Trypanosomatid Diseases (Molecular Routes to Drug Discovery) || Proteases of Trypanosoma brucei

20Proteases of Trypanosoma bruceiDietmar Steverding�

AbstractHuman African trypanosomiasis (HAT) or sleeping sickness is a parasitic infectioncaused by the protozoan Trypanosoma brucei. The disease occurs in sub-SaharanAfrica where it is a major cause of morbidity and mortality in man. Combinations oftoxicity and poor efficacy of current anti-sleeping sickness drugs means that new,effective, and better tolerated chemotherapies are needed for the treatment ofhuman African trypanosomiasis. Proteases play a key role in the life cycle ofT. brucei and in the pathogenesis of sleeping sickness. In vitro and in vivo studiesover the last decades have shown that proteases are valid targets for the developmentof new drugs against T. brucei. Here, the major proteases of T. brucei and theircellular roles and potential as drug targets will be reviewed.

Introduction

The flagellated protozoan Trypanosoma brucei is the etiological agent of humanAfrican trypanosomiasis (HAT) or sleeping sickness. The parasite is transmitted bythe bite of infected tsetse flies (Glossina spp.), and lives extracellularly in the bloodand tissue fluids of humans. The occurrence of sleeping sickness is restricted to thedistribution of tsetse flies, which are exclusively found in sub-Saharan Africabetween 14� North and 20� South latitude [1]. In this so-called tsetse belt, millionsof people living in 250 rural foci scattered over 36 African countries are at risk ofcontracting the disease [2,3].Sleeping sickness occurs in two disease patterns caused by two subspecies of

T. brucei. The chronic form of sleeping sickness is caused by T. b. gambiense andoccurs in west and central Africa. This form of the disease accounts for about 95% ofall reported cases of the infection [3]. The remaining cases are due to the acute formof sleeping sickness caused by T. b. rhodesiense, which is found in east and southernAfrica. During the course of sleeping sickness, two disease stages can be distin-guished. In the first stage, the parasites are restricted to the blood and lymph system.This hemolymphatic phase is characterized by irregular fever, headaches, joint

� Corresponding Author

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

j365

pains, and itching [3]. In the second stage, parasites infect the central nervoussystem. Patients entering this neurological phase display the more obvious signsand symptoms of the disease, which are confusion, disturbed sleep pattern, sensorydisturbances, extreme lethargy, poor condition, and coma [3]. If left untreated,sleeping sickness patients die within months when infected with T. b. rhodesiense orwithin years when infected with T. b. gambiense [3]. Sadly, only a few drugs (suramin,pentamidine, melarsoprol, and eflornithine (DL-a-difluoromethylornithine)) andone drug combination (eflornithine/nifurtimox) are available for chemotherapy ofsleeping sickness [4]. All these drugs have major drawbacks, including poor efficacy,significant toxicity, requirement for parenteral administration, and being increasinglysubject to drug resistance [5–7].As proteases have been shown to perform important vital functions in pathogens

and/or can be involved in the pathogenesis of infectious diseases, they have quicklyattracted much attention as potential drug targets. Indeed, one promising line ofresearch towards the development of new anti-sleeping sickness drugs has been thetargeting of proteases. This chapter will discuss some important proteases ofT. brucei, including their cellular role and their potential as drug targets.

Classes of Proteases

The first characterizations of proteolytic activities in T. brucei were carried out in the1980s [8–14]. Subsequent research revealed that these proteolytic activities are fromproteases belonging to the cysteine, serine, threonine, and metallo family ofpeptidases. Table 20.1 summarizes the proteases of T. brucei discussed in thischapter.

Cysteine Peptidases

Cysteine proteases are characterized by a common catalytic mechanism thatinvolves a nucleophilic thiol group of a cysteine residue that is part of a catalyticcysteine–histidine dyad.

Cathepsin B- and L-Like ProteasesCysteine cathepsins are the best-characterized cysteine peptidases and belong tothe C1 family of papain-like enzymes (Table 20.1). They play a key role amonglysosomal proteases and are widely distributed among living organisms, includingprotozoan parasites.Perhaps the best-characterized cysteine peptidases in T. brucei are two proteases

homologous to mammalian cathepsin B and L [15,16]. Based on the recentlyproposed nomenclature system for kinetoplastid C1 peptidases [15], T. bruceicathepsin B- and L-like proteases have been named TbCATB and TbCATL,respectively. In common with other C1 peptidases, both TbCATB and TbCATLcomprise a signal peptide, a propeptide containing an I29 inhibitor domain, and aPepC1 catalytic domain, and have conserved cysteine, histidine, and asparagine

366j 20 Proteases of Trypanosoma brucei

residues in the active site that are embedded in highly conserved peptide motifs[15,17]. In addition, other amino acids and peptide sequences are also conservedin both TbCATB and TbCATL, including several cysteine residues that formdisulfide bonds [15].The length of the prepro-TbCATB is 340 amino acids and thus similar to the

length of human cathepsin B (339 amino acids) [15]. The enzyme is encoded by asingle-copy gene for which expression is regulated at the level of mRNA stability andis greatest in bloodstream forms [18].At 450 amino acids, the length of the prepro-TbCATL is significantly longer than

that of human cathepsin L (333 amino acids) [14]. This is due to a 108-amino-acidC-terminal extension on the parasite enzyme [19] for which a definitive biologicalfunction is not yet known. TbCATL is encoded by more than 20 genes arranged inlong tandem array [19,20]. The expression of TbCATL is developmentally regulatedwith the highest activity levels found in bloodstream forms [21,22].

Table 20.1 Summary of T. brucei peptidases.

Family Peptidase Geneticallyvalidated

Chemicallyvalidated

Drugtarget

C1 (papain family) TbCATB yesa) ? ?TbCATL nob) yes yes

C2 (calpain family) TbCALP1.1 no NDc) ?TbCALP1.2 yes ND ?TbCALP1.3 no ND ?TbCALP1.4 no ND ?TbCALP4.1d) yes ND noTbCALP8.1 yes ND ?

C13 (legumain family) TbGPI8 yes ND ?C14 (caspase family) TbMCA1 ND ND no

TbMCA2 yese) yes ?TbMCA3 yese) yes ?TbMCA4 ND ND noTbMCA5 yese) ND no

M8 (leishmanolysinfamily)

TbMSP-B no ? ?

S9 (prolyl oligopeptidasefamily)

TbOPB ND yes yesTbPOP nof) ? ?

T1 (proteasome family) TbPSB1 yes ND ?TbPSB2 yes yes yesTbPSB5 yes yes yes

a) Incomplete RNAi: 60–65% of TbCATB remained.b) Incomplete RNAi: 30–35% of TbCATL remained.c) ND, not determined.d) Procyclic forms.e) Only simultaneous RNAi against TbMCA2, TbMCA3, and TbMCA5 showed a phenotype.f) Incomplete RNAi: 20% of TbPOP remained.

Classes of Proteases j367

Calpain-Like ProteinsCalpains are a large family of calcium-dependent cysteine proteases (C2 family ofcalpain-like enzymes) that are involved in a wide range of differentiation and cellregulatory processes. They are heterodimeric proteins consisting of an 80-kDa subunitand a 28-kDa subunit [23]. The large subunit is typically divided into an N-terminaldomain of unknown function, a protease domain containing the catalytic triad ofcysteine, histidine, and asparagine, a linker domain, and a calcium-bindingdomain [23].Systematic analysis of the genome of T. brucei revealed a large and diverse family

of calpain-like proteins in this parasite (TbCALPs; Table 20.1) [24]. Based on theirstructural features, TbCALPs were categorized into five groups [24]. Only TbCALPsof group 1 and 2 are four-domain proteins resembling the general structure ofmammalian calpains, while TbCALPs of group 3 consist only of the N-terminaldomain, and TbCALPs of group 4 and 5 are highly divergent proteins [24,25].Interestingly, the catalytic triad is only conserved in one member of the TbCALPs,suggesting that most of these proteins probably do not act as cysteine proteases [24].In addition, the C-terminal domain of all TbCALPs does not show any similarity tothe calcium-binding domain of conventional calpains [24]. Several TbCALPs containN-terminal dual myristoylation/palmitoylation signals, indicating that theymight bemembrane-associated, and some TbCALPs are differentially expressed in blood-stream and procyclic forms [25].

MetacaspasesMetacaspases are a new family of cysteine proteases (C14 family of caspase-likeenzymes) that are homologous to mammalian caspases, and are found in plants,fungi, and protozoa [26]. Metacaspases are predicted to have a similar structure tocaspases with conserved histidine and cysteine residues in the active site [26].However, they have a strict arginine or lysine substrate specificity and lack aspara-gine specificity, which is a characteristic of caspases [27,28].To date, fivemetacaspases (TbMCA1–TbMCA5; Table 20.1) have been identified in

T. brucei [29]. Interestingly, TbMCA1 and TbMCA4 have a serine residue in place of acysteine residue in their catalytic site [30], suggesting that these metacaspases mightbe inactive. In addition, TbMCA4 is both myristoylated and palmitoylated, andprimarily expressed in bloodstream forms [31]. For TbMCA2 it has been shown thatcysteine peptidase activity is calcium dependent [28]. TbMCA2 and TbMCA3, whichare located in a tandem repeat on chromosome 6, are only expressed in bloodstreamforms, while TbMCA5 is also expressed in procyclic forms [32].

GPI : Protein TransamidaseGPI : protein transamidases are enzyme complexes that catalyze the attachmentof GPI (glycosylphosphatidylinositol) anchors to proteins. The subunit responsiblefor the protein–GPI anchoring reaction is GPI8, a cysteine peptidase containingcysteine and histidine residues in its active site. GPI8 belongs to the C13 family oflegumain-like enzymes.The homolog of GPI8 in T. brucei, TbGPI8 (Table 20.1), has been identified, and

the corresponding gene cloned and expressed [33,34]. TbGPI8 is encoded by a


single-copy gene, and is expressed by both procyclic and bloodstream forms [34].The enzymatic activity of recombinant TbGPI8 was shown to be strongly affected bythe sulfhydryl alkylating agent p-chloro-mercuriphenyl-sulfonic acid, indicating thatthe enzyme is a cysteine protease [33].

Serine Peptidases

In serine proteases, the hydroxyl group of a serine residue in the active site acts as anucleophile that attacks the carbonyl carbon of the scissile peptide bond of thesubstrate protein. So far, two serine proteases, oligopeptidase B (TbOPB) and prolyl-oligopeptidase (TbPOP) have been characterized in T. brucei, both belonging to theS9 family of prolyl oligopeptidase-like enzymes (Table 20.1). Oligopeptidases areendopeptidases that cleave peptides but not proteins. That oligopeptidases cannotcleave proteins is due to their structure: their active site is located at the end of anarrow cavity, which is only accessible for peptides.TbOPB is a soluble serine oligopeptidase that is released into the bloodstream

during infection [35,36]. TbOPB is optimally active at alkaline pH and does nothydrolyze proteins larger than 4 kDa [35]. The protease exhibits activity towardstrypsin-like enzyme substrates but not towards prolyl oligopeptidase substrates [36].Unlike most serine peptidases, the activity of TbOPB is inhibited by thiol-blockingreagents and enhanced by reducing agents and polyamines [36]. The reactivecysteine residues for thiol-inhibiting and -activating TbOPB activity have beenidentified as C256, and as C559 and C597, respectively [37].TbPOP is also a soluble serine oligopeptidase that is discharged into the blood-

stream of T. brucei-infected mice [38]. TbPOP hydrolyzes peptide bonds at theC-terminal side of proline and alanine residues at slightly alkaline pH [38]. Unlikemost other prolyl oligopeptidases, TbPOP can also cleave collagen [38].

Metalloproteases

The catalytic mechanism of metalloproteases involves a metal, which in most casesis zinc. The metal ion is coordinated by three ligands, which can be histidine,glutamate, aspartate, lysine or arginine. The fourth coordination position is taken upby a labile water molecule.The genome of T. brucei contains three gene families encoding major surface

proteases, TbMSP-A, TbMSP-B, and TbMSP-C, which belong to the M8 family ofleishmanolysin-like enzymes (Table 20.1) [39]. Each of these proteases displaysabout 33% sequence identity with the major surface protease of Leishmaniaspecies, GP63 [39]. In all three sequences the positions of 20 cysteine residues,10 proline residues, and the zinc-binding motif HEXXH are conserved [39].TbMSP-A and TbMSP-B families comprise five and four genes, respectively,whereas the TbMSP-C family contains only one gene [39]. TbMSP-B is tran-scribed in both bloodstream forms and procyclic forms [39] but its mRNAaccumulates to a 50-fold higher steady level in bloodstream trypanosomes [40].

Classes of Proteases j369

In contrast to TBMSP-B, TbMSP-A and TbMSP-C are transcribed only inbloodstream forms [39].

Threonine Proteases

Like serine proteases, threonine proteases also have a hydroxyl group in their activesite that acts as a nucleophile to cleave a peptide bond. However, the hydroxyl groupcomes from a threonine residue and not from a serine residue. The archetypemembers of this class of proteases are the catalytic subunits of the proteasome,which belong to the T1 family of proteasome b subunit-like enzymes.The proteasome is a multifunctional enzyme complex that plays an important role

in the degradation of intracellular proteins [41]. The proteasome of T. bruceiresembles structurally and functionally those of other eukaryotic cells [42]. The20S core particle of the trypanosomal proteasome has a barrel-shape structure and ismade up of four rings [43]. By using mass spectrometric techniques and bio-informatics, all essential seven a-subunits and seven b-subunits have been identi-fied as part of the trypanosomal 20S proteasome [44]. This suggests that the twoouter and the two inner rings of the trypanosomal 20S proteasome are eachmade upof the seven a-subunits and seven b-subunits, respectively, like any other eukaryotic20S proteasome. Affinity-labeling experiments confirmed that also for the trypano-somal 20S proteasome the trypsin-like activity is associated with the b2 subunit(TbPSB2; Table 20.1) and the chymotrypsin-like activity with the b5 subunit(TbPSB5; Table 20.1) [45]. With respect to substrate specificity and inhibitorsensitivity, the trypanosomal proteasome differs from the mammalian proteasome.Whereas the proteasome of T. brucei exhibits a high trypsin-like activity but a lowchymotrypsin-like activity, the reverse is true for the proteasome ofmammalian cells[43]. Likewise, the trypanosomal and mammalian proteasome are particularlysensitive to inhibition of the trypsin-like activity and the chymotrypsin-like activity,respectively [46,47].

Cellular Functions

TbCATB and TbCATL

Both TbCATB and TbCATL are found in the lysosome of bloodstream forms[22,48], whereas their subcellular localization in procyclic forms is unknown. Inbloodstream forms of T. brucei, both enzymes are involved in the degradation ofphagocytosed host proteins and are essential for survival [18,48]. In addition,TbCATL seems to facilitate trans-endothelial entry of bloodstream forms into thebrain. The ability of bloodstream forms of T. brucei to cross an in vitro model of ahuman blood–brain barrier (BBB) was reduced by RNA interference (RNAi)against TbCATL and blocked by the cysteine protease inhibitor N-methylpiper-azine-urea-phenylalanyl-homophenylalanine-vinylsulfone-benzene (K11777; Fig-ure 20.1) [49,50].


TbCALPs

Three principle types of subcellular localization for TbCALPs were identified: theflagellum, the cell body and the subpellicular cytoskeleton [25]. TbCALP1.3 islocalized to the tip of the flagellum, which suggests a possible involvement insensory functions [25]. TbCALP1.4, TbCALP4.2, and TbCALP6.1 occur throughoutthe cell body, but their functions remain to be determined [25]. TbCALP4.1 andTbCALP8.1 are associated to the cytoskeleton, and are essential and required forcorrect cell morphogenesis and organelle positioning in the parasite [51].TbCALP4.1 fulfils this function in procyclic forms, whereas TbCALP8.1 is respon-sible for this role in bloodstream forms [51].

TbMCAs

TbMCA2, TbMCA3, and TbMCA5 are found to colocalize with RAB11, a marker forrecycling endosomes, whereas TbMCA4 is associated with the flagellum [32]. Thesubcellular localization of TbMCA2, TbMCA3, and TbMCA5 rules out any involve-ment of these metacaspases in programmed cell death [32]. Simultaneous RNAiagainst TbMCA2, TbMCA3, and TbMCA5 led to an immediate growth arrest inbloodstream forms, indicating that they play an essential role for the parasite [32].Recently, it has been shown that TbMCA3 is involved in the processing of TbMCA4

Figure 20.1 Structures of peptidyl cysteine protease inhibitors with in vitro and in vivo activityagainst bloodstream forms of T. brucei [48,58].

Cellular Functions j371

during which the latter metacaspase is released from the parasite [31]. Reversegenetics revealed that TbMCA4 has roles in both cell cycle progression and parasitevirulence during murine infection [31]. In addition, overexpression of TbMCA4 inyeast led to growth inhibition, mitochondrial dysfunction, and cell death, supportingthe role of this metacaspase in controlling cellular proliferation in connection withmitochondrial biogenesis [29].

TbGPI8

TbGPI8 localizes within the tubular structures of the endoplasmic reticulum [33],where the addition of GPI anchors usually occurs. TbGPI8 plays an essential role inboth procyclic and bloodstream forms [34]. Procyclic mutants deficient in TbGPI8lack the major GPI-anchored surface protein, procyclin, but accumulate a pool ofunlinked GPI molecules [34]. Although viable in culture, these mutants are unableto establish an infection in the midgut of tsetse flies, confirming the important roleof GPI-anchored proteins for the insect–parasite interaction [34]. Inducible RNAiagainst TbGPI8 in bloodstream forms results in a severe growth deficiency of theparasites followed by cell death [34]. The development of multinuclear, multi-kinetoplast and multiflagellar phenotypes in these cells suggests a block in cyto-kinesis [34]. These data indicate that TbGPI8 is important for correct cell cycleprogression in T. brucei.

TbOPB and TbPOP

Both TbOPB and TbPOP are released into the host bloodstream and contribute to thepathogenesis of African trypanosomiasis [35,36,38].TbOPB is not secreted by live trypanosomes but released from dead or dying

parasites into the circulation where it retains full activity [36]. This serinepeptidase has been implicated in the hydrolysis of the atrial natriuretic factor[36], a peptide hormone involved in the homeostatic control of body water,sodium, potassium, and fat. The cleavage of the atrial natriuretic factor by TbOPBhas been suggested from the observations that the peptide hormone is a substratefor the enzyme in vitro [35] and that its level is reduced in the plasma of T. brucei-infected dogs [52]. A reduction of the atrial natriuretic factor would explain theobserved hypervolemia and, thus, the cardiomyopathy known to occur in Africantrypanosomiasis [53].Like TbOPB, released TbPOP retains its activity in the plasma of infected rodents.

In the bloodstream the enzyme mediates the degradation of several hormones andneuropeptides, including bradykinin, b-endorphin, gonadotropin-releasing hor-mone, neurotensin, and thyrotropin-releasing hormone [38]. The collagenolyticactivity of TbPOP could help the parasite to cross the BBB and thus to establish aninfection of the central nervous system [38]. These features may indicate that TbPOPplays an important role in the development and maintenance of a T. brucei infectionwithin a mammalian host [38].


TbMSP-B

The metalloprotease TbMSP-B is an integral membrane protein that is involved inthe release of the variant surface glycoprotein (VSG) during differentiation ofbloodstream forms to procyclic forms [54]. Using RNAi, it was shown thatTbMSP-B can release a recombinant VSG from transgenic procyclic trypanosomes[39]. In bloodstream-form mutants deficient in TbMSP-B, the VSG was removedmore slowly and in a non-truncated form during differentiation [54]. This latterresult, however, indicated that TbMSP is not solely responsible for the release ofVSG during the differentiation process. In fact, it has been shown that TbMSP-B anda phospholipase C (GPI-PLC) act synergistically in removal of VSG molecules fromthe cell surface during the differentiation of bloodstream trypanosomes to procyclicforms [54].

Proteasome

The main function of the proteasome is the degradation of regulatory proteinstargeted for breakdown by ubiquitin conjunction. In T. brucei, the proteasome fulfilsa similar role. For example, inhibition of the activity of the proteasome by lactacystinblocks the turnover of ubiquitinated proteins in intact cells of T. brucei [55]. Inaddition, inhibition of the proteasome activity arrests procyclic forms in G2 andbloodstream forms in both G1 and G2 phases of the cell cycle, indicating that theproteasome is essential for driving cell cycle progression in T. brucei [56].

Proteases as Drug Targets

TbCATB and TbCATL

The lysosomal cysteine protease activity of T. brucei has been chemically validated asa drug target (Table 20.1). Studies with peptidyl diazomethylketones, peptidylchloromethylketones, peptidyl fluoromethylketones, and peptidyl vinyl sulfoneshave shown that these irreversible cysteine protease inhibitors kill cultured blood-stream forms at low micromolar concentrations [48,57]. Furthermore, treatment ofT. brucei-infected mice with carbobenzyloxyphenylalanyl-alanine diazomethylketone(Cbz-Phe-Ala-CHN2; Figure 20.1) and N-methylpiperazine-urea-phenylalanyl-homophenylalanine-vinylsulfone-benzene (K11777; Figure 20.1) leads to a reduc-tion in parasitemia and a prolongation of survival [48,58]. The killing of the parasitesin vivowas found to be correlated with the inactivation of lysosomal cysteine proteaseactivity [48]. However, genetic validation of TbCATB and TbCATL requirement forsurvival has been confounded by a lack of complete RNAi silencing of the targettranscript (Table 20.1). Although RNAi against TbCATB was shown to be toxicin vitro [18] and to rescue mice from an otherwise lethal T. brucei infection [49], theobserved modest reduction of 32% of TbCATB protein [18] raises the question as to

Proteases as Drug Targets j373

whether the RNAi-induced lethality had anything to do with the cysteine protease.RNAi against TbCATLwas shown not to produce any phenotype in vitro [18] and notto rescuemice from a lethal T. brucei infection [49]. This is probably not surprising asRNAi of TbCATL never resulted in a complete ablation of the enzyme in vitro andin vivo (only 60–65% reduction of the protein) [18,49]. On the other hand, recentchemical validation using specific peptidyl inhibitors revealed that TbCATL, ratherthan TbCATB, is essential to the survival of bloodstream forms of T. brucei and,therefore, the appropriate drug target [59]. However, considering that non-specificcysteine protease inhibitors are therapeutic in vivo and show excellent selectivityindices in vitro [48,58], it may not be necessary to design compounds that actselectively against either TbCATB or TbCATL.

TbCALPs

A systematic RNAi study of chromosome 1 genes revealed growth phenotypes forTbCALP1.2 but not for TbCALP1.1, TbCALP1.3, and TbCALP1.4 in bloodstreamforms of T. brucei (Table 20.1) [25,60]. Depletion of TbCALP4.1 and TbCALP8.1 usingRNAi (Table 20.1) was shown to interfere with cytokinesis, organelle positioning andcell growth in procyclic and bloodstream forms, respectively [51], demonstrating therequirement of these calpain-related proteins in distinct life-cycle stages of T. brucei.However, because of the lack of chemical validation, it remains to be shown whetherTbCALPs are good drug targets for the treatment of sleeping sickness.

TbMCAs

Their absence in humans and their marked difference from the orthologous humancaspases make TbMCAs attractive new drug targets for anti-trypanosomal chemo-therapeutics. This suggestion is corroborated by the observation that TbMCA2,TbMCA3, and TbMCA5 are essential for bloodstream forms of T. brucei (Table 20.1)[32]. The apparent essentiality of metacaspases in T. brucei has led to the develop-ment of a series of inhibitors for TbMCA2 and TbMCA3 on the basis of knownsubstrate specificity and the predicted catalytic mechanism of these enzymes. Someof the newly developed compounds (derived from a-amino-protected arginine withan a-ketoheterocyclic P10 warhead; Figure 20.2) inhibit the enzymatic activity ofTbMCA2 and TbMCA3with IC50 values in the low micromolar range and displaymodest trypanocidal activities in vitrowith low cytotoxicity [61]. Further optimizationand in vivo testing of the compounds are necessary before TbMCAs can be regardedas validated drug targets for HAT.

TbGPI8

Although TbGPI8 has been shown to be essential for bloodstream forms of T. brucei(Table 20.1) [34], it remains to be demonstrated whether the differences between


substrate specificities and functions of mammalian and parasite enzymes aresufficient for the development of selective inhibitors.

TbOPB and TbPOP

As yet, TbOPB has not been genetically validated to be essential for the survival ofT. brucei (Table 20.1). Studies with peptidyl chloromethylketones and peptidylphosphonate diphenyl esters, two groups of irreversible serine peptidase inhibitors,have been shown to inhibit trypsin-like protease activity of bloodstream forms of T.brucei and to display modest trypanocidal activities in vitro [62]. Active-site labelingstudies revealed that both groups of inhibitors primarily target an 80-kDa protein,indicative of TbOPB [62]. One of these inhibitors, carbobenzyloxyglycyl-4-amidino-phenylglycine phosphonate diphenyl ester (Cbz-Gly-(4-AmPhGly)p(OPh)2; Fig-ure 20.3), was able to cure a T. brucei infection in mice at a dosage of5mg/kg/day [62]. These findings indicate that TbOPB may represent a potentialchemotherapeutic target in T. brucei.Prolylisoxazoles (Figure 20.4), prolylisoxazolines (Figure 20.4), and JTP-4819,

specific inhibitors of prolyloligopeptidases were shown to inhibit the growth ofbloodstream forms of T. brucei in vitro with 50% growth inhibition values in thelow micromolar range [38,63]. As the inhibitors are not toxic to mammalian cells

Figure 20.2 Structure of an a-amino-protectedarginine with an a-ketobenzothiazole P10

warhead and a leucine residue at the level of theP2 position with inhibitory activity against

TbMCA2 and in vitro trypanocidal activityagainst bloodstream forms of T. brucei. Thedesignation “compound 18” refers to notationused in [61].


at the concentrations used in the tests [38], it is likely that the observedtrypanocidal activities of the compounds are due to the inhibition of TbPOP.On the other hand, RNAi against TbPOP did not produce any phenotype inbloodstream and procyclic forms (Table 20.1) [38], raising questions about therequirement of TbPOP for T. brucei. However, as the ablation of TbPOP wasincomplete, the remaining activity of about 20% of the protein could explain thesurvival of the parasites [38]. Further research on the action of mechanism ofTbPOP inhibitors is necessary to establish structure–activity relationships of thesecompounds.

Figure 20.4 Structure of prolylisoxazole and prolylisoxazoline compounds with in vitrotrypanocidal activity against bloodstream forms of T. b. rhodesiense. The designations “compound3d” and “compound 3f” refer to denotations used in [63].

Figure 20.3 Structure of a peptidyl serine peptidase inhibitor with inhibitory activity againstTbOPB, and in vitro and in vivo activity against bloodstream forms of T. brucei [62].


TbMSP-B

Bloodstream form Dmsp-bmutants, in which all four tandem TbMSP-B genes fromboth chromosomal alleles were deleted, show no significant difference in growthin vitro compared to wild-type bloodstream forms (Table 20.1) [54]. This findingindicates that TbMSP-B is not essential for normal growth of T. brucei bloodstreamforms. Nevertheless, some peptidomimetic inhibitors of mammalian zinc metal-loproteases (SmithKline Beecham Pharmaceuticals compounds BRL29808,BRL49244, BRL57240, and SB201140; Figure 20.5) were found to be trypanocidalin vitro with growth inhibitory activities in the low micromolar range [64]. Althoughthree of these compounds (BRL29808, BRL57240, and SB201140) were able toinactivate GP63, the TbMSP-B homolog of Leishmania major [64], this observationdoes not prove that the TbMSP-B is the inhibition target in bloodstream-formtrypanosomes. It is also possible that these peptidomimetic inhibitors exert theirtrypanocidal activities against bloodstream forms of T. brucei by targeting otherenzymes than metalloproteases.

Proteasome

The necessity of the proteasome for survival of T. brucei has been genetically andchemically validated (Table 20.1). By using RNAi against all b-subunits genes of the

Figure 20.5 Structures of peptidomimetic zinc metalloproteases inhibitors with in vitrotrypanocidal activity against bloodstream forms of T. brucei [64].


proteasome, it was shown that the three catalytic active b1-, b2-, and b5-subunits(TbPSB1, TbPSB2, and TbPSB5) are vital for T. brucei [55]. All proteasome inhibitorstested so far displayed substantial trypanocidal activities with 50% growth inhibitionvalues in the nanomolar range [46,47,65–68]. In addition, the peptide boronateinhibitor MG-262 (Figure 20.6) was shown to slow the growth of T. brucei in theblood of infected mice [42] indicating that proteasome inhibitors display trypano-cidal activity in vivo. The observed differences in peptidase activity, substratespecificity, and inhibitor sensitivity between the trypanosomal and the mammalianproteasome [42] makes this enzyme complex an interesting drug target for sleepingsickness. As the trypanosomal proteasome is particularly sensitive to inhibitors ofthe trypsin-like activity [46,47,66], agents targeting this activity would be the rationalchoice for future anti-trypanosomal drug development.

Conclusion

Proteases have been validated as targets for the development of new drugs againstHAT. Importantly, proteases are druggable targets as verified by the developmentof anti-protease drugs as effective therapies for many human diseases. The mostpromising targets for anti-trypanosomal drugs are the lysosomal cysteine proteaseTbCATL and the proteasome. The research to target TbCATL and the proteasomeshould benefit from the intense interest by pharmaceutical companies to designprotease inhibitors as therapeutics for arthritis, osteoporosis, and various cancers.In addition, the development of new inhibitors to target proteases for thetreatment of other protozoan infections will provide new avenues for noveltrypanocidal agents.

References

1 Molyneux, D.H., Pentreath, V., and Doua,F. (1996) African trypanosomiasis in man,inManson’s Tropical Diseases (ed. G.C.Cook), Saunders, London, pp. 1171–1196.

2 World Health Organization (2005) Controlof human African trypanosomiasis: a strategyfor the African region. AFRO(AFR/RC55/11), WHO, Geneva.

Figure 20.6 Structure of the proteasome inhibitor MG-262 with promising in vitro and in vivoactivity against bloodstream forms of T. brucei [42].


3 World Health Organization (2010) Africantrypanosomiasis (sleeping sickness). FactSheet 259, WHO, Geneva.

4 Steverding, D. (2010) The development ofdrugs for treatment of sleeping sickness: ahistorical review. Parasit. Vectors, 3, 15.

5 Fairlamb, A.H. (2003) Chemotherapy ofhuman African trypanosomiasis: currentand future prospects. Trends Parasitol., 19,488–494.

6 Matovu, E., Seebeck, T., Enyaru, J.C., andKaminsky, R. (2001) Drug resistance inTrypanosoma brucei ssp., the causative agentsof sleeping sickness in man and nagana incattle. Microbes Infect., 3, 763–770.

7 Delespaux, V. and de Koning, H.P. (2007)Drugs and drug resistance in Africantrypanosomiasis. Drug Resist. Updat., 10,30–50.

8 Steiger, R.F., Van Hoof, F., Bontemps, J.,Nyssens-Jadin, M., and Druetz, J.E. (1979)Acid hydrolases of trypanosomatidflagellates. Acta Trop., 36, 335–341.

9 North, M.J., Coombs, G.H., and Barry, J.D.(1983) A comparative study of theproteolytic enzymes of Trypanosoma brucei,T. equiperdum, T. evansi, T. vivax,Leishmania tarentolae and Crithidiafasciculate.Mol. Biochem. Parasitol., 9,161–180.

10 Lonsdale-Eccles, J.D. and Mpimbaza, G.W.(1986) Thiol-dependent proteases ofAfrican trypanosomes. Analysis byelectrophoresis in sodium dodecylsulphate/polyacrylamide gels co-polymerized with fibrinogen. Eur. J.Biochem., 155, 469–473.

11 Lonsdale-Eccles, J.D. and Grab, D.J. (1987)Lysosomal and non-lysosomal peptidylhydrolases of the bloodstream forms ofTrypanosoma brucei brucei. Eur. J. Biochem.,169, 467–475.

12 Boutignon, F., Huet-Duvillier, G.,Demeyer, D., Richet, C., and Degand, P.(1990) Study of proteolyitc activitiesreleased by incubation of trypanosomes(Trypanosoma brucei brucei) in pH 5.5 and7.0 phosphate/glucose buffers. Biochim.Biophys. Acta, 1035, 369–377.

13 Robertson, C.D., North, M.J., Lockwood, B.C., and Coombs, G.H. (1990) Analysis ofthe proteinases of Trypanosoma brucei.J. Gen. Microbiol., 136, 921–925.

14 Mbawa, Z.R., Gumm, I.D., Fish, W.R., andLonsdale-Eccles, J.D. (1991) Endopeptidasevariations among different life-cycle stagesof African trypanosomes. Eur. J. Biochem.,195, 183–190.

15 Caffrey, C.R. and Steverding, D. (2009)Kinetoplastid papain-like cysteinepeptidases.Mol. Biochem. Parasitol., 167,12–19.

16 Caffrey, C.R., Lima, A.P., and Steverding,D. (2011) Cysteine peptidases ofkinetoplastid parasites. Adv. Exp. Med.Biol., 712, 84–99.

17 Lecaille, F., Kaleta, J., and Br€omme, D.(2002) Human and parasitic papain-likecysteine proteases: their role in physiologyand pathology and recent developments ininhibitor design. Chem. Rev., 102,4459–4488.

18 Mackey, Z.B., O’Brien, T.C., Greenbaum,D.C., Blank, R.B., and McKerrow, J.H.(2004) A cathepsin B-like protease isrequired for host protein degradation inTrypanosoma brucei. J. Biol. Chem., 279,48426–48433.

19 Mottram, J.C., North, M.J., Barry, J.D., andCoombs, G.H. (1989) A cysteine proteinasecDNA from Trypanosoma brucei predicts anenzyme with an unusual C-terminalextension. FEBS Lett., 258, 211–215.

20 Berriman, M., Ghedin, E., Hertz-Fowler,C., Blandin, G., Renauld, H., Bartholomeu,D.C., Lennard, N.J. et al. (2005) Thegenome of the African trypanosomeTrypanosoma brucei. Science, 309, 416–422.

21 Pamer, E.G., So, M., and Davis, C.E. (1989)Identification of a developmentallyregulated cysteine protease of Trypanosomabrucei.Mol. Biochem. Parasitol., 33, 27–32.

22 Caffrey, C.R., Hansell, E., Lucas, K.D.,Brinen, L.S., Alvarez Hernandez, A.,Cheng, J., Gwaltney, S.L.2nd et al. (2001)Active site mapping, biochemicalproperties and subcellular localization ofrhodesain, the major cysteine protease ofTrypanosoma brucei rhodesiense.Mol.Biochem. Parasitol., 118, 61–73.

23 Goll, D.E., Thompson, V.F., Li, H., Wei, W.,and Cong, J. (2003) The calpain system.Physiol. Rev., 83, 731–801.

24 Ersfeld, K., Barraclough, H., and Gull, K.(2005) Evolutionary relationships andprotein domain architecture in an

References j379

expanded calpain superfamily inkinetoplastid parasites. J. Mol. Evol., 61,742–757.

25 Liu, W., Apagyi, K., McLeavy, L., andErsfeld, K. (2010) Expression and cellularlocalisation of calpain-like proteins inTrypanosoma brucei.Mol. Biochem.Parasitol., 169, 20–26.

26 Uren, A.G., O’Rourke, K., Aravind, L.A.,Pisabarro, M.T., Seshagiri, S., Koonin, E.V.,and Dixit, V.M. (2000) Identification ofparacaspases and metacaspases: twoancient families of caspase-like proteins,one of which plays a key role in MALTlymphoma.Mol. Cell, 6, 961–967.

27 Vercammen, D., van de Cotte, B., DeJaeger, G., Eeckhout, D., Casteels, P.,Vandepoele, K., Vandenberghe, I. et al.(2004) Type II metacaspases Atmc4 andAtmc9 of Arabidopsis thaliana cleavesubstrate after arginine and lysine. J. Biol.Chem., 279, 45329–45336.

28 Moss, C.X., Westrop, G.D., Juliano, L.,Coombs, G.H., and Mottram, J.C. (2007)Metacaspase 2 of Trypanosoma brucei is acalcium-dependent cysteine peptidaseactive without processing. FEBS Lett., 581,5635–5639.

29 Szallies, A., Kubata, B.K., and Duszenko,M. (2002) A metacaspase of Trypanosomabrucei causes loss of respirationcompetence and clonal death in the yeastSaccharomyces cerevisiae. FEBS Lett., 517,144–150.

30 Mottram, J.C., Helms, M.J., Coombs,G.H., and Sajid, M. (2003) Clan CDcysteine peptidases of parasitic protozoa.Trends Parasitol., 19, 182–187.

31 Proto, W.R., Castanys-Munoz, E., Black, A.,Tetley, L., Moss, C.X., Juliano, L., Coombs,G.H., and Mottram, J.C. (2011)Trypanosoma bruceimetacaspase 4 is apseudopeptidase and a virulence factor.J. Biol. Chem., 286, 39914–39925.

32 Helms, M.J., Ambit, A., Appleton, P.,Tetley, L., Coombs, G.H., and Mottram,J.C. (2006) Bloodstream form Trypanosomabrucei depend upon multiple metacaspasesassociated with RAB11-positiveendosomes. J. Cell Sci., 119, 1105–1117.

33 Kang, X., Szallies, A., Rawer, M., Echner,H., and Duszenko, M. (2002) GPI anchortransamidase of Trypanosoma brucei:

in vitro assay of the recombinant proteinand VSG anchor exchange. J. Cell Sci., 115,2529–2539.

34 Lillico, S., Field, M.C., Blundell, P.,Coombs, G.H., and Mottram, J.C. (2003)Essential roles for GPI-anchored proteinsin African trypanosomes revealed usingmutants deficient in GPI8.Mol. Biol. Cell,14, 1182–1194.

35 Troeberg, L., Pike, R.N., Morty, R.E., Berry,R.K., Coetzer, T.H., and Lonsdale-Eccles,J.D. (1996) Proteases from Trypanosomabrucei brucei. Purification, characterisationand interactions with host regulatorymolecules. Eur. J. Biochem., 238, 728–736.

36 Morty, R.E., Lonsdale-Eccles, J.D., Mentele,R., Auerswald, E.A., and Coetzer, T.H.(2001) Trypanosome-derivedoligopeptidase B is released into theplasma of infected rodents, where itpersists and retains full catalytic activity.Infect. Immun., 69, 2757–2761.

37 Morty, R.E., Shih, A.Y., F€ul€op, A., andAndrews, N.W. (2005) Identification of thereactive cysteine residues in oligopeptidaseB from Trypanosoma brucei. FEBS Lett.,579, 2191–2196.

38 Bastos, I.M., Motta, F.N., Charneau, S.,Santana, J.M., Dubost, L., Augustyns, K.,and Grellier, P. (2010) Prolyl oligopeptidaseof Trypanosoma brucei hydrolyzes nativecollagen, peptide hormones and is active inthe plasma of infected mice.MicrobesInfect., 12, 457–466.

39 LaCount, D.J., Gruszynski, A.E.,Grandgenett, P.M., Bangs, J.D., andDonelson, J.E. (2003) Expression andfunction of the Trypanosoma bruceimajorsurface protease (GP63) genes. J. Biol.Chem., 278, 24658–24664.

40 El-Sayed, N.M. and Donelson, J.E. (1997)African trypanosomes have differentiallyexpressed genes encoding homologues ofthe Leishmania GP63 surface protease.J. Biol. Chem., 272, 26742–26748.

41 Coux, O., Tanaka, K., and Goldberg, A.L.(1996) Structure and function of the 20Sand 26S proteasome. Annu. Rev. Biochem.,65, 801–847.

42 Steverding, D. (2007) The proteasome as apotential target for chemotherapy ofAfrican trypanosomiasis. Drug Dev. Res.,68, 205–212.


43 Hua, S., To, W.Y., Nguyen, T.T., Wong,M.L., and Wang, C.C. (1996) Purificationand characterization of proteasomes fromTrypanosoma brucei.Mol. Biochem.Parasitol., 78, 33–46.

44 Huang, L., Jacob, R.J., Pegg, S.C., Baldwin,M.A., Wang, C.C., Burlingname, A.L., andBabbitt, P.C. (2001) Functional assignmentof the 20 S proteasome from Trypanosomabrucei using mass spectrometry and newbioinformatics approaches. J. Biol. Chem.,276, 28327–28339.

45 Wang, C.C., Bozdech, Z., Liu, C.L.,Shipway, A., Backes, B.J., Harris, J.L., andBogyo, M. (2003) Biochemical analysis ofthe 20 S proteasome of Trypanosoma brucei.J. Biol. Chem., 278, 15800–15808.

46 Glenn, R.J., Pemberton, A.J., Royle, H.J.,Spackmann, R.W., Smith, E., Rivett, A.J.,and Steverding, D. (2004) Trypanocidaleffect of a0,b0-epoxyketones indicates thattrypanosomes are particularly sensitive toinhibitors of proteasome trypsin-like activity.Int. J. Antimicrob. Agents, 24, 286–289.

47 Steverding, D., Pemberton, A.J., Royle, H.,Spackman, R.W., and Rivett, A.J. (2006)Evaluation of the anti-trypanosomal activityof tyropeptin A. Planta Med., 72, 761–763.

48 Scory, S., Caffrey, C.R., Stierhof, Y.D.,Ruppel, A., and Steverding, D. (1999)Trypanosoma brucei: killing of bloodstreamforms in vitro and in vivo by the cysteineproteinase inhibitor Z-Phe-Ala-CHN2. Exp.Parasitol., 91, 327–333.

49 Abdulla, M.H., O’Brien, T., Mackey, Z.B.,Sajid, M., Grab, D.J., and McKerrow,J.H. (2008) RNA interference ofTrypanosoma brucei cathepsin B and Laffects disease progression in a mousemodel. PLoS Negl. Trop. Dis., 2, e298.

50 Nikolskaia, O.V., Lima de A., A.P., Kim,Y.V., Lonsdale-Eccles, J.D., Fukuma, T.,Scharfstein, J., and Grab, D.J. (2006)Blood–brain barrier traversal by Africantrypanosomes requires calcium signallinginduced by parasite cysteine protease.J. Clin. Invest., 116, 2739–2747.

51 Olego-Fernandez, S., Vaughan, S., Shaw,M.K., Gull, K., and Ginger, M.L. (2009) Cellmorphogenesis of Trypanosoma bruceirequires the paralogous, differentiallyexpressed calpain-related proteins CAP5.5and CAP5.5V. Protist, 160, 576–590.

52 Ndung’u, J.M., Wright, N.G., Jennings,F.W., and Murray, M. (1992) Changes inatrial natriuretic factor and plasma renninactivity in dogs infected with Trypanosomabrucei. Parasitol. Res., 78, 553–556.

53 Antoine-Moussiaux, N., B€uscher, P., andDesmecht, D. (2009) Host-parasiteinteractions in trypanosomiasis: on the wayto an antidisease strategy. Infect. Immun.,77, 1276–1284.

54 Grandgenett, P.M., Otsu, K., Wilson, H.R.,Wilson, M.E., and Donelson, J.E. (2007) Afunction for a specific zinz metalloproteaseof African trypanosomes. PLoS Pathog., 3,1432–1445.

55 Li, Z., Zou, C.B., Yao, Y., Hoyt, M.A.,McDonough, S., Mackey, Z.B., Coffino, P.,and Wang, C.C. (2002) An easilydissociated 26 S proteasome catalyzes anessential ubiquitin-mediated proteindegradation pathway in Trypanosomabrucei. J. Biol. Chem., 277, 15486–15498.

56 Mutomba, M.C., To, W.Y., Hyun, W.C., andWang, C.C. (1997) Inhibition ofproteasome activity blocks cell cycleprogression at specific phase boundaries inAfrican trypanosomes.Mol. Biochem.Parasitol., 90, 491–504.

57 Troeberg, L., Morty, R.E., Pike, R.N.,Lonsdale-Eccles, J.D., Palmer, J.T.,McKerrow, J.H., and Coetzer, T.H. (1999)Cysteine proteinase inhibitors kill culturedbloodstream forms of Trypanosoma bruceibrucei. Exp. Parasitol., 91, 349–355.

58 Caffrey, C.R., Scory, S., and Steverding, D.(2000) Cysteine proteinases of trypanosomeparasites: novel targets for chemotherapy.Curr. Drug Target, 1, 155–162.

59 Steverding, D., Sexton, D.W., Wang, X.,Gehrke, S.S., Wagner, G.K., and Caffrey,C.R. (2012) Trypanosoma brucei: chemicalevidence that cathepsin L is essential forsurvival and a relevant drug target. Int.J. Parasitol., 42, 481–488.

60 Subramaniam, C., Veazey, P., Redmond,S., Hayes-Sinclair, J., Chambers, E.,Carrington, M., Gull, K. et al. (2006)Chromosome-wide analysis of genefunction by RNA interference in theAfrican trypanosome. Eukaryot. Cell, 5,1539–1549.

61 Berg, M., Van der Veken, P., Joossens, J.,Muthusamy, V., Breugelmans, M., Moss,

References j381

C.X., Rudplf, J. et al. (2010) Design andevaluation of Trypanosoma bruceimetacaspase inhibitors. Bioorg. Med. Chem.Lett., 20, 2001–2006.

62 Morty, R.E., Treoberg, L., Powers, J.C.,Ono, S., Lonsdale-Eccles, J.D., and Coetzer,T.H. (2000) Characterisation of theantitrypanosomal activity of peptidyla-aminoalkyl phosphonate diphenyl esters.Biochem. Pharmacol., 60, 1497–1504.

63 Bal, G., Van der Veken, P., Antonov, D.,Lambier, A.M., Grellier, P., Croft, S.L.,Augustyns, K., and Haemers, A. (2003)Prolylisoxazoles: potent inhibitors ofprolyloligopeptidase with antitrypanosomalactivity. Bioorg. Med. Chem. Lett., 13,2875–2878.

64 Bangs, J.D., Ransom, D.A., Nimick, M.,Christie, G., and Hooper, N.M. (2001)In vitro cytocidal effects on Trypanosomabrucei and inhibition of LeishmaniamajorGP63 by peptidomimetic metalloprotease

inhibitors.Mol. Biochem. Parasitol., 114,111–117.

65 Nkemgu-Njinkeng, J., Rosenkranz, V.,Wink, M., and Steverding, D. (2002)Antitrypanosomal activities of proteasomeinhibitors. Antimicrob. Agents Chemother.,46, 2038–2040.

66 Steverding, D., Spackman, R.W., Royle,H.J., and Glenn, R.J. (2005) Trypanocidalactivities of trileucin methyl vinyl sulfoneproteasome inhibitors. Parasitol. Res., 95,73–76.

67 Steverding, D., Baldisserotto, A., Wang, X.,and Marastoni, M. (2011) Trypanocidalactivity of peptidyl vinyl ester derivativesselective for inhibition of mammalianproteasoem trypsin-like activity. Exp.Parasitol., 128, 444–447.

68 Steverding, D., Wang, X., Potts, B.C., andPalladino, M.A. (2012) Trypanocidal activityof b-lactone-c-lactam proteasomeinhibitors. Planta Med., 78, 131–134.


Documents

Trypanosomatid Diseases (Molecular Routes to Drug Discovery) || Proteases of Trypanosoma brucei