Current Enzyme Inhibition, 2007, 3, 19-48
Trypanosomatidae Peptidases: A Target for Drugs Development Alane Beatriz Vermelho*,1 , Salvatore Giovanni De Simone2 , Claudia Masini d’Avila-Levy1,2 , André Luis Souza do Santos1 , Ana Cristina Nogueira de Melo1 , Floriano Paes Silva Jr.2 , Elba Pinto da Silva Bon3 and Marta Helena Branquinha1 1Departamento
de Microbiologia Geral, Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), Centro de Ciências da Saúde (CCS), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil 2Departamento
de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ and Departamento de Biologia Celular e Molecular, Universidade Federal Fluminense, Rio de Janeiro, Brazil 3Departamento
de Bioquímica, Instituto de Química, Centro de Tecnologia (CT), UFRJ, Rio de Janeiro, Brazil
Abstract: In most organisms around 2% of the genes code for peptidases being this number only surmounted by the genes that code for transcriptional factors. This ubiquitous presence is almost unequaled and has for long fascinated biochemists. Although peptidases have been classified in four mechanistic classes (aspartic-, cysteine-, serine- and metallo-peptidases), the more recent MEROPS database recognizes 42 evolutionary distinct peptidase structures corresponding to 42 different families. As peptidases are involved in several physiological processes they are an obvious target for the development of therapeutic agents to treat infectious disease. The Trypanosomatidae family includes etiologic agents for human and veterinary diseases, such as Trypanosoma cruzi, Leishmania spp. and the African trypanosomes that are responsible for the Chagas disease, for a wide spectrum of clinical manifestations known as leishmaniasis, and for the “sleeping sickness”, respectively. These microorganisms present a complex life cycle that includes dimorphic developmental stages in distinct hosts and by extension show nutritional adaptation. This review covers the recent advances in the biochemical characterization of trypanosomatid proteolytic enzymes and that of specific inhibitors to block their hydrolytic activity, in accordance to the peptidases potential role as target to the treatment of the aforementioned illnesses.
Keywords: Trypanosomatidae family, Trypanosoma, Leishmania, chagas disease, leishmaniasis, sleeping sickness, trypanosomatidae peptidases, peptidases, proteolysis, peptidases inhibitors, chemotherapy. 1. THE TRYPANOSOMATIDAE FAMILY The Trypanosomatidae family, which encompasses a diverse group of parasitic protozoa, presents a characteristic single flagellum, a sub-pellicle microtubule cytoskeleton and a relatively small kinetoplast containing densely packed DNA that is organized in mini- and maxi-circles [1-3]. Trypanosomatids combine its typical morphology to the ability of infecting a diverse range of hosts. The family includes four genera of digenetic organisms that parasite vertebrate (Trypanosoma, Leishmania and Endotrypanum) or plant (Phytomonas) hosts, with insects or leeches as vectors, and the monogenetic genera (Leptomonas, Crithidia, Blastocrithidia, Herpetomonas and Rhynchoidomonas) that are found largely in hemipteran and dipteran insect hosts with a limited distribution in seven other orders of insects, as well as in ciliates [1, 2]. The African and American trypanosome species and microorganisms of the Leishmania and Phytomonas genera are the etiologic agents of major human, animal and plant parasitic diseases worldwide, being responsible for large socio-economic losses, especially in developing countries . Leishmania spp. are protozoan parasites that live as promastigotes in the digestive tract of sandflies and as amastigotes in the phagolysosomes of *Address correspondence to this author at the Instituto de Microbiologia Prof. Paulo de Góes (IMPPG), CCS, Bl. I, UFRJ, Rio de Janeiro, 21941590, Brazil; E-mail: [email protected]
mammalian macrophages. At least 20 species of Leishmania are known to infect humans, being the cause of a spectrum of clinical manifestations that result in significant morbidity and mortality. It is estimated that 10% of the world’s population is vulnerable to this infection which severity ranges, pending on the causative specie and immunological state of the host, from a simple cutaneous lesion to the disfiguring mucocutaneous leishmaniasis and the visceralized form (kala-azar) that is fatal if untreated . The increase of the incidence and morbidity rates of this disease, the spread of some leishmaniasis forms to new geographical areas and the Leishmania-HIV co-infection, have become a major public health concern worldwide [5, 6]. Trypanosoma cruzi is the etiological agent of the Chagas’ disease, also known as American trypanosomiasis. The development of its life cycle involves an hematophagous insect vector and a mammalian host where it displays an obligate intracellular amastigote replicative form, and the trypomastigote nonreplicative form in the bloodstream. In the insect vector it is also observed the epimastigote replicative form and a nonreplicative infective metacyclic trypomastigote form. Sixteen to eighteen million people are currently infected by the Trypanosoma cruzi and one hundred million are at risk of infection throughout Central and South America, being the Chagas disease responsible for fifty thousand deaths per year . This illness is one of the most serious parasitic diseases in Latin America and is amongst its leading health problems [8, 9]. © 2007 Bentham Science Publishers Ltd.
Current Enzyme Inhibition, 2007, Vol. 3, No. 1
Vermelho et al.
Recent reports have included this disease as an important health problem in the United States because of the possibility of transmission through blood transfusion . Infection in humans normally progresses from an acute stage characterized by systemic parasitemia to an almost asymptomatic stage that can last for several decades. Eventually, one-third of those chronically infected develops severe cardiac and/or gastrointestinal illness. Although there is no cure, the drugs nifurtimox and benznidazole show some effectiveness during the initial stages of the infection. However, the use of these drugs is limited by their extreme toxicity and availability [8, 9]. African trypanosomes are transmitted by the bite of infected tsetse flies. They are the causative agents of sleeping sickness in humans and nagana disease in cattle, where they live in the extracellular environment of the blood and tissue fluids. The protozoan Trypanosoma brucei is divided three subspecies: T. brucei gambiense, which causes a chronic form of sleeping sickness in west and central Africa, T. brucei rhodesiense, which causes an acute form of this desease in east and southern Africa, and T. brucei brucei, not infectious to humans . Over sixty million people from thirty six countries are vulnerable to the sleeping sickness and it is estimated that three to five hundred thousand are already infected with fifty five thousand deaths per year . Trypanosoma congolense, the agent of bovine trypanosomiasis (nagana disease), threats a cattle population of forty six million. The economic loss is estimated in $1340 million dollars annually. In some areas, as subSaharan Africa, this infection has precluded the use of domestic animals as food [13, 14]. The effective treatment of diseases caused by trypanosomatids is still an open issue, nevertheless of paramount importance. Chemotherapy still relies on drugs developed decades ago showing limited efficiency and toxic side effects . On the other hand, new treatments based on the use of lipid formulations of amphotericin B or miltefosine, are too expensive considering the target population. Moreover, it has been reported the emergence of resistance [15, 16]. Efforts to tackle these diseases require research on the molecular components regulating the Table 1.
infection initiation, which is critical for a better understanding of the diseases’ pathogenesis. Another promising line of research targets the peptidases of these parasites aiming the establishment of novel, effective and selective chemotherapies. This review presents the current status and development of peptidase inhibitors as potential drugs to treat trypanosomatid diseases within a background assessment on Trypanosomatidae peptidases. 2. PEPTIDASES The name “peptidase” has been recommended by The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (http://www.chem. qmul.ac.uk/iubmb/enzyme/index.html) for the subset of hydrolases, also known as proteolytic enzymes or proteases, that are able to cleave peptide bond (subclass EC 3.4). The enzymes that hydrolyze peptide bonds at the amino- or carboxy-terminus are classified as exopeptidases (EC 3.4.1119), and those that cleave peptide bonds inside the polypeptide are endopeptidases (EC 3.4.21-25 and EC 3.4.99). The majority of peptidases are endopeptidases and are classified into sub-subclasses according to their catalytic mechanism: serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metalloendopeptidases (EC 3.4.24) and threonine endopeptidases (EC 3.4.25). Endopeptidases that are not assigned to the aforementioned sub-subclasses EC 3.4.21-25 are grouped in the sub-subclass EC 3.4.99. The MEROPS database (http://merops.sanger.ac.uk) is a tool to address the difficult task of peptidases classification. It provides a catalogue and a structure-based classification whereby homologous sets of peptidases are grouped into families and the homologous families into clans . Every peptidase, family and clan is identified by a capital letter that shows the catalytic type of the peptidases contained in the group: ‘A’ (aspartic), ‘C’ (cysteine), ‘G’ (glutamic), ‘M’ (metallo), ‘S’ (serine), ‘T’ (threonine), ‘U’ (unknown type) or in certain clan names, ‘P’ (protein nucleophile: for clans of mixed - C, S or T - catalytic type). The identifier of a
MEROPS Identifiers and NC-IUBMB Classification of Trypanosomatid Peptidases Investigated as Drug Targets
Cruzipain Oligopeptidase B
Trypanosoma congolense Trypanosoma brucei rhodesiense Trypanosoma brucei brucei
Congopain Rhodesain Brucipain
CPA CPB CPC Leishmanolysin / Gp63 Methionyl aminopeptidase 2 Leucyl aminopeptidase
Catalytic Type Endo/Exopeptidase Clan
Merops ID Identifier
126.96.36.199 * *
C01.075 C01.072 C01.072
* * *
C01.076 C01.074 C01.098
Endopeptidase Exopeptidase Exopeptidase
MA MG MF
M8 M24 M17
188.8.131.52 184.108.40.206 220.127.116.11
M08.001 ** **
Nomenclature for peptidases described in the MEROPS database ( http://www.merops.sanger.ac.uk), including name, catalytic type, reaction NC-IUBMB number (Nomenclature Committee of International Union of Biochemistry and Molecular Biology) and MEROPS identifiers . *Not yet included in IUBMB recommendations. **Not included in MEROPS.
catalysed, clans and families,
clan consists of two letters indicating the catalytic type followed by a serial letter. A family name consists of the catalytic type letter followed by a serial number of up to two digits. This classification forms a framework around which a wealth of supplementary information on peptidases is presented. Table 1 shows trypanosomatid peptidases with potential use as drug target, its MEROPS identification code, and other MEROPS database relevant informations. This database has been recently expanded to include peptidases inhibitors that are similarly classified. 3. TRYPANOSOMATID PEPTIDASES Over the past few years it has become clear that peptidases from the pathogenic trypanosomatids play an important role in several steps of the host infection: absorption, penetration, intracellular survival, replication, differentiation, infectivity, immune evasion and nutrition. Trypanosomatids present a large and varied array of intracellular and/or extracellular peptidases whose regulated expression entails specific functions in the parasite life-cycle stages. Many peptidases have been purified and characterized and their genes cloned and sequenced. It is generally recognized that the parasite peptidases are previleged targets for novel antiparasitic agents due to their unusual structural features (Table 1). Besides, the immunodominant nature of many peptidases is valuable for serodiagnosis and vaccine development [19-28]. Trypanosomatid peptidases may also contribute as a model to study the evolution and function of peptidases in general through the comparison of amino acid sequences, molecular three-dimensional structures and their biochemical mechanism of action as changes may have arisen from new demands throughout evolution [18, 22, 29, 30]. 3.1. Cysteine Peptidases (EC 3.4.22) Cysteine peptidases present a catalytic cysteine residue, which mediates protein hydrolysis via a nucleophilic attack on the carbonyl carbon of a susceptible peptide bond. Clan CA contains all the families of cysteine peptidases showing a structure similar to that of papain although other families are assigned to this clan based on their sequence motifs. In addition to the residues Cys158 and His292 of the catalytic dyad, the other functionally important residues are Gln152, that helps the formation of the ‘oxyanion hole’, an electrophilic center that stabilizes the tetrahedral intermediate, and Asn308 (sometimes Asp in families C12, C19, C28 and C39), which is thought to orientate the imidazolium ring of the catalytic His. The order of these residues in the sequence is Gln, Cys, His, Asn/Asp, that corresponds in mature papain to Gln19, Cys25, His159 and Asn175 (MEROPS database, http://merops.sanger.ac.uk). Members of Clan CA are either targeted to intracellular vesicle compartments or are secreted. Family C1, a group typified by the plant enzyme papain but that also includes the mammalian cysteine peptidases cathepsin B, C, F, L and S, belongs to the Clan CA and is the most studied family of peptidases in trypanosomatids. Cysteine peptidases from trypanosomatids show homology with cathepsins B and L [18, 24, 31]. Hydrolysis of small peptides that contain arginine at P 2 has often been used to discriminate capthepsin
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 21
B from cathepsin L activity. Specificity at the S2 pocket has been ascribed to the chemical properties of the residue corresponding to position 205 in the papain numbering, and identified in the human cathepsin B and cathepsin L as glutamate and alanine, respectively. In addition, cathepsin L has a conserved inter-space motif in the pro-region, Glu-X3Arg-X2-(Ile/Val)-Phe-X2-Asn-X3-Ile-X3-Asn (“ERFNIN”, named after the single letter code for amino acids, being “X” any amino acid). Cathepsin B lacks the ERFNIN motif but does have an inserted peptide loop in the catalytic domain referred as the “occluding loop”, which is related to an additional dipeptidyl carboxypeptidase activity. The cathepsin B and L subfamilies can be further delineated by the length and sequence similarity with the respective proregions as well as the number and order of cysteine residues involved in the disulfide bond formation. In contrast to the cysteine peptidases of higher plant and mammalian cells, the enzymes of early branched eukaryotes and metazoan play physiological roles in a variety of chemical environments and cellular compartments. Thus, the biological importance and distribution of cysteine peptidases is larger than originally proposed for the cathepsins [32, 33]. In trypanosomatids, cysteine peptidases are crucial for a range of important biological processes, including general catabolic functions, intracellular protein degradation (endosomal/lysosomal system), protein processing, adhesion, immunoevasion and pathogenesis. This group includes cruzipain of T. cruzi, congopain, rhodesain and brucipain of African trypanosomes and the CPA, CPB and CPC multigene peptidase family of Leishmania spp. . It has also been established that cysteine peptidases are promising chemotherapeutic and vaccine targets [23, 24, 35, 36, 37]. Indeed, inhibitors of cruzipain, a specific and essential cysteine peptidase from T. cruzi, are already in preclinical studies . Members of clan CA are either targeted to intracellular vesicle compartments or are secreted, and thus possess a leader peptide. Cruzipain-Cruzain-gp57/51 from Trypanosoma cruzi Cruzipain, the major cysteine peptidase in Trypanosoma cruzi (Fig. (1)), also called cruzain (the recombinant catalytic domain of cruzipain) or gp57/51, is differentially expressed in the four main stages of the parasite life cycle, with 10fold higher levels in the reservosome of epimastigotes . This enzyme, with a molecular mass ranging from 40 to 60 kDa pending on the electrophoretic conditions, is an endopeptidase able to digest several protein substrates including casein, bovine serum albumin, denatured hemoglobin and also small peptides . Although it has been reported to be specific to the lysosomal compartment, plasma membrane-bound isoforms of cysteine peptidases have been shown in the different developmental stages of T. cruzi [42, 43, 44, 45]. The cruzipain is encoded by numerous genes (up to 130 in Tul 2 strain) that contain no introns, encodes a signal peptide, a pro-peptide, the mature enzyme and, as for all C1 family from trypanosomatids, a characteristic C-terminal domain. As in cysteine peptidases from Trypanosoma rangeli and Crithidia fasciculata but in contrast to similar enzymes from Leishmania and T. brucei, cruzipain N-terminal is retained in the natural mature form of the enzyme. This extension is 130 amino acids residues long, contains a number of post-translation modifications
Current Enzyme Inhibition, 2007, Vol. 3, No. 1
Vermelho et al.
Fig. (1). Cruzipain and MSP three-dimensional structures. A. Cruzipain is folded into two domains: one mainly α-helical, the Ldomain (left), and the other composed of β-sheets, the R-domain (right). Residues Cys25, His159 and Asn175 (papain numbering system) make up the “catalytic triad” and are clustered in the cleft between the two domains. B. Close-up of the marked area in “A” showing hydrogen bonding (HB) interactions (dashed lines) in the covalent adduct with benzyloxycarbonyl-tyrosinyl-alanylfluoromethyl ketone (Cbz-Tyr-Ala-FMK, or Cbz-YA adduct). HB partners and the catalytic triad residues are shown in sticks while the protein backbone is depicted in ribbons. For clarity, picture elements far from the central plane in the axis perpendicular to the page are either shown in lighter tones or simply omitted. C. MSP is a compact molecule containing predominantly β-sheet secondary structure. The crystallized protein consists of three domains: the N-terminal (top-right dark grey), central (light grey) and C-terminal (bottom dark grey) domains. D. MSP is clearly a metzincin class zinc peptidase, but does not satisfy the defining sequence motif HExxHxxGxxH because of the insert between the glycine (Gly271 ) and the last histidine (His 334 ). Metzincins are characterized by the presence of an unusual tight 1,4 ‘met-turn’ with a conserved methionine at the base of the active site (Met345 ). The zinc atom (represented as a solid sphere) is coordinated to the nitrogen atoms of His264 , His 268 and His334 in a similar fashion to other zinc metallopeptidases.
and most antibodies present in serum from patients with chronic Chagas' disease are directed against it . Recently, sulphated high-mannose type oligosaccharides were identified in cruzipain . Considering the possible functions of this enzyme, it has been implicated in a number of cellular processes including cell invasion, in addition to its role in parasite nutrition as its major lysosomal peptidase [24, 48]. It is also an immunodominant antigen recognized during human infection, with a possible participation in the defense of the parasite against the immune system of the mammalian host. The enzyme also displays a role in the parasite differentiation process that leads from one stage to the next in its life cycle [49, 50]. Previous studies have shown that cruzipain is able to initiate the cell death program by direct activation of caspase zymogens 3 and 6 . Selective inhibitors of this peptidase block the proliferation of both extracellular epimastigotes and intracellular amastigotes and arrest metacyclogenesis in vitro, indicating that the enzyme
performs essential functions for parasite survival, differentiation and growth [33, 52, 53]. Other possible role for cruzipain has been proposed based on its ability to induce the production of the proinflammatory peptide Lysbradikinin directly by proteolysis of kininogen or by activation of plasmatic pre-kallikrein [54, 55]. In adittion to the cathepsin L-like cruzipain, all T. cruzi life cycle forms express a cathepsin B-like cysteine peptidase with a molecular mass of 30 kDa, which may also be considered a virulence factor, as evidenced by its overexpression in a parasite strain resistant to the cruzipain inhibitor . Recently, specific antibodies against the cathepsin B-like cysteine peptidase from T. cruzi were detected in chagasic patients and in patients with mucocutaneous leishmaniasis and kala-azar patients. However, the activity of T. cruzi cysteine peptidase was not inhibited by the chagasic patient’s sera. To date, no study has been done using the 30 kDa cysteine peptidase as drug target for peptidases inhibitors has been done .
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 23
Rhodesain, Brucipain and Congopain from African Trypanosomes
neutral/alkaline pH, and strictly requires acidic conditions for its activity .
The major lysosomal cysteine peptidases of African trypanosomes are a target for new chemotherapy agents using synthetic peptidase inhibitors. These cathepsin L-like enzymes are termed rhodesain in T. brucei rhodesiense and brucipain, also known as trypanopain–tb, in T. brucei brucei [58, 59]. Brucipain from T. brucei is a group of lysosomal cathepsin L-like cysteine peptidases. The enzyme is developmentally regulated, displaying 10 to 15 fold higher levels of expression in the short stumpy form than in either the long slender or in the procyclic (insect) forms. The enzyme has been purified, cloned and expressed, showing homology with other cathepsin L-like enzymes in trypanosomatids . Anti-rhodesain antibodies localized the enzyme in the lysosome in the long slender form of T. brucei brucei and also in all life-cycle stages of T. brucei rhodesiense. In this latter parasite, the expression of cysteine peptidase was five fold higher in short stumpy trypanosomes than in the other stages. Both enzymes have a molecular mass of 40 kDa. Other peptidases showing 33 and 29 kDa may represent differentially glycosylated forms of cysteine peptidases lacking the C-terminal domain, or other minor cysteine peptidases. The 40 kDa value is consistent with the mature glycosylated peptidase bound to its C-terminal domain .
High titres of anti-congopain IgG were detected in trypanosome-infected trypanotolerant cattle (N’Dama, Bos taurus), and this antibody inhibited the enzyme activity . Cattle were immunized with CP1 and CP2 in order to test the role of these enzymes in the pathology of trypanosomiasis, but immunization had no effect on the establishment of infection and in the development of acute anaemia. However, immunized cattle, unlike control, maintained or gained weight during infection. Their hematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. So, trypanosome cysteine peptidases may play a role in anaemia and immune suppression; conversely, anti-cysteine peptidases antibodies may modulate the trypanosome-induced pathology . Leishmania Cysteine Peptidases: CPA, CPB and CPC
Besides the major cathepsin L-like cysteine peptidase from T. brucei, genomic analysis has identified TbcatB, a cathepsin B-like cysteine peptidase gene, as a target for chemotherapy. Moreover, it is possible that this enzyme, but not rhodesain, is essential for T. brucei survival in culture . Trypanosoma congolense possesses at least two families of closely related cysteine peptidases, named CP1 and CP2. The deduced amino acid sequence of both peptidases is 90% identical; however, they differ in their N-terminal sequence, Ala-Pro-Pro-Ala for CP1 and Ala-Pro-Glu-Ala for CP2. The latter, named congopain, is a cathepsin L-like cysteine peptidase, and has been purified and biochemically characterized from bloodstream forms [62-66]. This peptidase differs only slightly from cruzipain in specificity due to the presence of Leu instead of Glu at position 205 (papain numbering), in the S2 subsite. Cruzipain and congopain have a long C-terminal extension that is linked to the catalytic domain by a repeated sequence (poly-Thr in cruzipain and poly-Pro in congopain), the function of which is unclear and not necessary linked to enzyme activity [65, 67]. Congopain has a marked preference for a proline residue at P2’ position, which is a known feature of trypanosomal cysteine peptidases, and it is active in a broad range of pH, from 4.0 to 8. Although CP1 and CP2 genes are highly homologous, as the pre- and pro-peptides are identical and the C-terminal extensions show 90% identity, the catalytic domain bear more variability (85% identity). Half of the 28 amino acid substitutions result in an increase in the negative charges of CP2, as compared with CP1. Moreover, four of these substitutions consist in the replacement of a basic amino acid (Arg or Lys) by an acidic (Asp or Glu). This is reflected in pI values, 8.3 for CP1 and 4.74 for CP2. These differences also reflect in the enzyme activity: while CP2 appears as a stable protein, CP1 is rapidly inactivated at
Leishmania spp. contain multiple, highly active cysteine peptidase activities. A detailed analysis of the recently completed Leishmania major genome database has revealed that there are genes encoding for a total of 65 cysteine peptidases, grouped into four Clans and 13 Families. Many of these enzymes are likely to play crucial roles in hostparasite interactions being by extension potential targets for drug development. However, the majority of the studies so far developed have dealt with only three types of cysteine peptidases, designated CPA, CPB and CPC, all of them are papain-like and belong to the Clan CA of the Family C1 . CPB are cathepsin L-like peptidases that correspond to the majority of the activity detected and so designated Type I cysteine peptidases. These enzymes, detected in L. major and L. mexicana, are encoded by multicopy genes (8 in the former, and 19 in the latter) arranged in tandem arrays with an unusual C-terminal extension, previously detected in trypanosomal cysteine peptidases. Although the highest level of activity occurs in the amastigote forms, the first two genes of the array in L. mexicana are predominantly transcribed in metacyclic promastigotes. A similar genomic organization of cathepsin L-like genes was described in L. pifanoi and L. donovani complexes . CPA (Type II) peptidase also presents homology to cathepsin L; this enzyme is encoded by a single copy gene and has been detected in the infective forms (amastigotes and metacyclic promastigotes) of L. mexicana and L. major. CPC, also known as Type III peptidase, is cathepsin B-like and is expressed by a single copy gene in all developmental stages of L. mexicana and L. major [22, 70, 72]. Cysteine peptidases appear to be relevant to several aspects of the Leishmania life cycle and of parasite-host relationship. Among others, cysteine peptidases participate in the nutrition of the parasite at the expense of the host, play a role in the successful invasion of host macrophages, and participate in the escape mechanisms of the parasite from the host’s immune system . The generation of null mutants for CPA, CPB and CPC genes in L. mexicana has provided the first genetic support for the key role of leishmanial cysteine peptidases in parasite virulence, and hence their validation as drug targets [22, 23, 70, 73]. Knockout of all the 19 genes of the CPB gene array resulted in a reduced intracellular survival of the null mutant and a
Current Enzyme Inhibition, 2007, Vol. 3, No. 1
delay in the time required for development of lesions in BALB/c mice, although these enzymes were not essential for growth or differentiation of the parasite in vitro. The reinsertion of the amastigote-specific CPB2.8 or metacyclicspecific CPB2 genes into the null mutant failed to restore sustained virulence, and only the re-expression of multiple CPB genes restored infectivity towards macrophages to wildtype levels, suggesting that some complementation of the phenotype has occurred . Interestingly, the CPA/CPB double null mutant (∆cpa/cpb) was not capable of forming lesions in BALB/c, which provides evidence that the enzymes play a complementary role in the interaction of the parasite with the mammalian host. In addition, the immune response of BALB/c mice exposed to ∆cpa/cpb parasites shifted from Th2 to a protective Th1-like response, which suggests a role for cysteine peptidases in the modulation of the host immune response. By contrast, the ∆cpb null mutants were not able to produce lesions in the L. majorresistant C57BL/6 or C3H mice: the virulence of L. mexicana for these strains of mice has been associated with the ability of CPB to inhibit Th1 responses . Wild-type amastigotes have a greater ability to inhibit IL-12 production than ∆cpb, implicating the multiple CPB isoenzymes as important species-specific virulence factors for the L. mexicana complex, and providing one possible explanation to why these parasites, unlike L. major, that has fewer CPB genes and so a lower overall activity, tend to induce chronic lesions in the majority of mouse strains. Furthermore, inserting multicopy L. mexicana CPB genes into L. major increased parasite numbers in C3H mice, and so CPB can be considered as a determinant of increased virulence of L. mexicana for most mouse strains . These findings showed that CPB peptidases somehow help L. mexicana to escape from the microbicidal activity of macrophages. In contrast to the importance of CPBs, the ∆cpa null mutant was phenotipically indistinguishable from the wild type, and the knockout of the CPC gene yielded parasites with reduced infection capacity, but the onset of lesions in mice occurred as rapidly as with wild-type parasites. This suggests that both gene products are not essential for parasite survival, perhaps because the role of the encoded enzymes may have been assumed by other cysteine peptidases, such as those encoded by CPB . Accordingly, it has recently been demonstrated that cysteine peptidases are preferentially expressed in virulent, as opposed to avirulent L. amazonensis promastigotes . In mammals, the major cysteine peptidases are the lysosomal cathepsins B and L and they are implicated in many physiological processes. The L. major cathepsin Blike sequence is 82% and 45% identical with those from L. mexicana and human cathepsin B, respectively. The L. major cathepsin L-like sequence shares 74-75% amino acid identity with the CPB sequence of L. mexicana and L. pifanoi cathepsin L-like enzyme, 45-47% identity with the T. cruzi, T. congolense and the T. brucei major cysteine peptidases, but only 34% identity with human cathepsin L. The high sequence identity shared among the Leishmania cathepsin-L and cathepsin-B sequences, compared to the human enzymes, suggests that inhibitors can be designed to take advantage of the amino acid differences between the host and the parasite in order to increase the specificity against the parasite’s enzymes .
Vermelho et al.
As previously described, CPBs are important virulence factors that modulate the host’s immune response, as well as facilitate the parasite’s survival and growth within the host macrophage. The multiplicity of these genes could be a mechanism of ensuring the synthesis of large amounts of the enzyme. The CPB isoenzymes characterized so far are highly conserved , but some activity differences are apparent, showing that the few amino acid variations at strategic positions are indeed important in modifying the substrate specificities of the isoenzymes, which may provide the parasite with a wide array of hydrolytic activities . Individual isoenzymes encoded by the CPB array differ not only in structure but also in their substrate preferences and their function in the parasite’s interaction with the host, as revealed by the re-expression of different copies of the CPB array in null mutants . This has profound implications for the design of specific inhibitors against these peptidases and their potential as antileishmanial drugs: any inhibitor would need to be applied in a high concentration to effect inhibition of this intense proteolytic activity and might have such a broad specificity in order to inhibit several peptidases that, in combination, are essential in host-parasite interactions. Nevertheless, some cysteine peptidase inhibitors have been found to be effective against Leishmania spp. . The CPBs of L. mexicana are mostly confined to megasomes in amastigote forms, but they are also found in the multi-vesicular tubule-lysosome in promastigotes. The Leishmania route of trafficking of cysteine peptidases to lysosomes is unusual, involving the flagellar pocket and the endosomal pathway [79, 80]. Studies on the effect of cysteine peptidase inhibitors showed that regardless of the chemistry of the inhibitor, the morphologic abnormalities produced in the parasite were the same for all inhibitors that were effective against the peptidase target . As mammalian cysteine peptidases are activated in lysosomes, the host and trypanosomatid parasites appear to have different default pathways for processing and trafficking, which could be exploited for the design of selective cysteine peptidase inhibitors as antiparasitic drugs . The cysteine peptidase inhibitors can selectively eliminate parasitic organisms without undue toxicity to the host. Despite the lack of specificity for Leishmania cysteine peptidases, toxicity is not seen much probably because the concentrations of host peptidases within the lysosomal or other cellular compartments may far exceed the concentration of the comparable parasite targets, and the parasites may also selectively take up and therefore concentrate small molecular mass inhibitors as part of their highly evolved adaptation to intracellular parasitism [23, 24]. 3.2. Serine Peptidases (EC 3.4.21) Serine peptidases with the classic Asp-His-Ser triad belonging to the trypsin and chymotrypsin families are the largest class of peptidases found in data banks. While the function of the catalytic triad and oxyanion hole can be rationalized in terms of electrostatic stabilization of charges developing in the transition state, how remote binding interactions facilitate catalysis remains a mystery. Catalysis and specificity are not simply controlled by a few residues, but are rather a property on the entire protein framework,
controlled via the distribution of charge across a network of hydrogen bonds and perhaps also by the coupling of domain motion to the chemical transformation. Serine peptidases present a unique opportunity to test these ideas . Although serine peptidases are thoroughly investigated, only few members of this class of peptidase have been well characterized in trypanosomatids. Out of these, only two have been used as chemotherapeutic targets: oligopeptidase B, a member of the prolyl oligopeptidase (POP) family found in the cytosol of T. cruzi, African trypanosomes and L. amazonensis, and POP Tc80, another member of the prolyl oligopeptidase family found in T. cruzi. Oligopeptidase B Oligopeptidase B (EC 18.104.22.168) is a member of the prolyl oligopeptidase family of serine peptidases (clan SC, family S9) . As the remaining components of this family, the proteolytic activity is restricted to hydrolysis of low-molecular mass peptides, but the subgroup of oligopeptidase B exhibits trypsin-like substrate specificity, cleaving peptides on the carboxyl side of two adjacent basic residues, not prolyl residues [84, 85]. This proteolytic activity is sensitive to the low-molecular mass serine peptidase inhibitors, as 4-(2-aminoethyl)benzenesulphonyl fluoride (AEBSF) and 3,4-dichloroisocoumarin (DCI), and insensitive to the high-molecular mass serine peptidase inhibitors soybean trypsin inhibitor (STI) and turkey ovomucoid, which indicates that the enzyme is a serine oligopeptidase. Trypsin-like enzymes with properties similar to those of oligopeptidase B have been described in T. cruzi , African trypanosomes of the T. brucei group , T. congolense  and in L. amazonensis . The comparison of the deduced amino acid sequences of the trypanosomal oligopeptidase B genes, including L. major sequence, demonstrated that the overall similarity is 80% and that these enzymes are closely related to the bacterial enzymes, sharing 30% identity with the Escherichia coli oligopeptidase B . No oligopeptidase B enzymes have been identified in or cloned from mammalian cells, but the P 3-P1 specificity of the trypanosomal enzymes parallels that of many mammalian plasma serine peptidases, which could hamper the development of highly specific inhibitors [84, 85, 90]. Nevertheless, the mechanistic features which distinguish oligopeptidase B from the human serine peptidases, as its substrate-dependent temperature sensitivity, the acute sensitivity to thiol-reactive agents, the irregular pH-rate profiles for several substrates and the specific carboxypeptidase activity for basic amino acids, may facilitate drug design to counteract the trypanosomal infections [85, 91, 92]. In T. cruzi, oligopeptidase B is a cytosolic enzyme, and it is directly involved with the parasite ability to invade a wide variety of mammalian cells, and consequently in the establishment of the infection . In mammalian nonphagocytic cells, the invasion by T. cruzi involves a highly localized clustering of host lysosomes, followed by their fusion at the invasion site . Trypomastigote-induced transient increases in the host intracellular calcium concentration are required for the localized lysosome-plasma membrane fusion events that mediate T. cruzi entry . The parasite calcium-signaling activity is linked to a cytosolic serine peptidase, the oligopeptidase B named Tc-
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 25
OP, which generates an active calcium agonist from a cytosolic precursor molecule [86, 96]. This finding was confirmed by the inhibition of the intracellular calcium concentration transients using specific antibodies to the recombinant oligopeptidase B , by the generation of oligopeptidase B null mutants  or by peptidase inhibitors . In African trypanosomes, the cytosolic enzyme, named Tb-OP, is released into the host bloodstream by disrupted parasites, where it persists and retains catalytic activity, and may promote disease pathogenesis through the anomalous degradation of host peptide hormones . The fact that the enzyme retains full catalytic activity when released into the host plasma, even in the presence of many peptidase inhibitors present in the bloodstream, reinforces the importance of its protein-processing activity . Trypanosoma cruzi POP Tc80 A proteolytic activity detected in all the developmental stages of T. cruzi is the POP Tc80 peptidase , being secreted by trypomastigotes and also associated with small vesicles surrounding the flagellar pocket of this infective form [98,99]. The enzyme was classified as a serine peptidase belonging to the prolyl oligopeptidase family (EC 22.214.171.124) due to its property to cleave peptide bonds at the carboxyl side of proline residues in small peptides and also due to its highly significant amino acid sequence identity within the catalytic domain to various bacterial and mammalian prolyl oligopeptidases . Additionally, the unusual inhibitory profile of this group is characterized by the inactivation by the serine peptidase inactivator diisopropyl fluorophosphate (DFP), but not by phenylmethanesulphonyl fluoride (PMSF) and DCI. Its inhibition by some cysteine reagents, such as pchloromercuribenzoate (pCMB), iodoacetamide and peptidyl diazomethanes suggests the presence of a cysteine residue near or at the active site of the enzyme [98,100]. This peptidase mediates the specific degradation of purified human types I and IV collagens, and native type I collagen in rat mesentery at physiological pH. The enzyme may thus facilitate T. cruzi migration through the extracellular matrix, gaining access to cells in virtually any part of the host body, or may be involved in the host cell invasion per se, cleaving collagen interacting with integrin receptors . More recently, Grellier et al.  demonstrated that POP Tc80 is also able to cleave another major extracellular matrix component, fibronectin, and is involved in nonphagocytic mammalian cell invasion. The action of this enzyme on the degradation of the extracellular matrix of the cell being infected led some authors to design specific inhibitors of this proteolytic activity. As POP Tc80 was shown to exhibit the unusual property of cleaving collagens I and IV, fibronectin, and small peptides used as substrates for human prolyl oligopeptidases, selectivity between parasitic and human enzymes towards inhibitors could be expected. 3.3. Metallopeptidases (EC 3.4.24) In trypanosomatids, the so-called gp63 from Leishmania spp. is the best characterized metallopeptidase . Distinct names have been used for this enzyme in the literature, as PSP (promastigote surface peptidase) and leishmanolysin,
Current Enzyme Inhibition, 2007, Vol. 3, No. 1
although the protein name MSP (major surface peptidase) was chosen to conform to the recommendations for gene and protein nomenclature in trypanosomatids . MSP (EC 126.96.36.199) is a metallopeptidase with the zinc-binding sequence His-Glu-X-X-His (HEXXH motif, named after the single letter code for amino acids), and a more C-terminal His residue that is the third ligand of zinc (Fig. (1)). This endopeptidase belongs to the peptidase family M8, and its tertiary structure places this family in the clan MA and in the subclan of metzincins, MA(M) . The mechanism of action and substrate specificity of this peptidase are similar to those of members of the matrix metallopeptidase family, being active over a broad range of pH. In addition, the P 1’ residue is often Leu, Ile or Val, and Lys in P3’ or P4’ may be favourable. All MSPs studied to date in the different species share high sequence similarity, and several Leishmania spp. contain distinct classes of MSP genes, which can be distinguished by their sequences and the stage specificity of their expression . The enzyme corresponds to the most abundant surface glycoprotein in promastigotes, being anchored via a glycosylphosphatidylinositol (GPI) anchor, and a significant proportion of MSP is released into the extracellular medium by autoproteolysis  or secretion . Although its expression by leishmanial amastigotes is much reduced, being detected both at the cell surface and within the lysosome of the parasites, MSP may contribute to Leishmania spp. virulence and pathogenicity. The expression of the enzyme is increased in metacyclic promastigotes and it may be a ligand involved in the interaction of the parasite with defensive systems of the host, including components of the complement system and the macrophage surface, as well as to play a role in intracellular amastigote survival within macrophage phagolysosomes. Nevertheless, data from MSP knockouts are conflicting . Homologues of MSP were also detected in other trypanosomatid protozoa , including the ones which develop their life cycles predominantly in insects, such as Blastocrithidia culicis , Crithidia deanei [106, 107], C. fasciculata [108,109], Crithidia guilhermei , Crithidia lucilae , Herpetomonas samuelpessoai , Herpetomonas megaseliae , Leptomonas seymouri , Phytomonas françai  and Phytomonas serpens . In addition, MSP homologues were also detected in T. brucei  and T. cruzi . The sequence motif of metzincin class peptidases and positionally conserved cysteine residues throughout the catalytic domain are conserved, although the sequences are considerably diverged in other regions . Several authors have suggested that the MSP-like molecules from lower trypanosomatids might fulfill a nutritional role in the insect midgut [105, 108, 112, 116, 117], since insect colonization is believed to be the only life cycle stage common to the monoxenous and heteroxenous flagellate trypanosomatids. Recently, our group showed a probable function for the MSP-like molecules on the adhesion of lower flagellates to the invertebrate epithelial cells [28, 107, 112]. T. cruzi possesses a family of MSP genes composed of multiple groups . Two of these groups, Tcgp63-I and Tcgp63-II, are present as high-copynumber genes and antibodies against Tcgp63-I partially blocked the infection of Vero cells by trypomastigotes,
Vermelho et al.
which suggests a possible role for this metallopeptidase during the infection process in vitro . Bangs et al.  demonstrated that a surface metallopeptidase activity of T. brucei is responsible for the shedding of variant surface glycoprotein (VSG) during cellular differentiation. 3.4. Proteasomes In eukaryotic cells, the turnover of intracellular proteins is mediated mainly by the machinery of the ubiquitin/adenosine-5’-triphosphate (ATP)-dependent proteasome pathway, which is a strictly controlled enzymatic complex [120, 121]. The mechanism of degradation is successive: protein substrates selected for destruction are marked by the covalent addition of poly-ubiquitin chains (E1-E3 ubiquitin conjugation pathway), which are recognized and proteolytically degraded by the multifunctional 26S proteasome complex, a large nonlysosomal multisubunit and multicatalytic peptidase complex located in the cytosolic and the nuclear compartments [122, 123]. This proteolytic pathway controls a broad array of cellular functions, including stage-specific gene transcription , antigen-processing , cell cycle progression , regulation of membrane-anchored and secretory pathway-compartmentalized proteins  and protein quality control . The functional proteasome (known as 26S proteasome) is an ATP-dependent, multifunctional proteolytic complex that differs in many respects from typical proteolytic enzymes . It consists of a proteolytic core particle, the 20S (720 kDa) proteasome, capped at one or both ends by a regulatory component termed the 19/22S complex (890 kDa) (regulatory particle), also called the proteasome activator (PA)700, composed of at least 18 peptides that determine substrates specificity for the selective degradation of ubiquitinated proteins . Ubiquitinated substrates are processed at the active sites located within the inner cavity of the core particle, whereas the regulatory particle is responsible for recognition, unfolding and translocation of the selected substrates into the lumen of the core particle. Mammalian cells and the protozoan T. brucei also contain an ATP-independent activator of the 20S proteasome (or 11S regulator), named the PA28 or PA26 complex, respectively, that can replace 19S and activates the proteolysis of short peptides . The PA28 activator is a 200 kDa ring-shaped heteromultimer composed of two isoforms of a 28 kDa subunit (PA28α and PA28β) and is present in the cytoplasm as a free complex or associated with the proteasome. In certain tissues and cell types the PA28 complex may also associate with the γ -interferon, which is believed to stimulate production of antigenic peptides by proteasomes by an unclear mechanism [132, 133]. The structure of the 20S proteasome has been recently described . Detailed crystallographic studies of the 20S proteasome revealed a complex cylindrical structure made up of four stacked heptameric rings composed of seven different α-subunits in the outer rings and seven different β-subunits in the inner ones. The active sites reside within the βsubunits, which provide the catalytic N-terminal threonine residues. The eukaryotic 20S proteasome shows several distinct proteolytic activities, since in vivo assays with
chromogenic substrates demonstrated the following activities: a chymotrypsin-like activity, a trypsin-like activity, a peptidyl-glutamyl-peptide-hydrolyzing activity, a branched chain amino acid-preferring activity and a small neutral amino acid-preferring activity. However, the chymotrypsin-like activity and the trypsin-like activity are considered the main activities . Trypanosoma brucei Proteasome T. brucei proteasome was the first to be purified and characterized from a parasite. Several differences between T. brucei and mammalian proteasomes have been found: (1) the 20S proteasome has a molecular mass of 630 kDa in trypanosomes compared with 700 kDa in mammals ; (2) two-dimensional gel electrophoresis of the trypanosome 20S proteasome yielded only 26 protein spots - far fewer than in rat liver 20S proteasome ; (3) polyclonal antibodies raised against the human 20S proteasome showed no appreciable cross-reaction with the trypanosome 20S proteasome (procyclic and bloodstream forms), but crossreacted strongly with the rat 20S proteasome, accordingly it was demonstrated that the T. brucei α 5 subunit shows only 50% sequence identity with the rat counterpart ; (4) biochemical analysis has revealed that the purified 20S proteasome from T. brucei differs in terms of substrate specificity from the eukaryote complexes, since the trypanosome proteasome exhibits high trypsin-like but low chymotrypsin-like activities compared with the mammalian counterparts [135, 138]. Earlier studies showed that an activator of the 20S proteasome was identified in T. brucei procyclic and bloodstream forms . This 26 kDa protein (PA26) polymerizes spontaneously in vitro with the 20S proteasome to yield the activated 20S proteasome. Structural analysis of T. brucei PA26 complexed with the yeast 20S proteasome core indicated that PA26 forms a heptameric ring and associates with the proteasome α subunits, which causes an opening of the channel leading to the catalytic chamber . The activated 20S proteasome from T. brucei has been show to act upon peptide but not protein substrates and therefore may not play a major role in the degradation of proteins . Recently it was demonstrated the 26S proteasome from T. brucei is not involved in the degradation of mammalian ornithine decarboxylaseantienzyme complex, which catalyzes the first step in the polyamine biosynthetic pathway. This characteristic constitutes the real difference between mammalian and trypanosomal 26S proteasome . In addition, the functional characterization of the 11 non-ATPase [regulatory particle non-ATPase (Rpn)] subunit proteins in the 19S regulatory complex showed that, when Rpn10 was deficient, a 19S complex without Rpn was still formed, while the cell growth was arrested. This structural dispensability, but functional indispensability, of Rpn10 constitutes another unique aspect of the T. brucei proteasome . Trypanosoma cruzi Proteasome The presence of the 26S proteasome and the ubiquitin pathway in T. cruzi was documented for the first time by de Diego et al. . The 26S proteasome of T. cruzi epimastigotes was identified as a high molecular mass complex (1400 kDa) with composition that resembles that of
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 27
the 26S proteasome isolated from other eukaryotic cells, and with an ATP-dependent chymotrypsin-like activity against the substrate Suc-Leu-Leu-Val-Tyr-AMC (Suc corresponding to Succinyl, AMC being 7-amino-4-methylcoumarin). Some members of the AAA family of ATPases were identified in the 19S complexes. These members cap the 26S proteasomes and are highly conserved . The characterization of the proteasomes from trypanosomes and its comparison with the rat proteasomes have shown that they share structural similarities but are functionally distinct . Therefore, it is of great interest to investigate further the subunit composition of trypanosome proteasomes and to compare it with that of proteasomes from other organisms, as well as to isolate genes coding proteasomal subunits. Recently, two genes encoding T. cruzi α proteasome subunits α 1  and α 6  were cloned and characterized, and the results showed that T. cruzi proteasome might be composed of the same set of subunits as in other eukaryotes. However, it is still not clear whether all the corresponding α- and β-family subunits are found in the proteasomes of early diverging eukaryotes, such as T. cruzi. Leishmania Proteasome In Leishmania genus, the proteasome has been studied in L. mexicana , L. major , L. donovani  and L. chagasi . In L. mexicana, the 20S proteasome has a molecular mass of around 670 kDa , which is consistent with the size of 20S proteasomes from other species , and it is composed of at least 10 distinct subunits in the 22 to 32 kDa size range. However, the molecular mass of the L. mexicana proteasome increases to 1200 kDa in the presence of ATP, consistent with there being a 26S proteasome in the parasite, extending, thus, the range of eukaryotic species known to have the 26S form of the proteasome. It is already known that kinetoplastid protozoa have genes for ubiquitin  and there are evidences for a functioning ubiquitination system in T. cruzi . It seems that the L. mexicana proteasome has a substrate preference profile more similar to proteasomes from higher eukaryotes than from the related protozoan T. brucei, which has an unusually high tryptic peptidase activity . The purified 20S proteasome from promastigote forms of L. chagasi possesses six proteins with molecular masses of 22-35 kDa. This data is consistent with the observed sizes for subunits α and β from T. cruzi , T. brucei  and L. mexicana proteasomes . The L. chagasi proteasome, like other 20S proteasomes, hydrolyzes substrates with basic and bulky P1 residues, exhibiting a higher trypsin-like activity compared to chymotrypsin-like activity. A similar finding was reported by Hua and colleagues for T. brucei . Thus, the L. chagasi proteasome has an activity pattern closer to the related protozoan T. brucei than to the proteasomes from higher eukaryotes and the protozoan L. mexicana, which has an unusually high chymotryptic peptidase activity . Since the proteasome is involved in a wide spectrum of cellular functions in eukaryotes, it represents a potential target for the development of therapeutic agents for the treatment of pathologies such as cancer, immune diseases and others [150, 151]. It is possible that proteasomes are involved in the regulation of important cellular processes in
Current Enzyme Inhibition, 2007, Vol. 3, No. 1
trypanosomes, including gene expression, cell cycle progression, differentiation and infectivity . This hypothesis is corroborated by experiments with specific inhibitors of proteasome activity such as peptide aldehydes [e.g. N-carbobenzoxyl-Leu-Leu-leucinal (MG132)], lactacystin and gliotoxin that have helped to define the role of the proteasome in trypanosomes as well as in other protozoan parasites . 3.5. Aminopeptidases Aminopeptidases, which catalyze the removal of Nterminal amino acid residues from peptides and proteins, can be considered as a novel drug target in trypanosomatid infections. The few reports found in the literature describing the inhibition of this class of exopeptidases in these microorganisms are related to leucyl aminopeptidase, methionine aminopeptidase 2 and basic aminopeptidases. A detailed study describing the cloning and biochemical characterization of the major, if not only, leucyl aminopeptidase (Lap) from L. amazonensis, L. donovani and L. major was reported by Morty and Morehead . The enzyme was found in the cytosol of all life cycle stages of the parasite, and the phylogenetic comparison of the Lap genes indicated that the Leishmania enzymes were the most evolutionarily divergent members of the M17 family of Laps, including homologues from prokaryotic and eukaryotic organisms. Besides the indication that zinc is the most likely natural Lap cofactor, its potent activity was demonstrated exclusively against synthetic aminopeptidase substrates containing leucine, methionine and cysteine residues, representing the most restricted substrate specificity for any known Lap. The proper characterization of methionine aminopeptidase 2 and basic aminopeptidases in trypanosomatids has never been attempted, although a few reports using inhibitors of these enzymes were described. Methionine aminopeptidase 2 (MetAP2) is a monomeric, cobalt-containing enzyme responsible for the hydrolysis of the initiator methionine molecule from the majority of newly synthesized proteins. This enzyme is found ubiquitously in all organisms, since failure to remove the initiator methionine can result in a protein product that is usually inactive, leading to slower growth or lethality . 4. PEPTIDASE INHIBITORS 4.1. Cysteine Peptidase Inhibitors Peptides Containing Natural and Non-Natural Amino Acids The kinetic properties of Leishmania and mammals homologous cysteine peptidases have been studied using a variety of substrates and inhibitors. It is well established that the primary determinant of specificity for papain and cathepsins B and L is the S2 subsite: hydrophobic residues are preferred at the P2 position of substrates for papain and cathepsin L, although cathepsin B also accepts basic residues. The S 1 subsites of papain and cathepsin L are not as selective as the S2, but a preference of the papain S1 subsite for basic residues is reported. The substrate specificity of only two members of the CPB array in L.
Vermelho et al.
mexicana, CPB2.8 and CPB3, has been studied in detail [78, 154]. CPB2.8 was overexpressed in Escherichia coli as an inactive pro-form lacking the characteristic C-terminal extension (CTE) of this class of cysteine peptidases in trypanosomatids, in order to facilitate the refolding of the recombinant enzyme . The recombinant enzyme, originally designated CPB2.8∆CTE, is also named rCPB2.8. The sudies of the substrate specificity of this enzyme revealed that rCPB2.8 has strict preferences for the S 1 to S3 subsites, accepting substrates containing basic amino acid residues in P1, hydrophobic residues in P 2 and basic residues in P3. In contrast, the specificity of the primed subsites was shown to be broad . Although structurally distinct, CPB (cathepsin L-like) and CPC (cathepsin B-like) overlap in substrate specificity . Based on many studies, several peptide inhibitors have been reported for rCPB2.8 and described below [157-163]. A series of peptides derived from Lys-Leu-Arg-Phe-SerLys-Gln, previously known as a good substrate for cruzipain and cathepsin L, were synthesized in which Arg at P1 was substituted by all natural amino acids, in order to compare the specificity of rCPB2.8 with that of the former enzymes . To this end, the quencher group o-aminobenzoic acid (Abz) was introduced at the N-terminal site and N-[2,4dinitrophenyl]-ethylenediamine (EDDnp) at the C-terminal. While the peptides from this series with charged side chains and Cys were well hydrolyzed, all other substitutions resulted in peptides that were hydrolyzed very slowly and inhibited CPB2.8 with K i values in the 9-400 nM range. It is noteworthy that the hydrolysis of the peptides containing Ile and Gly by cathepsin L occurred with significantly higher K m values than the K i obtained with CPB2.8, suggesting a selective binding to the leishmanial enzyme. Alves et al.  investigated as substrates for rCPB2.8 a series of fluorogenic peptides based on the sequence Benzoyl-Phe-Arg-AMC, in which Phe (P2) residue was substituted for by non-natural amino acids that combine hybrid hydrophobic-basic side chains: the presence of this kind of amino acid would indicate whether hydrophobic interactions prevail at S2, and whether this subsite accepts basic groups attached to aromatic or bulky hydrophobic groups. This work indicated that the S2 subsite specificity of CPB2.8 is more restrictive than that of cathepsin L. The peptides with the basic side chains residues 4-aminomethylphenylalanine (Amf) and 4-aminomethyl-cyclohexyl-alanine (Ama) presented higher affinity to CPB2.8, and the latter was an inhibitor of the enzyme (K i = 7.6 µM). The combination of hydrophobicity and small size of side chain at P2 position seems to be crucial for Leishmania CPB inhibitor design. Non-natural amino acids were also used by the same group  in the synthesis of one series of internally quenched fluorogenic peptides derived from the sequence Abz-Phe-Arg-Ser-Arg-Gln-EDDnp, based on the human kininogen sequence at the C-terminal region of bradykinin. P 1 position was then substituted by amino acids that combine a large hydrophobic group with a positively charged group at their side chains, so that these residues would take the advantage of both the high hydrolytic susceptibility of substrates with basic amino acids at P1 and
the high affinity of peptides with hydrophobic residues at this position. The peptides with the residues Ama and 4aminocyclohexyl-alanine (Aca) were very resistant to hydrolysis and inhibited the enzyme with K i values of 23 nM and 30 nM, respectively. The substrate containing Aca was efficiently hydrolyzed by human cathepsin L, which suggests that efficient inhibitors can be designed with this amino acid at the P1 position. Successful peptidase inhibitors must have a good binding affinity but also display high selectivity among the numerous peptidases present in biological systems to avoid serious side effects. To this end, it is necessary to screen many putative inhibitors to find a potent yet selective compound. Combinatorial libraries offer a large number of compounds that can be selected by biological activity and provide new lead structures. With the development of one bead-two compounds libraries, it became possible to screen a great number of compounds in a competitive manner, in which each inhibitor in a single bead competes with a fluorogenic quenched substrate for binding to the peptidase . Peptidomimetics prepared by solid-phase peptide synthesis combined with various organic reactions can provide compounds that bind to peptidases as a natural peptide substrate but are more resistant to hydrolysis because of a structural mimetic within the peptide backbone. In this way, rCPB2.8 inhibitors have been identified with intramolecularly quenched fluorogenic substrates  and by screening of combinatorial bicyclic ketone , reduced peptide bond inhibitor libraries  and peptidotriazoles  on solid support. Resynthesis and kinetic evaluation in solution validates the libraries and the screening/sorting process. These informations may be utilized for the synthesis of second-generation inhibitor libraries that better conform to the criteria for an effective drug. The combinatorial fluorescence quenched peptide library previously used to identify efficient substrates of L. mexicana rCPB2.8  was analyzed in more detail by Alves et al.  in the search for peptides with a high affinity for the enzyme but slowly hydrolyzed. All these peptides possessed the amino acid sequence motif basichydrophobic-hydrophobic in positions P3-P1, and the peptides containing the sequences Lys-Tyr-Leu and Lys-LeuLeu inhibited CPB2.8 with the highest affinity (K i values of 7 and 12 nM, respectively). These sequences mirror a region of the pro-peptide that is known to interact with and inhibit the catalytic site of cathepsins B and L, cruzipain and congopain. In order to identify which segments of the proregion inhibit the enzyme, 9-mer and 14-mer peptidyl amides spanning the C-terminal 57 amino acids were synthesized and assayed. The nine-amino acid stretch PheAla-Ala-Arg-Tyr-Leu-Asn-Gly-Ala was identified as the part of the pro-region most likely to interact with the active site of the parasite enzyme, and the extension of this sequence at both the N- and C-termini and the introduction of Abz at the N-terminal site and EDDnp at the C-terminal reduced the K i value up to 30 nM. Of particular interest was the finding related to the high value of K i for cathepsin L in comparison to CPB2.8 obtained with the peptide Lys-Leu-Ser-Lys-TyrLeu-Ser-Lys, which presents the motif basic-hydrophobichydrophobic, a promising feature in the design of specific inhibitors of leishmanial cysteine peptidases .
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 29
A combinatorial split-and-mix library of peptide isosters was formed in a solid-phase reaction, placing the isoster at an internal position . When the library was screened with rCPB2.8, several beads containing compounds with inhibitory activity could be selected from the library and analyzed by MALDI-TOF mass spectrometry for structure elucidation. Selected hit sequences and constructed consensus sequences, based on the observed frequencies of amino acids in different subsites that were resynthesized and assayed in solution for inhibitory activity, displayed IC50 values in the high nanomolar to low micromolar range. Two novel classes of inhibitors were revealed: the first class had the full-length bicyclic Diels-Alder product isosteric element incorporated internally in a peptide, and the second type was an N-terminal α,β-unsaturated ketone Michael acceptor used as starting material for the Diels-Alder reaction. A substratelike orientation of the inhibitors was assumed and the amino acid subsites assigned as X2, X1, X1’, X2’ and X3’ in analogy to the standard nomenclature of peptidase substrates. Within the former class of inhibitors, hydrophobic residues were highly preferred in X 2 and X1’, while subsites X1 and X2’ were nonspecific. The subsistes of inhibitors of the second class were labeled X1’-X3’, hence assuming interaction with only half of the active site. Leu was highly preferred in subsite X1’, whereas the remaining subsistes were more nonspecific, although with a preference for hydrophobic residues as β-cyclohexyl alanine (Cha). This library was validated when structures exhibiting inhibitory activity against rCPB2.8 on the solid phase presented the same profile in solution. The synthesis and screening of a library of potential cysteine peptidase inhibitors comprising octapeptides with a reduced peptide bond at the putative P1-P1’ was reported by Hilaire et al. . The reduced bond peptides could be inhibitory for this class of peptidases if the side chains of the amino acids flanking the nonhydrolyzable secondary amine led to tight binding of the inhibitor to the enzyme subsites, thus sterically preventing substrate access to the reactive thiol. The inhibitors displayed great specificity in the subsites flanking rCPB2.8 catalytic triad with Cha and Ile-/Leu preferred in P2, Phe in P1, Cha and Ile/Leu in P 1’ and Ile/Leu in P2’. The surprising preference for a hydrophobic amino acid in P1, in contrast to the substrate specificity of the enzyme, could confer the advantage of a tight binding by utilizing the cavity at S1, thus inducing a peptide conformation not conducive to catalysis, as previously determined . Most of these inhibitors had micromolar K i values, and a few inhibited the enzyme at nanomolar concentrations. One inhibitor, Asp-Lys-HisPhe(CH2NH)-Leu-Leu-Val-Lys, was shown to affect L. braziliensis survival with an IC50 of approximately 50 µM, being effective in decreasing both the number of macrophages infected and the number of amastigotes per infected cell. There was no apparent adverse effect on the macrophages, suggesting that cysteine peptidase inhibitors impair the parasites’ ability to successfully invade host cells. A library of peptidotriazoles was generated by Tornoe et al. , by virtue of the small, rigid and aromatic structure of triazoles combined with its hydrogen-bonding capabilities and resistance to enzymatic hydrolysis. In all cases, the basic construct corresponded to X1-X2-Triazole-X3-X4, and the screening against rCPB2.8 afforded novel inhibitors, with
Current Enzyme Inhibition, 2007, Vol. 3, No. 1
five sequences displaying K i values in the low micromolar range. The observed selectivity in the peptidotriazole library for cationic residues (arginine-derived triazoles and Arg in X3) and Leu in X4 correlated well to the known substrate specificity of the enzyme . Sequences including four amino acids from mass-ionization spacer, Thr-Ile-Ser-Arg, were the best inhibitors, with K i values between 76 and 240 nM. The lack of selectivity for X1 and X2 substantiated the proposed binding mode where only the spacer, the positions X3 and X4 and the triazole were recognized. This study underlined the importance of selecting the proper massionization spacer. Pseudopeptide inhibitors, prepared by solid phase synthesis, was also designed and developed for cruzipain and congopain . The 121 tetrapeptides synthesized share the framework: Cha-X1-X2-Pro (X1 and X2 were phenylalanyl analogs), where Cha and Pro occupied the putative P2 and P 2’ positions, respectively. All peptides are water soluble at a concentration of 10 mM. The substrate specificity of trypanosomal cysteine peptidases is primarily determinated by P 2/S 2 interactions, with a marked preference for aromatic residues such as Phe at P2. Five peptides containing a nitro– substituted aromatic residue (Tyr/Phe) and one 4-chlorophenylalanine at the position X1, and 3-(2-naphthyl)-alanine, homocyclohexylalanine or nitro-tyrosine (3-NO2-Tyr) at the position X2 were tested as reversible inhibitors of trypanosomal congopain and cruzipain. They were more effective to congopain than cruzipain except for Cha-4-NO2Phe-3-NO2-Tyr-Pro, which bound both enzymes similarly. Ho-Cha, NO2, and 3-NO2-Tyr-containing peptides bound quite well to the S1’ subsite of cruzipain, while D-Phe and phenyl glycine were poorly accepted. Cha-3-NO2-TyrHoCha-Pro and Cha-4-NO 2-Phe-3-Tyr-Pro were respectively the most selective for congopain relative to cathepsin B and L. Cha-4-NO2-Phe-3-NO2-Tyr-Pro was the weakest inhibitor of human cathepsins L and B due to the presence of 3-NO2Tyr at X2. No hydrolysis of these peptides was detected upon 5 hours with cathepsin B and L, congopain, cruzipain or unrelated peptidases, such as trypsin and chymotrypsin. The stability of the pseudopeptide Cha-4-NO2-Phe-3-Tyr-Pro was tested in rat plasma and fetal calf plasma, and preliminary results showed that 25% of the peptide was recovered after 1 hour. Even though the affinity of such reversible inhibitors, containing unencoded amino acids in their peptide framework, remains weak in part due their short length, their increased properties in terms of stability and resistance to proteolysis are promising to the development of new inhibitory compounds . In a different approach, Scheidt et al.  used the crystal structure of cruzipain, covalently inactivated by the fluoromethyl ketone (FMK) inhibitor Cbz-Phe-Ala-FMK (described below, Cbz corresponding to benzyloxycarbonyl), as a template to design conformationally constrained cysteine peptidase inhibitors. The peptide aldehyde Cbz-Phealanal was then converted to the pyrrolidinone-containing aldehyde inhibitor, and this compound exhibited notable activity against L. major cysteine peptidases, with an IC50 of 100 nM. The homophenylalanine derivative, prepared to increase the hydrophobic interactions with the S1 binding site, displayed an IC50 of 30 nM, and the glycine analogue, prepared in order to determine whether a substituent at P1 was necessary for activity, gave an IC50 of 600 nM. The
Vermelho et al.
synthesis of a pyrrolidinone inhibitor by replacement of the Cbz group by a phenylsulfonamide ester was performed in order to acquire additional binding interactions with the S 3 site, and gave an IC50 of 20 nM. These results confirmed that peptide aldehydes are potent in vitro inhibitors of cysteine peptidases and that the pyrrolidinone ring is an effective conformational constraint for leishmanial peptidase inhibitors. Peptidyl Fluoromethylketones Fluoromethylketones (FMK)-derivatized peptides are substrate analogues that irreversibly inhibit cysteine peptidases. The first demonstration of the effect of irreversible cysteine peptidase inhibitors on T. cruzi was the work of Ashall et al.  that showed the lysis of trypomastigotes by Cbz-Phe-Ala-FMK. Studies with peptidyl FMKs showed that some derivatives such as CbzPhe-Ala-FMK and Cbz-Phe-Arg-FMK arrested intracellular replication of the parasite, as well as the differentiation of trypomastigotes to amastigotes and also from amastigotes to trypomastigotes in J774 and LLC-MK2 cultures . In addition, peptidyl acylomethylketone (AMK), Cbz-Phe-LysCH2-OCO–(2,4,6-Me3)Ph.HCl lysed epimastigotes and trypomastigotes and reduced infection and intracellular multiplication of the amastigote forms . McKerrow  reported that FMK peptides, while useful in earlier studies concerning the function of parasite peptidases, were dropped from drug development because of the potential toxicity from fluoridated amino acids. If a FMK dipeptide is cleaved, releasing the FMK-amino acid, it could inhibit the Krebs cycle, shutting down cellular ATP production . The effect of such toxicity was shivering and hypothermia in mice. Nevertheless, even within the FMK series, inclusion of a non-natural side chain as part of the peptide significantly reduced these toxic effects [23,170]. Peptidomimetics containing at least one non-natural amino acid such as homophenylalanine (hPhe) do not undergo this cleavage and toxicity . The morpholineurea-phenylalanine-homophenylalanineFMK derivative (Mu-Phe-hPhe-FMK) arrested growth of epimastigotes of T. cruzi Y strain at 5 µM . In order to determine the cellular localization of the parasite enzymeinhibitor complexes in living epimastigotes, the parasites were treated with biotinylated Phe-Ala-FMK for 1 hour. In parallel, a fluorescent acidic probe specific for lysosomes and an anti-cruzipain antibody were used in these studies. Both the biotinylated Phe-Ala-FMK and the anti-cruzipain antibody localized the cysteine peptidases into membranebound organelles, consistent with lysosomes or reservosomes in epimastigotes . In T. cruzi treated with the inhibitors for 40 hours, the antibody bound to cruzipain not only in the lysosome but also in the Golgi complex region, which is anterior to the nucleus in T. cruzi. These results suggested that the inhibitor is transported into epimastigote organelles, where it binds to cruzipain and alters its subcelullar localization. The treatment of T. cruziinfected mice with FMK-derivatized peptides rescued mice from a lethal infection. Peptidyl Vinyl Sulphones and Derivatives Vinyl sulphones (VS) represent a new class of small, electrophilic inactivators of cysteine peptidases. Members of
this inhibitor class contain a Michael acceptor functional group and react irreversibly with the cysteine residues in the active site. They act by electrophilic capture of Cys nucleophiles via 1,4 conjugate addition, forming a stable thioeter adduct that irreversibly inactivate the enzyme [166, 171-173]. Engel et al. [52, 169] studied the effect of VS derivatives in the cysteine peptidases of T. cruzi. At first, the VS inhibitor morpholineurea-phenylalanine-homophenylalanine-vinylsulphonyl-phenyl (Mu-Phe-hPhe-VSph), also known as K11002 , arrested the growth of epimastigote forms, altered the intracellular localization of the enzyme and induced alterations in the Golgi complex. The presence of unprocessed cysteine peptidases accumulated in peripheral dilatations of the Golgi cisternae, and a reduction (70%) of cruzipain transported to lysosomes was observed, suggesting that VS derivatives prevent the normal autocatalytic processing and trafficking of cruzipain in the Golgi. After inhibitor treatment, major morphological alterations were also observed in the nuclear membrane and endoplasmatic reticulum. The accumulated enzyme decreased the mobility of Golgi membranes, resulting in a distention of cisternae in parallel with the death of the parasite . The ultrastructural alterations in the Golgi complex of K11002treated amastigotes resemble those described for epimastigotes. A marked decrease of cysteine peptidase expressed on the cell surface of K11002-treated amastigotes was evident, compared with untreated cells . This derivative was the most effective compound and inhibited 100% of the intracellular cycle of T. cruzi in macrophages in vitro. At the concentration of 20 µM, T. cruzi-infected macrophages were cured. No intracellular amastigotes or released trypomastigotes were observed in cultures treated for 12 days and maintained without the inhibitor for up to 30 days, indicating that K11002 not only blocked the intracellular development of T. cruzi but eliminated all parasites as well . Selzer et al.  tested K11002 against L. major. The VS inhibitor prevented promastigote growth at concentrations over 20 µM, and also reduced the rate of amastigote replication in cultured macrophages. Targeting of cathepsins B- and L-like by K11002 was confirmed by inhibition of 80% of the total Leishmania cysteine peptidase activity measured by the substrate Cbz-Phe-Arg-AMC (which detects both activities) after treatment of promastigotes with 50 µM K11002 for 24 hours. In addition, the use of radioactively labeled vinyl sulphone demonstrated that the cathepsin B-like enzyme is the predominant, but not exclusive, target of K11002. Treatment of promastigotes with 50 µM K11002 resulted in the accumulation of multivesicular and dense bodies, lipid inclusions and myelin figures within dilated parasite lysosomes and in the flagellar pocket. The cathepsin B-like peptidase was localized inside the dilated lysosomes and the flagellar pocket of treated parasites, whereas in normal cells the peptidase was found solely in lysosomes. Because of the selective arrest of parasite versus host cell growth by the vinyl sulphone inhibitors added to cultures, the efficacy of these compounds in vivo was evaluated in L. major-infected BALB/c mice. A significant delay on the onset of footpad lesions and a reduction in the parasite burden was observed after the injection of the VS inhibitor into infected mice at a dose of 100 mg/kg of body weight, although a complete
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 31
cure was not observed. No detectable toxic effects in mice were observed . The determination of K i values indicated that selectivity of this inhibitor for the L. major cathepsin B-like, T. cruzi cruzipain and papain versus mammalian cathepsin B is significant. No alteration in host cells replication or ultrastructural appearance was detected at concentrations up to 50 µM, and the lack of toxicity at the doses used in mice is consistent with results of a similar study in the treatment of T. cruzi infection . The selectivity of the effects caused by the inhibitor on the parasite suggests that cysteine peptidases are crucial to L. major, whereas host cells are less sensitive. As pointed out by Selzer et al. , besides the fact that parasites appear to take up and concentrate the inhibitor much more effectively, the concentration of peptidases within host cells is substantially higher, and consequently the phenotypic effect in the latter may be reduced. The irreversible cysteine peptidase inhibitor K11777, with broad activity against cathepsins B and L, is a pseudopeptide substrate analogue with the structure Nmethylpiperazine-phenylalanyl-homophenylalanyl-VSph (NPip-Phe-hPhe-VSph) that is studied as an anti-Chagas’ disease drug . In comparison to K11002, for which the oral bioavailability in Sprague-Dawley rats was 3%, K11777 possesses a N-methyl piperazine ring instead of a morpholine-urea ring, which increases solubility and thereby oral bioavailability . The latter was used in experimental Chagas` disease in CH3 mice; 10 mice survived at least 7 days longer than untreated mice, and 6 out of 10 mice survived over 270 days after infection. No toxicity was observed when the inhibitor was used orally or intraperitoneally in uninfected mice . K11002- and K11777-resistant T. cruzi epimastigotes were generated through a gradual drug increase. After one year of parasite treatment, resistance to 20-fold the lethal inhibitor concentration was achieved, and a marked increase in the number of vesicles trafficking from the Golgi complex to the flagellar pocket occurred in resistant cells, and no mature peptidases reached the lysosomes. High molecular mass cruzipain species, consistent with the presence of cruzipain precursors complexed with the inhibitor, were secreted into the culture medium. In addition, the enzyme activity was negligible in these epimastigote cells, but was restored following the withdrawal of the inhibitor. These findings suggest that one of the mechanisms of resistance was an enhancement of the Golgi secretory pathway, which led to the release of the precursor before the enzyme reaches its site of biological activity . K11777 also blocked L. major and L. tropica promastigote replication . Concentrations of 5, 20, and 50 µM inhibited L. tropica growth 3-fold, 7-fold and 49fold, respectively, and at 100 µM completely arrested growth. The inhibitor had similar effects on L. major promastigotes, but not on L. mexicana promastigotes, which is refractory to this compound. When L. tropica-infected mice were treated by intraperitoneal or subcutaneous injections of the inhibitor at 100 mg/kg and 4 mg/kg, respectively, a reduced footpad thickness (that correlates well with the number of amastigotes) was observed up to 1-2 months after the end of treatment. The increase of footpad
Current Enzyme Inhibition, 2007, Vol. 3, No. 1
swelling after termination of the treatment suggests that at the concentrations used in the study, the inhibitor did not eliminate all of the amastigotes at the site of infection. A second generation of vinyl sulfonamide inhibitors of T. cruzi cysteine peptidases was developed by Roush et al. . N-alkoxyvinyl sulfonamide derivatives act as irreversible inhibitors of cruzipain, increasing the survival of T. cruzi-treated macrophages. One of them was also a potent inhibitor of rhodesain . In an effort to synthesize epoxy sulphone inhibitors, allyl sulphones (AS) were produced from vinyl sulphones . The dipeptidyl allyl sulphone Cbz-Leu-Phe-AS-Ph (isomer B) was synthesized from the precursor Cbz-Leu-D-PheVSph. When the double bond isomerizes, the original Z stereochemistry of the vinyl sulphone was lost and the product has an unknown ratio of E to Z isomers at the new allyl double bond. The isomer A Cbz-Leu- Phe-AS-Ph, derived from the vinyl precursor Cbz-Leu-L-Phe-VSph, was more potent for cruzipain and rhodesain than isomer B, and both isomers were more potent than the corresponding CbzLeu-D-Phe-VSph. The possible mechanisms for the inhibition are: a) the active site cysteine could directly displace the phenyl sulphinic acid in SN2 reaction; b) the active site cysteine could attack the allylic double bond with loss of the phenyl sulphinic acid; or c) the re-isomerization of the peptidyl allyl sulphone to the vinyl sulphone. The first two mechanisms result in the alkylation of the active site cysteine residue. This new inhibitor shows moderate inactivation rates with calpain and papain and faster rates of inactivation with rhodesain and cruzipain. The dipeptidyl allyl sulphones do not inhibit cathepsin B . Peptidyl Diazomethanes Peptidyl-diazomethanes (PDAMs) are irreversible inhibitors of cysteine peptidases, having the general structure peptidyl-C(=O)-CHN2 and sometimes are referred as peptidyl diazomethyl ketones. The initial finding that CbzPhe-CHN2 inactivates papain by a stoichiometric alkylation of the active site cysteine residue has been followed by variations in the peptidyl portion of the inhibitor, providing high reactivity and some degree of specificity . The effect of PDAMs on cardiomyocytes infected with T. cruzi was studied for the first time by Meirelles et al. , who determined that the compound inhibited the infection of cells by T. cruzi in vitro. Using a panel of derivatives of this inhibitor, it was found that the most potent compounds had a bulky hydrophobic residues in P1, in addition to P2, such as Cbz-(Bz)-Cys-Phe-CHN2 (benzyloxycarbonyl benzyl-cysteinyl-phenylalanyl-diazomethane). The best inhibitors of cruzipain interfered with the parasite capacity to invade cardiomyocytes, and blocked the intracellular development of the amastigote forms . Franke de Cazzullo et al.  tested two PDAMs, CbzPhe-CHN2 and Cbz-Phe-Ala-CHN 2 against T. cruzi. These compounds inhibited the cruzipain activity in living parasites. These PDAMs had no effect on epimastigotes, but they inhibited the differentiation to metacyclic trypomastigotes, as demonstrated previously by Bonaldo et al. , and reduced the infection of Vero cells and the intracellular multiplication of T. cruzi amastigotes. These inhibitors were also tested against the cysteine peptidases from T. brucei , and it was demonstrated that Cbz-Phe-
Vermelho et al.
Ala-CHN2 decreased the parasitemia of infected mice to undetectable levels from the third to the sixth day following treatment with 250 mg/kg after infection. Although parasitemia returned thereafter to control levels, infected mice treated with the inhibitor survived approximately twice as long as those treated with placebo. Cbz-Phe-Ala-CHN2 inhibited proteolysis in lysosomes in vitro and almost completely blocked cysteine peptidase activity in vivo. However, these inhibitors have a limited selectivity for the parasite enzymes and are embryotoxic . As these compounds readily permeate through cell membranes, living L. amazonensis amastigotes were incubated with Cbz-Tyr-Ala-CHN2. The inhibitor specifically labeled certain amastigote cysteine peptidases, but parasites remained viable even after 48 hours of drug exposure . Whereas certain diazomethanes were shown to be toxic for T. cruzi, as Cbz-(S-benzyl)-Cys-Phe-CHN2, Cbz-Phe-Phe-CHN2 and Cbz-Tyr-Phe-CHN2 , no toxicity was detected for L. amazonensis amastigotes with Cbz-Phe-Ala-CHN2, Cbz-Tyr-Ala-CHN2 or Cbz-Phe-PheCHN2 [182, 183]. Nevertheless, these compounds proved to be useful probes for active cysteine peptidases and helped to elucidate the enzymes functions in Leishmania spp., as demonstrated by Antoine et al. . Frame et al.  showed that CPB-deficient L. mexicana mutants (∆cpb) were phenotypically different from the parental line using cell-permeant peptidyl-CHN2 inhibitors. Inhibition of CPBs in situ using 10 µg/ml CbzPhe-Ala-CHN2 and Cbz-Leu-Val-Gly-CHN2 had no measurable effect on parasite growth or axenically differentiation in vitro, as previously demonstrated for the null mutants. These findings clearly showed that L. mexicana differs in this respect from T. cruzi, since the differentiation of this parasite from epimastigote to metacyclic forms is susceptible to the same PDAMs due to the presence of the CPB homologue, cruzipain . Cbz-Phe-Ala-CHN2, but not Cbz-Leu-Val-Gly-CHN2, reduced the infectivity of the wild-type L. mexicana parasites to macrophages . Time-course experiments demonstrated that irrespective of whether promastigotes were preincubated with Cbz-Phe-Ala-CHN2 or the inhibitor was added 4-24 hours after infection, the inhibitor had a great anti-parasite effect over 7 days, after which the number of parasite-infected cells was found to be less than 10% of the control. To determine whether the use of this inhibitor mimicked the deletion of the CPB array, the effect of the compound on the infectivity of wild-type lesion-derived amastigotes to macrophages was examined: in this case, the number of infected macrophages was again reduced after 7 days to a very low level, compared with the control parasites. In contrast, the lesion-derived amastigotes of ∆cpb were as infective as wild type amastigotes are to macrophages, and their intracellular multiplication was similar. As found with ∆cpb promastigotes, ∆cpb amastigotes produced lesions in BALB/c mice much more slowly than the wild-type line, indicating that the situation in vivo is more complex than in vitro. These evidences suggested that CPB peptidases not only aid the initial survival of promastigotes as they differentiate into amastigotes in macrophages but also play other roles that facilitate infection in mice, probably in the destruction
of effector molecules, helping the parasite circumvent the host’s immune system. The differentiation process requires considerable cell remodeling that also involves a large amount of protein turnover, and consequently it is possible that the lack of these enzymes compromises the ability of the parasite to survive the microbicidal action of the macrophage during the differentiation. As there are important differences between the results obtained with ∆cpb and the use of PDAMs, a possibility is that the inhibitors are affecting other cysteine peptidases in addition to CPB, which would offer additional targets for chemotherapeutic targets . Amino Acid Esters A distinct approach for the use of specific inhibitors of parasite peptidases is through their ability to activate a prodrug. In this sense, intracellular and isolated L. amazonensis amastigotes are destroyed by amino acid and peptide methyl esters, such as L-leucine methyl ester (Leu-OMe) and Lleucyl-L-leucine methyl ester. Morphological studies pointed to megasomes as the probable site of action of these compounds. Killing involves ester trapping within the acidified megasome content, owing to its weak base properties, and ester hydrolysis by peptidases may lead to the accumulation of less permeant, osmotically active amino acids, which results in swelling, fusion and decrease in electron density of these compartments. These effects are abolished by the pre-treatment of the parasites with the peptidase inhibitors antipain and chymostatin . Since these are relatively nonspecific inhibitors, the use of the peptidyl diazomethane Cbz-Phe-Ala-CHN 2, an irreversible and specific inhibitor of cysteine peptidases that was also protective against Leu-OMe, supported the role of parasitic cysteine peptidases . L. amazonensis amastigotes are more sensitive to Leu-OMe than L. major and L. donovani amastigotes, and this correlates with the amount of cysteine peptidase activity expressed by these different species . Whereas high concentrations of these compounds are required to cause toxicity in mammalian cells, L. amazonensis amastigotes are markedly more susceptible, and the concentration of Leu-OMe at 0.8 mM, which does not affect macrophages, kills more than 90% of intracellular amastigotes, suggesting that the esters may be more efficiently cleaved by enzymes within the parasite’s endocytic pathway . Although the antileishmanial amino acid esters showed good potency in vitro and some in vivo, their hydrolysis by host lysosomal enzymes makes the potential usefulness of these compounds to be limited . Epoxy Ketones The first epoxysuccinyl peptide discovered was L-transepoxysuccinyl-leucylamido(4-guanidino)butane (E-64), which was initially isolated from Aspergillus japonicus by Hanada et al. . E-64 inhibits cysteine peptidases by the irreversible alkylation of the active site cysteine residue. Other epoxysuccinyl peptides such as cathestatin and circinamid are potent and selective inhibitors . For cruzipain tests, dipeptidyl epoxy ketones were designed and synthesized by combining features of the known inhibitor, E-64, and the dipeptidyl fluoromethyl ketone Cbz-Phe-Ala-FMK using molecular modeling and the examination of the X-ray structure of cruzipain complexed
Current Enzyme Inhibition, 2007, Vol. 3, No. 1 33
with Cbz-Phe-Ala-FMK, and papain complexed with E-64 and chloromethyl ketones. One of the derivatives, a dipeptidyl α’,β’-epoxy ketone, is a potent and irreversible inhibitor of cruzipain . Recently, Roush et al.  developed new derivatives, such as the D-homophenylalanyl epoxy succinate. One of the most potent derivatives contains an O-benzyl hydroxamate and D-homotyrosine, but is inactive against the parasite in vivo. Only two compounds, containing either D-Phe or D-hPhe, displayed activity against T. cruzi in tissue culture. Most of the compounds showed low water solubility, which may contribute to their lack of activity in vivo, and further structural modifications must be done in order to improve their low bioavailability . Mercaptomethyl, Cyclic and Hydroxymethyl Ketone Reversible inhibitors have been developed in order to minimize the toxicity that is observed with irreversible inhibitors . In this sense, mercaptomethyl  and cyclic  ketones were synthesized and screened against cruzipain. The mercaptomethyl and cyclic ketone series, synthesized using solid–phase combinatorial strategy, are selective for cruzipain. They are very poor inhibitors of cathepsin B and some of them shared similar K i values for cruzipain. The most active cyclic ketone inhibitors contained the side chain present in the most potent mercaptomethyl ketones. The potency and selectivity of these inhibitors are quite promising, since in contrast to the acyclic mercaptomethyl ketone inhibitors, the cyclic inhibitors do not interact with the S1 pocket of the enzyme . Huang et al.  synthesized two hydroxymethyl ketones containing a phenylalanine residue in P2 that moderately inhibited cruzipain. The crystal structure of them complexed with cruzipain showed that the active site cysteine residue does not form a covalent bond with the ketone, but forms a strong bond between the active site imidazole and the hydroxyl group of the inhibitor. PS28, A Derivative of Folic Acid The application of structure-based drug design by the use of computational screening of available chemical databases was explored as an inexpensive shortcut to identify potential chemotherapeutic agents against Leishmania cysteine peptidases, mainly because of the relationship between these target enzymes and cysteine peptidases of known structure and catalytic mechanism, as T. cruzi cruzipain, papain and human cathepsin B. To address this question, Selzer et al.  produced structural models of the two major cysteine peptidases from L. major, the cathepsin L-like and cathepsin B-like enzymes. It was noticed that L. major cathepsin Blike enzyme belongs to the cathepsin B subfamily by virtue of its structural homology, although the replacement of glutamic acid 205 by a glycine results in the S2 site having a much more hydrophobic pocket, and consequently a more “cathepsin L-like” activity. The predictive active site binding regions of both enzymes were then used to screen the Available Chemicals Directory (ACD), a public domain database. ACD contains small molecular mass, commercially available compounds for potential chemotherapeutic leads and is distributed by Molecular Design Limited Information System (San Leandro, CA). This approach, performed with the software DOCK3.5
Current Enzyme Inhibition, 2007, Vol. 3, No. 1
(University of California, San Francisco) , led to the selection of 69 compounds. Of these, 18 showed IC50’s between 50 and 100 µM and 3 had IC50’s below 50 µM. The most promising lead compound was a derivative of folic acid, known as PS28 (3,5-dichlorofolic acid, or L-glutamic acid, N-[4[[(2-amino-1,4-dihydro-4-oxo-6-pteridinyl)methyl] amino]-3,5-dichlorobenzoyl), as it is a reversible inhibitor of both peptidases and is easily derivatized, with an IC50 of 10 µM. The most likely orientation showed the pteroic acid half of the compound close to the catalytic triad, and the hydrophilic glutamic acid was predicted to be as pointing into either the hydrophobic S1-site or S2-site, which suggests that compounds having a more hydrophobic moiety may be better inhibitors. Hydrazides and Acyl Hydrazides In the original work of Selzer et al. , a second approach to the discovery and design of novel inhibitors for the L. major cysteine peptidases was to test a library of compounds, synthesized by combinatorial chemistry and originally derived from a structural screen against falcipain, the homologous peptidase of Plasmodium falciparum. This library was based on the original lead compound oxalic bis[(2-hydroxy-1-naphthyl)methylene]hydrazide, which generated a second group of compounds, CbzL’s. The enhanced inhibitory activity of these second-generation molecules is the result of specific synthetic modifications based on computer predictions made with DOCK. The results emphasized that L. major cathepsin B-like enzyme shares substrate-binding similarities with cathepsin L-like enzymes, as falcipain, and that both cathepsin B-like and cathepsin L-like enzymes from L. major were inhibited, overcoming the redundancy in activity suggested by the null mutant studies . The hydrazide inhibitors and the vinyl sulphones produced very similar effects . At 20 µM, the three hydrazides tested – ZLIII43A, ZLIII115A and ZLIII133A – totally inhibited parasite growth, and exchanging the culture media every day for a total of 3 days, thereby keeping the inhibitor concentrations stable, led to death of all the parasites. The reversible inhibitors ZLIII43A and ZLIII115A displayed a 2- to 10-fold higher inhibitory activity toward the L. major cathepsin B-like enzyme (IC50 10 µM and 2 µM, respectively) versus papain or mammalian cathepsin B, as demonstrated by Selzer et al. . The lack of toxicity to J774 cells, a mammalian macrophage cell line, at the inhibitor concentrations which kill parasites may reflect a greater peptidase redundancy in mammalian lysosomes or differential uptake of inhibitor by the parasites . To determine the effects of hydrazide inhibitors on Leishmania amastigotes, irradiated J774 cells were infected with metacyclic promastigotes, and after 12 hours macrophages were treated with a single dose (40 µM) of the inhibitor and cultured for another 5 days. Replication of the parasites in the amastigote form was decreased in cultures treated with the hydrazide inhibitors, and no macrophage was infected after the treatment. Treatment of the parasites with 50 µM ZLIII115A or vinyl sulphones produced very similar effects: the cathepsin B-like peptidase was localized in dilated lysosomes and flagellar pocket of treated parasites, suggesting that the cellular alterations seen are due specifically to inhibition of Leishmania cysteine peptidases.
Vermelho et al.
The inhibition of lesion development in vivo was detected at the same dose (100 mg/kg of body weight) previously tested for vinyl sulphones in L. major-infected BALB/c mice . These results demonstrated that there are differences in the susceptibility of specific stages in distinct Leishmania spp. to specific cysteine peptidase inhibitors: L. major seems to be sensitive to these compounds in all life cycle stages, since promastigote replication was arrested and flagellar pocket-endosomal pathway abnormalities were detected , but L. mexicana showed little or no effect of loss of proteolytic activity on promastigotes . In contrast, cysteine peptidases are virulence factors for the amastigote forms of both L. major  and L. mexicana . The implication of the cysteine peptidases in protein degradation was initially demonstrated by their localization in lysosomes. The inhibitors may have prevented peptidase precursor processing, resulting in accumulation and organelle damage along the trafficking pathway between the flagellar pocket and the lysosome/endosome compartment. The localization of the peptidase in secretory vesicles destined for the flagellar pocket and in the pocket itself suggested this is one pathway by which peptidase may reach the lysosome . Non-peptidyl acyl hydrazides, which are reversible inhibitors of brucipain (trypanopain –Tb), kill bloodstream forms of T. brucei in vitro at micromolar concentrations . Caffrey et al.  tested a library of 500 peptidyl acyl hydrazide derivatives against brucipain. Eight acyl hydrazide showed 50% or more inhibition of trypanosome replication at