has side effects,including irritation. Injectable formulations of PM have beenused in the treatment of VL in Europe, India and Africa butspecies dependent sensitivity has been observed [14].

Other compounds under investigation include allopurinol,bis-triazole derivatives, miltefosine and licochalcone A [11].

The spread of resistance against several of these compoundsin many countries has highlighted the need for a new generationof drugs. Focussed drug discovery over the past two decades hasconcentrated on the identification and characterisation of differ-ent biochemical and molecular targets. Several targets have beenidentified and some validated by gene knockout experiments[15]. The targets investigated include the sterol biosyntheticpathway in T. cruzi, ODC in T. brucei, S-adenosylmethionine[16], trypanothione reductase and nucleoside phosphotransferase

in Trypanosoma and Leishmania spp., glycosomal enzymes [17]

such as triose phosphate isomerase of T. brucei, cysteine pro-teases in T. cruzi and Leishmania, folate metabolism, DNAtopoisomerase [18, 19], the purine salvage pathway, metalloprotei-nases, kinases and phospholipases of Leishmania, N-myristoyltransferase [20, 21] and ribonucleotide reductase of T. brucei [22].Several of these enzymes and pathways are common to all thepathogenic trypanosomatids, which opens up the possibilitythat a clinically useful drug, active against all these parasitescould be developed. One such unique enzyme system for drugdevelopment is the trypanothione cycle.

2. Trypanothione cycle

A major biochemical difference between parasites of the orderKinetoplastida and nearly all other eukaryotes, as well asprokaryotes, is that their main thiols are covalent conjugates ofglutathione and spermidine principally glutathionyl-spermi-dine, N1,N8-bis(glutathionyl)spermidine (trypanothione,T(SH)2) and N1,N9-bis(glutathionyl)aminopropylcadaverine(homotrypanothione) [23]. The flavoenzyme trypanothionereductase (TR [TS2 + NADPH + H+ → T(SH)2 + NADP+])maintains the thiol in the reduced form as opposed to glutath-ione reductase (GR; GSSG + NADPH + H+ → 2GSH +NADP+), the analogous enzyme present in eukaryotes.

In contrast to mammalian enzymes, Trypanosomes and Leish-mania have iron-containing superoxide dismutases but lack glu-tathione peroxidase [24]. The function of glutathione peroxidasein these parasites is replaced by the tryparedoxin/tryparedoxinperoxidase enzyme system which is of broader specificity but canutilise peroxides and alkylhydroperoxides as substrates at a lowerefficiency. In the case of South American trypanosomes (T. cruzi)recent studies have shown that the tryparedoxin/tryparedoxin

Figure 2. Structures of currently evaluated antiparasitic drugs.

O

OH

O

OHNH2

O

OOH

OH OH

OH

OH OH O OH

H

OH

O

OH

Amphotericin B

NH3+

COO-

NH3+CHF2

DL-α-Difluoromethylornithine

O

OH

OH

NH2

CH2OH

O

O

OH

CH2OH

NH2

OH NH2

OH

O

O

O

OHNH2

CH2NH2

Paromomycin

Expert Opin. Investig. Drugs (2002) 11(2) 219


peroxidase and glutathione peroxidase enzyme systems are bothpresent [25]. The trypanosomatids have been compared to cancercells [26] due to their capacity for rapid growth. Both also havesensitivity to oxidative stress, which has led to TR being consid-ered the main target for the development of a new generation ofantiparasitic drugs [27,28]. Validation of the enzyme as a drugdesign target has been undertaken using gene-knockout experi-ments [15] in which its levels in T.b. brucei were reduced to < 5%of normal. Cells consequently stopped growing, became hyper-sensitive to oxidative stress and were unable to infect mice.

3. Trypanothione reductase

TR activity has been found in all trypanosomatid speciesexamined to date, including the parasites, T. brucei, T. cruziand Leishmania. The enzyme has been purified from Crithidiafasciculata [29-33], T. cruzi [34], T. brucei [31, 35] and isolated as a

recombinant protein from Trypanosoma congolense [36, 37] andT. cruzi [38].

Trypanothione reductase catalyses the NADPH-dependentreduction of oxidised trypanothione TS2 (Figure 3), but notoxidised glutathione GSSG. This enzyme, like GR, belongs toa large well characterised family of FAD-dependent NADPHoxidoreductases, both of which share close structural andmechanistic similarities. They are both homodimeric proteinsof ∼ 50 kDa per subunit, contain FAD as a coenzyme, act asan electron acceptor from NAD(P)H and have a redox-activecystine disulfide in the active site essential for catalytic activity[39,40]. Both TR and GR share many physical and chemicalproperties [27. The main difference between the two is theirmutually exclusive specificity for their respective disulfidesubstrate and the binding region for the disulfide substratebeing more spacious in TR than in GR.

3.1 Substrate specificityThe x-ray crystal structures of TRs complexed to trypan-othione [41], glutathionylspermidine [42] and the inhibitormepacrine [43] have been reported. Crystallographic studiesindicate that the specificity of the TS2 substrate is controlledby 25 amino acid residues, implicated in binding of the pep-tide by hydrogen bonding and/or van der Waals interactions(Table 1). A hydrophobic patch in the binding site has beenidentified and associated with binding of the N4-secondaryamine of the spermidine (Spm) bridge by van der Waals andcation-π interactions [41]. In addition, a second hydrophobicpocket (not used by the trypanothione substrate) was discov-ered in TR [44]. This second hydrophobic pocket has becomethe target for the design of TR inhibitors [45] based on leadstructures resembling the tricyclic neuroleptics, the imi-pramines and compounds of the phenothiazine type [46].

3.2 Peptide/polyamines/natural product inhibitorsA range of peptides have been demonstrated to competitivelyinhibit TR [47], the strongest of which are N-benzoyl-Leu-Arg-Arg-β−naphthylamide [45,47] and Z-Ala-Arg-Arg-4-methoxy-β-naphthylamide [47] with Ki values of 13.8 and 2.4 µM,respectively [47]. Non-reducible acyclic substrate analogues ofTR incorporating amine-bearing chains in place of the sper-

Table 1. The interacting residues of T[S]2 and TR [41].

Number of contacts

T[S]2residue A site B site Trypanothione-reductase residues

γGluI 10 24 Pro336, Ile339, Gly459', His461', Glu466'

CysI 8 5 Val54, Tyr111, Thr335, Ile339, His461'

GlyI 7 8 Ser15, Leu18, Tyr111, Ile339

Spm 18 14 Leu18, Glu19, Trp22, Ser110, Tyr111

γGluI 25 20 Val54, Val59, Lys62, Phe396, Lys399, His461', Pro462', Thr463', Ser464'

Glu466',Glu467'

CysII 5 5 Val59, Ile107, His461'

GlyII 1 0 Ile107

The number of contacts excluding hydrogen bonds with a distance restriction of 4.0 Å are listed ; 'signifies a residue from the partner subunit.

Figure 3. Trypanothione-mediated hydroperoxide metabolism. ROOH: Alkyl hydroperoxide; TR: Trypanothione reductase; T(SH)2Trypanothione; TXN: Tryparedoxin; TXNPx: Tryparedoxin peroxidase.

NADPH

NADP+

TRox

TRred

T(SH2)

TS2

TXNox

TXNred

TXNPxred

TXNPxox

ROOH, H2O2

ROH, HOH


D’Silva & Daunes

midine group and a CBz moiety in place of the γ-glutamylmoiety have been reported [48, 49] (Figure 4 [1 - 6]) to be com-petitive inhibitors of TR with Ki values ranging from 30 - 90µM [49]. Tricyclic ester and amide trypanothione stereologues(7) derived from bimane have been synthesised as inhibitorsof TR [50] (Figure 4) but were found to be inactive althoughthey did exhibit weak in vitro antitrypanosomal activityagainst L. donovani.

Natural products such as lunarine (8), a polyamine derivedfrom European garden plants Lunaria biennis and L.rediviva,have been reported to be slow binding inhibitors of TR withan apparent Ki of 144 ± 30.5 µΜ [41] whilst Kukoamine A, [9] apolyamine from the root bark of Lycium chinensea, is a mixedinhibitor of TR (Ki = 1.8 µM, Kii = 13 µM) [51]. A variety ofsynthetic polyamine based inhibitors of spermidine and sper-

mine derivatives (10, 11) have been reported to be competi-tive inhibitors of TR with Ki values of 3.5 [52] and 0.61 µΜ[53] for (10) and (11) respectively. These compounds showgood in vitro trypanocidal activity which is lost in vivo [54].The use of combinatorial chemistry has recently been appliedto the preparation and screening of peptide polyamine inhibi-tors with the identification of a potent inhibitor of TR (12)with a Ki ~ 0.1 µΜ [55].

3.3 Tricyclic inhibitorsOut of 30 phenothiazine derivatives and tricyclic antidepres-sants investigated, clomipramine (13) was identified as themost potent competitive inhibitor of TR (Ki = 6 µΜ) [56].Other tricyclics identified as inhibitors of TR include crystalviolet (14) [57], acridine derivative (15) [58], mepacrine [43], N-

NH

NH

O

NH

O

CBz

R

NH

NH

NNH

CBz

O

O

N

1: R = S- S2: R = CH2SCH2SCH2-3: R = CH2SCH2-4: R = CH2SCH2CH2-5: R = CH = CH-6: R = CH2CH2-

NH

O

OH

OH

NH

NH

NH

O

OH

OH

9: Kukoamine A

N

N

O O

X X

(CH2)n (CH2)nS

O O

7

X = O, NH

n = 1 - 6

NH

O

NH

NHO

O

O

8: Lunarine

NNHRN NHR

10: R= H11: R = (CH2)3Ph

H-Trp-Arg/Arg(Pmc)-NH

NHH-Trp-Arg/Arg(Pmc)-NH

12

Figure 4. Structures of peptide/polyamines/natural product inhibitors of TR.



acyl promazines, 2-substituted phenothiazines (16), trisubsti-tuted promazines and the imipramines [59-61] (Figure 5). Thephenothiazines show considerable scope for substitution onthe nitrogen ring [59] with improvements in inhibitorypotency on quaternisation of the ω−(Ν,Ν−dimethylamino)propyl substitutent (17, Ki ~ 0.12 µΜ) [62]. Permanentlycharged analogues of phenothiazines are frequently less bio-logical active against the dopamine D2 receptor [63] so leadingto a reduction in the depressant side effects of such com-pounds. These quaternary tricyclic compounds are activein vitro against T. brucei at 1 µΜ but are toxic to macrophagesat a 10-fold higher concentration [47].

The two hydrophobic regions of TR play a role in thebinding of phenothiazine inhibitors. The first recognises thespermidinyl sector of the TR substrate [64] whilst the secondsite binds the tricyclic compound not the substrate. However,the hydrophobic pockets can exhibit multiple binding modeswith tricyclic inhibitors [61], confirming their structural sensi-tivity to such interactions.

3.4 2-AminodiphenylsulfidesMicroplate screening assays led to the discovery that the 2-aminodiphenysulfides act as inhibitors of TR (Figure 6).These compounds are structurally related to the phenothi-azines but are of much lower neuroleptic activity [65]. Substi-tution of the side chain of these compounds by linear orbranched amines based on spermine and spermidine (18 - 20)improved their activity. The best inhibitor of the series had aKi = 25 µΜ [66]. Dimerisation further improved the Ki of thisseries to 0.55 µΜ (21,22) [67]. This may indicate that more

than one 2-aminodiphenylsulfide nucleus can be accomo-dated per active site (perhaps by drug stacking), a propertycommonly seen in drugs containing a tricyclic nucleus [68].The addition of an extra sidechain to the dimeric compoundsresulted in the discovery of the most potent compound identi-fied to date with Ki = 0.2 µΜ [69].

3.5 Subversive substrates/irreversible inhibitorsSubversive substrates (turncoat/sabotage inhibitors) affect TS2(Figure 3) by promoting a non-physiological reaction whereTS2 reduction is inhibited, NADPH and O2 are wasted, theSH/SS ratio becomes lowered and superoxide radical forma-tion can consequently trigger lipid peroxidation, H2O2 pro-duction and other chain reactions. These compounds arecatalysts and undergo redox cycling to produce oxidativestress which can be exploited in the chemotherapy of parasiticdiseases [27]. Quinone based compounds such as menadione(23), plumbagin (24) [70] and a variety of 1,4-naphthoqui-nones [27, 71, 72] have been reported to act as subversive sub-strates of TR (Figure 7). The best subversive compoundagainst TR identified to date is (25), which is an uncompeti-tive inhibitor versus TS2 and NADPH [73]. The compoundhas in vitro activity against T. brucei (ED50 1.1 µΜ), L. dono-vani (ED50 11.1 µΜ) and T. cruzi (ED50 4.3 µΜ).

Allicin, the major sulfur component of garlic, is spontane-ously degraded to (E, Z)-ajoene (4,5,9-trithiadodeca-1,6,11-triene 9-oxide) (26) [74] on bulb maceration. This compoundis well known for its antifungal, antiviral, antitrypanosomal[75] and antimalarial activity. Ajoene has been shown to be asubversive substrate and covalent inhibitor of GR and TR.

Figure 5. Structures of tricyclic inhibitors.

13

N+

N

N

14

N

S

N

R1

17

N

NHR

R1 R2

15

N

S

R1

R

16

NCl

N


D’Silva & Daunes

The latter property it shared with the nitrosourea drug car-mustine (BCNU) [72]. Ajoene creates oxidative stress in thecell as a result of its reduction products which include 4,5,9-trithisdodeca-1,6,11-triene (deoxyajoene), 4,8,9,13-tetrathia-hexadeca-1,6,10,15-tetraene, allyl mercaptan, the oxidationproduct thioacroleine and superoxide anion radicals [76].(2,2′:6′,2′′ -Terpyridine)platinum (II) complexes (27 - 29)represent a new class of organometallic irreversible inhibitorsof TR but not GR which also has pronounced cytostaticactivity against trypanosomes and leishmania [77].

4. Tryparedoxins

Tryparedoxin I (TXN1) is a hydrogen donor protein fortryparedoxin peroxidase (TXNPx), an enzyme of the peroxi-doxin family of Mr = 22 kDa [78] which is involved in thereduction of hydroperoxides and alkylhydroperoxides(Figure 3). These are enzymes presumed essential to the sur-vival of parasitic protozoa and have been isolated from thetrypanosomatid species C. fasciculata (5% of the total solubleprotein) [79,80] and as a recombinant protein from the samespecies [81] and T. brucei [82].

The name ‘tryparedoxin’ was chosen for these enzymesbased on their similarity and function with the hostenzymes thioredoxin [83] and glutaredoxin [84] (Figure 8).However, despite its thioredoxin-like active site and reac-tivity [85], this protein shows little similarity to thioredoxin.With a molecular mass of 16 kDa, tryparedoxin I is sub-stantially larger than vertebrate thioredoxin [86] and T. bru-cei thioredoxin [87], both 12 kDa in size. Moreover, thespecificity of tryparedoxin is accentuated by a low limitingKm value for trypanothione (130 µM, as compared to990 µM for glutathione disulfide in the host system). Thecatalytic properties of tryparedoxins are also intermediatebetween those of the classical thioredoxins and glutaredox-ins and so may represent a new class of thiol-disulfide oxi-doreductase.

The isolation of a new gene from C. fasciculata whichencodes a tryparedoxin differing substantially from the partialsequence described earlier for tryparedoxin I led to the desig-nation of this protein as tryparedoxin II (TXN2;Mr = 18 kDa) [86]. Although these two tryparedoxins wereshown to mutually substitute for each other in the trypano-somal peroxidase system [86], their main biological roles werenot assigned until tryparedoxin II was shown to serve as a sub-

Figure 6. Structures of 2-Aminodiphenylsulfides.

S

NHCl

NNH2

NH2

18

S

NHCl

NH

NH2

19

S

O NH Cl

O

N

S

NHCl

O

N

NH2NH2

O

20

S

X NH

Y

R

Y Y S

XNH

Y

R

21R = Me2N or

S

NHY

NH

S

NHX

Y

NH

X

22 X = H, Cl or BrY = O or 2H

N

N



S

O

SS

NH

NH

NH

Pt2+

L

Cl

NH

NH

NH

Pt2+

R

S

OH

NH

NH

NH

NH

Pt2+

R

OH

O

O

(CH2)n-(CH2)nCONH R NHCO-(CH2)n

OH

O

O

R= NH2 NH NH2

25

27 28 29

26: Ajoene

OH

O

O

O

O

23: Menandione 24: Plumbagin

Figure 7. Structures of subversive substrates and irreversible inhibitors of TR.

Figure 8. Mammalian cells have two general mechanisms for the reduction of intracellular protein disulfides and otherdisulfides; GR, glutathione, and glutaredoxin (also known as thioltransferase) and thioredoxin reductase and thioredoxin. A: general purpose NADPH-dependent disulfide-reducing system : thioredoxin reductase and thioredoxin; B: general purpose NADPH-dependent disulfide-reducing system : glutathione reductase, glutathione, glutaredoxin.

NADPH + H+

NADP+

FAD

FADH2

HS SH

SS

2RSH

RSSR

SS

HS SH

Thioredoxin reductase Thioredoxin

NADPH + H+

NADP+

FAD

FADH2

HS SH

SS

RSSR

2RSH

HS SH

SS

GSSG

2GSH

Glutathione reductase Glutathione Glutaredoxin

A

B


D’Silva & Daunes

strate of ribonucleotide reductase from T. brucei [88], thus per-forming the analogous role of thioredoxin in the host system.Tryparedoxin II was then assigned the function of ribonucle-otide reductase substrate whereas tryparedoxin I was classifiedas a peroxidase substrate. Tryparedoxin I and II are locatedpredominantly in the cytosol of C. fasciculata [89] whilsttrypanothione reductase exists in the cytosol of T. brucei [90],T. cruzi and in the mitochondrion of T. cruzi [91]. Trypare-doxin peroxidase has recently been found to exist in thecytosol and mitochondrion of T. cruzi [92] and T. brucei [93].

4.1 Substrate specificityThe x-ray crystal structures of native [94] and recombinantTXN1 and TXN2 [95] as well as the TXN2 complex of N1-glutathionylspermidine [95] have been reported. The residuesassociated with trypanothione binding are conserved in bothTXN1 and TXN2 (Table 2). Modelling studies suggestArg45 (44), Glu73 (72), the Ile110 (109) cis-Pro111 (110)-bond and Arg129 (128) are involved in the binding oftrypanothione to TXN2 (TXN1) [95]. Arg 129 (128) andArg 45 (44) are both able to form a salt bridge with theglutamyl α-carboxylate group of trypanothione [94] whilstthe Ile110 (109) and cis-Pro111 (110) bond may H-bondwith the backbone of the substrate. In the case of the glu-tathionylspermidine complex of TXN2, crystallographicstudies indicate that the binding is controlled by electro-static interaction of Arg-129 with the Glu-αCOO- of thesubstrate and H-bonds between the NH amide group of theγ-glutamyl group and the CO group of cis-Pro-111 (110)whilst the NH group of the Ile-110 (109)-bond interactswith the amide bond of the Cys-Gly group of the glutathio-nylspermidine substrate (Figure 9).

The binding of glutathionylspermidine to TXN2 is pri-marily via the peptide backbone with the amino group andspermidyl side chain contributing negligibly, apart from aloose association with water molecules.

4.2 Glutathione-based inhibitorsS-Blocked [96-100], N,S-blocked [96-100] and N,S-blocked glu-tathione monoesters/amides and diamides/diesters [99,100]

have been used to identify the molecular determinants forglutathiones to bind to proteins. These studies identified Nand S-blocked glutathiones as weak inhibitors of E. coli glu-taredoxin [98,101] a hydrogen donor protein related to thiore-doxin and tryparedoxin (Figure 8). The first indication thatglutathione derivatives had antiparasitic activity came from asystematic investigation of S-blocked, N,S-blocked and N,S-blocked glutathione monoesters/amides, diamides anddiesters [201, 104] against T.b. brucei. These studies revealedthat N-benzyloxycarbonyl-S-Brbenzylglutathione dibutylester (30) and N-acetyl-S-benzyloxylcarbonylglutathionedimethyl ester (31) were potent inhibitors of T.b. brucei withKi values of 1.9 and 3.2 µM respectively (Figure 10). It wasinitially assumed that the in vitro activity of these peptideswas a result of inhibition of TR which led to the furtherdevelopment of a solution based combinatorial library ofN,S-blocked glutathione mono and diesters based around S-2,4-dinitrophenyl glutathione a known inhibitor of glutath-ione reductase [105] an enzyme structurally and chemicallyrelated to TR [27,104]. N,S-blocked glutathione diesters per seare not inhibitors of TR based on the known binding deter-minants of this enzyme and were considered prodrugs [104]

in their mode of action. The pharmacological importance ofthe diester groups to activity was determined from a QSARstudy [106] which demonstrated that the in vitro activity ofthese compounds against T.b. brucei and T.b. rhodesiense wasrelated to membrane penetration (log P) and steric factors(Es) via eqn 1 & 2 respectively. The optimum value for logP against T.b. brucei and T.b. rhodesiense were 5.6 and 5.03respectively. The differences in the QSAR equation observedbetween these two species of parasite:

1. log(1/ED50) = -2.57*log P+1.87*Es+0.20*MW-0.002*W- 102

Figure 9. A putative model of the glutathionylspermidine (GS) complex of tryparedoxin II obtained from its crystal structure[95], showing the main H-bonding interactions with the backbone and electrostatic interaction with Arg 129.



2. log(1/ED50) = -1.02*log P+0.21*Es+0.1*MW-0.0014*W- 50.6

reflect structural differences between the membranes of thetwo strains, as demonstrated by drug penetration characteris-tics and steric factors [107]. The best compounds identifiedfrom this study (32-35) showed in vitro activities in the range0.18-0.38 µM against T.b. brucei and T.b. rhodesiense, with 32& 33 exhibiting the lowest host toxicity [106]. Compound 32displayed activity against T.cruzi at 6.7 µM and compound34 displayed activity against L. donovani at 29.6 µM. Thehydrophobic nature of N,S-blocked glutathione diestersallowed them to enter parasitic cells but their fate within T.b.brucei cells had to be established by HPLC studies [108]. Themain degradation products of diesters were identified as N,S-blocked glutathione monoester and diacid which were theinhibitory forms of these peptides active against the targetenzyme. Exposure of TR to diesters, monoester or diacid[104,106] failed to produce inhibition of this enzyme and so

implicated another trypanothione dependent enzyme as thetarget. Qualitative identification of the target proteins wasachieved by the use of a quartz crystal AT-cut sensor device[104,106,108] on which an N,S-blocked glutathione was attached[108,110,111]. Exposure of the device to different MW fractionsof proteins isolated from C. fasciculata indicated that the tar-get protein was a low MW protein associated with the 60 -100% (NH4)2SO4 extract [104]. Gel electrophoresis of thisextract showed it to contain primarily three protein bands ofMW’s 22, 18 and 16 kDa corresponding to glutathione per-oxidase, TXN2 and TXN1 of which only the latter two pro-teins contained a trypanothione binding site [108]. Thetryparedoxins have been assigned the function of DNA syn-thesis [88] or peroxidase substrates. Treatment of T.b bruceicells with diesters causes the rapid onset of cell death. Withinan hour of exposure the cell contents exhibited erraticmotions and the cell structure underwent gross morphologicalchanges (Figure 11). These observations may indicate that oxi-dative stress is the primary mechanism of cell death, whenusing these compounds, rather than the inhibition of DNAsynthesis. Although the N-site of glutathione in the glutathio-nylspermidine complex of tryparedoxin protrudes from theprotein surface of tryparedoxin (Figure 9) this group may stillplay a role in the activity of these compounds by alteringmembrane penetration and host toxicity. A study of sometwenty N-substituted-glutathione diester derivatives appearsto substantiate these conclusions, whilst there are no signifi-cant improvements in in vitro activity there was a decrease inhost toxicity due to the ability of this site to accommodate alarge variety of structurally diverse groups, due to its presenceon the surface of the protein (Figure 9). An investigation of

Table 2. Sequence of TXN1 and TXN2 in the vicinity of residues implicated in trypanothione binding.

Residue no TXN1a TXN2b

41-50 PPCRGFTPQL PPSRAFTPQL

71-80 DEEEDGFAGY DESAEDFKDY

101-110 SKHFNVESIP TTGFDVKSIP

121-130 DVVTTRARAT NIITTQARTMa Sequence from structures 1EWX:A and 1QK8:A [96].b Sequence from structure 1I5G [96].

Figure 10. Structures of N,S-blocked glutathione receptors as antiparasitic inhibitors.

CbzNH

NH

OS

BzBr

O

NH

OBu

O

BuO O

30

NH

S

Cbz

NH

Ac

O O

O

O

NH

O

O

31

NH

O

NH

RO O

Cbz

S

DNP

O

NH

OR

O

32: R = (CH2)4CH3

33: R = CH(CH3)CH2CH3

34: R = cyclopentyl35: R = cyclohexyl


D’Silva & Daunes

the S-site of glutathione using a series of thirty different S-substituted-glutathione diesters have identified the presenceof an additional binding site on the target protein and themost active compound to date with Ki = 0.039 µM againstT.b. rhodesiense. Many of the N,S-blocked glutathione diesterderivatives have achieved significant levels of activity underin vitro conditions. Evaluation of these compounds againstT.b. brucei- or T.b. rhodesiense-infected mice failed to prolonglife but showed no signs of host toxicity, indicating problemswith in vivo stability. Studies on the use of S-blocked glutath-ione diesters to treat tumours in inbred mice identified thesecompounds to have a t1/2 < 30 s [112] in serum samples but at1/2 = 9.1 h in human serum samples.

5. Conclusion and expert opinion

There are approximately 27,000 new cases of African sleepingsickness reported each year but it is estimated that this figure isapproximately 13 times higher [113]. One of the highest priori-ties with regards to sleeping sickness control is the reduction ofmortality in the late stages of this disease, especially with respectto T.b. rhodesiense infections where deficiencies in treatment

currently exist. Extensive studies on TR over the last decadehave generated a wide variety of potential drugs with someshowing low toxicity and in vitro activity. To induce phenotypicchanges in TR, compounds have to achieve a 90% level of inhi-bition [24], which requires that they have inhibitory activities inthe nanomolar range. The most active compounds (~ 0.1 µΜ)contain cationic groups, such as the currently prescribed drugpentamidine which is unable to enter the CNS and so cannotkill parasites in the late stages of the disease when the mosthuman mortalities occur. The identification of the tryparedox-ins as a target for the design of antiprotozoal drugs opens upnew avenues for research where hydrophobic compounds oflow toxicity and significant activity can be prepared with thepotential to enter the CNS without major side effects. Theproblems of serum biostability and bioavailability now is thepenultimate obstacle in the realisation of a new drug for thetreatment of HAT.

Acknowledgements

Research work cited in this review was supported by grantsfrom EPSRC, WHO and HEFCE.

(a) (b) (c)

Figure 11. T.b.brucei cells incubated with N-CBzBrBzGSHMe2 (50 mM) (magnification 1000x) [108].(a) 1 min (b) 5 min (c) 30 min.

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97. DOUGLAS KT, SEDDON AP, D’SILVA C, BUNNI M: Photoaffinity labels for glutathione and glutathione disulphide-utilizing systems. Biochem. Soc. Trans. (1982) 124-125.

98. HOOG J-O, HOLMGREN A, D’SILVA C, DOUGLAS KT, SEDDON AP: Glutathione derivatives as inhibitors of glutaredoxin and ribonucleotide reductase from Escherichia coli. FEBS Lett. (1982) 13:59-61.


D’Silva & Daunes

99. D’SILVA C: Reinvestigation of the roles of the carboxyl groups of glutathione with yeast glyoxalase I. FEBS Lett. (1986) 202:240-244.

100. D’SILVA C: Inhibition and recognition studies on the glutathione-binding site of equine liver glutathione S-transferase. Biochem. J. (1990) 271:161-165.

101. HOOG JO, DOUGLAS KT, D’SILVA C, HOLMGREN A: Photoaffinity labelling of glutaredoxin from Escherischia coli. Biochem. Biophys. Res. Commun. (1982) 107:1475-1481.

102. D’SILVA C, DOUGLAS KT: Photoaffinity labelling of yeast glutathione reductase by a bifunctional aryl azide derivative of oxidised glutathione. Photobiochem. & Photobiophys. (1983) 5:151-158.

103. DOUGLAS KT, TIMARI AA., D’SILVA C, GOHEL DI: Role of the N-terminus of glutathione in the action of yeast glyoxalase I. Biochem. J. (1982) 207:323-329.

104. D’SILVA C, DAUNES S, ROCK P, YARDLEY. V, CROFT S: Structure-activity study on the in vitro antiprotozoal activity of glutathione derivatives. J. Med. Chem. (2000) 43:2072-2078.

105. BLIZER M, KRAUTH-SIEGEL RL, SCHRIMER RH, AKERBOOM TPM, SIES H, SCHULZ GE: Interaction of a glutathione S-conjugate with glutathione reductase: Kinetic and x-ray crystallographic studies. Eur. J. Biochem. (1984) 138:373-

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106. DAUNES S, D’SILVA C, KENDRICK H, YARDLEY V, CROFT SL: A QSAR Study on the Contribution of Log P and Es to the in vitro antiprotozoal activity of glutathione derivatives. J. Med. Chem. (2001) 44:2976-2983.

107. DAUNES S, D’SILVA C: Glutathione derivatives active against T.b.rhodesience & T.b.brucei in vitro. Antimicrob. Agents Chemother. (2002) 46:434-437.

108. D’SILVA C, DAUNES S: The mode of action of glutathione derivatives as antiprotozoal agents active against trypanosomes. (in Preparation).

109. D’SILVA C: Receptor based biosensors. In: Biosensors (Current Topics in Biophysics); FRANGOPOL. P.T., SANDULOVICIU. M., Eds.; Iasy University Press: Iasy, Romania. (1995) 5: 1-19.

110. BARNES C, D’SILVA C, JONES JP, LEWIS TJ: A concanavilin a coated piezoelectric crystal biosensor. Sensors & Actuators. B. (1991) 3:295-304.

111. FARRELL C, ROWEL FJ, CUMMING RH, D’SILVA C: Enzyme-linked immunosorbent assay for ceftazidime in airborne samples. Analyst (1994) 119:2411-2316.

112. KAVARANA MJ, KOVALEVA EG, CREIGHTON DJ, WOLLMAN MB,

EISEMAN JL: Mechanism-based competitive inhibitors of glyoxalase I: intracellular delivery, in vitro antitumor activities, and stabilities in human serum and mouse serum. J. Med. Chem. (1999) 42:221-228.

113. The Global Parasite Contol for the 21st Century; WHO Centre for Health Development (WHO Kobe Centre), May 1998, pp 35-40.

Patent

201. MANCHESTER METROPOLITAN UNIV.: GB2332676 (2001).

Website

301. Protein Data Bank at the Research Collaboratory for Structural Bioinformatics, New Brunswick, NJ, USA (http://www.rcsb.org/)

AffiliationClaudius D’Silva, BA, PhD, CChem, FRSCDepartment of Chemistry & Materials, The Manchester Metropolitan University,John Dalton Building, Chester Street, Manchester M1 5GD, UKhttp://www.CHEM-MATS.mmu.ac.uk/stafflist.html


Documents

The therapeutic potential of inhibitors of the trypanothione cycle