Synthetic and Natural Protease Inhibitors Provide Insights into Parasite Development, Virulence and Pathogenesis

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Current Medicinal Chemistry, 2013, 20, 3078-3102

Synthetic and Natural Protease Inhibitors Provide Insights into Parasite Development, Virulence and Pathogenesis A.A. Rascón Jr.* and J.H. McKerrow Department of Pathology and the Center for Drug Discovery and Innovation in Parasitic Diseases, University of California, San Francisco, San Francisco, CA 94158 USA Abstract: Protease function is essential to many biological systems and processes. In parasites, proteases are essential for host tissue degradation, immune evasion, and nutrition acquisition. Helminths (worms) depend on several classes of proteases for development, host tissue invasion and migration, and for degradation of host hemoglobin and serum proteins. The protozoa, which cause malaria, depend on both cysteine and aspartic proteases to initiate host hemoglobin digestion. Other types of proteases are involved in erythrocyte cell invasion and cell exit. Surface metalloproteases in kinetoplastids are implicated in the evasion of complement-mediated cell lysis and cell entry. Cysteine proteases in Entamoeba facilitate invasion of the host colon. Giardia utilizes a cysteine protease for both encystation and excystation. This review will summarize published data using protease inhibitors as tools to identify the function of parasite proteases in the development, virulence, and pathogenesis of parasites; as well as the role of endogenous parasite protease inhibitors in regulation.

Keywords: Development, digestion, helminths, hemoglobin, kinetoplastids, nutrition acquisition, parasites, pathogenesis, proteases, protease inhibitors, protozoa, virulence factors. 1. INTRODUCTION Proteases play important roles in many biological processes, from protein regulation to cell signaling [1-4]. Regardless of their specific roles, this class of enzymes catalyzes the cleavage of peptide bonds through a common mechanistic pathway. The aspartic, glutamic, and metalloproteases activate a water molecule that nucleophilically attacks the peptide bond of the protein substrate (Fig. 1A). The serine, cysteine, and threonine proteases utilize their respective amino acids (Ser, Cys, and Thr) as the nucleophile to attack the peptide bond (Fig. 1B), and since the nucleophiles come from the protease active site, these proteases can be covalently modified (either reversibly or irreversibly) [1]. Cleavage of the peptide bond by any of these proteases is an irreversible process and so the expression of these enzymes must be highly regulated. When misregulation of protease action occurs in humans, a broad range of pathologic conditions may manifest, from cardiovascular disease to cancer [1, 3, 4]. In addition, many microorganisms utilize proteases as virulence factors for pathogenesis [5, 6]. Parasitic organisms are among the most common infections of humans and domestic animals. Humans have evolved elaborate immune mechanisms to prevent disease and minimize infection by parasites [7]. In response to these defenses, parasites have also evolved a set of virulence factors to circumvent the human immune response [6]. Parasite proteases are essential for tissue degradation, evasion of host immune responses, and nutrient acquisition from the host *Address correspondence to this author at the Department of Pathology and the CDIPD, QB3, Byers Hall 509, 1700 4th St., University of California, San Francisco, San Francisco, CA, 94158, USA; Tel: (415) 502-8196; Fax: (415) 502-8193; E-mail: [email protected]. 1875-533X/13 $58.00+.00

[5, 6, 8]. Previous reviews are available describing the role of proteases in parasite metabolism of host proteins for helminths (parasitic worms) [5, 8-10] and protozoa (Plasmodium [11, 12], Leishmania, Trypanosoma, Entamoeba [12, 13]). Interest in parasitic diseases stems not only from their interesting biology, but also because of the global health burden they impose. Recently acquired biochemical knowledge and structural details of parasitic proteases provide a foundation for the development of parasite protease-specific inhibitors [14]. When a given protease has been shown to play a key role in a given infection and/or pathology, one potential therapeutic approach is to target the protease with a small molecule protease-specific inhibitor [3]. For proteases that utilize active site amino acids as the nucleophilic moiety (the Ser, Cys, and Thr proteases), an electrophilic group can be used to modify, either irreversibly or reversibly, the catalytic residue, resulting in proteolytic inhibition [1]. This approach has been used successfully to design protease inhibitors as drugs for HIV infection, diabetes, hypertension, and hepatitis C [3, 4]. However, challenges may arise when developing protease inhibitors as drugs against the highly conserved mechanistic action of parasitic and host proteases. Mammalian hosts express protease homologues and attempts at inhibiting parasitic proteases could lead to unwanted inhibition of the host enzyme, with undesirable side effects [1]. Previous reviews have discussed this issue and describe approaches to developing and improving protease inhibitor design, see references [1, 3, 4, 12]. It should also be noted that parasitic proteases required for virulence (especially cysteine proteases) are often extracellularly secreted, while homologues in the host are localized intracellularly [14, 15].

© 2013 Bentham Science Publishers

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Fig. (1). General catalytic mechanism of metallo-, cysteine, and serine proteases. A) Metalloproteases activate a water molecule for noncovalent (acid-base) catalysis. B) Cysteine, serine, and threonine proteases utilize their respective (active site) amino acid residues as the nucleophile, forming an acyl-enzyme intermediate. Histidine functions as a base, deprotonating the catalytic amino acid residue.

“Druggable” serine, cysteine, and metalloproteases have been identified in various parasitic organisms and are discussed in previous reviews [5, 6, 11-14]. This review article will therefore focus on proteases and the effects of protease inhibitors in the development, stage-specific transitions, virulence, and pathogenesis of parasitic organisms. 2. MOLTING, INVASION, AND FEEDING OF PARASITIC WORMS (HELMINTHS) Helminths are large complex multicellular worms that include the nematode (roundworm), trematode (fluke), and cestode (tapeworm) parasites. These parasites infect hundreds of millions of people worldwide, as well as farm and range animals [16, 17]. They live and migrate through various host tissues and organs. The lifecycles vary among helminths, and may involve distinct stages cycling between different hosts, or direct transmission from one definitive host to another [17]. Regardless of the mode of transmission, most of the migratory stages of these parasites are larval [5]. 2.1. Nematodes In nematodes, the lifecycle always involves larval molting. As nematodes transition from one larval stage to an-

other, they rapidly outgrow their cuticle (exoskeleton). This cuticle serves to support the nematode body shape and provides a protective barrier [18]. During the four different molting stages, the nematodes synthesize and form a new larger cuticle [18, 19]. The most important molt in the host occurs from the infective third larval stage (L3) to the fourth larval stage (L4) [19]. The overall molting process may include short periods of inactivation (lethargus), separation of the cuticle from the epidermis (apolysis), movement to loosen the cuticle, and periodic shedding of the exoskeleton (ecdysis) [18-20]. Both apolysis and ecdysis have been shown to require proteases to degrade cuticle proteins. In addition, proteases are believed to be essential for the activation of pro-proteins that are then incorporated into the newly synthesized cuticle [19]. Early work on Brugia pahangi [21], Onchocerca volvulus [22, 23], and Ascaris suum [24], respectively, implicated a metallo-aminopeptidase, a cysteine protease (cathepsin L and Z-like), and a possible combination of an aminopeptidase and a cysteine protease in the L3 to L4 molt. Specific protease inhibitors blocked the transition from L3 to L4, resulting in larval degeneration [21-24]. RNA interference (RNAi) targeting filarial cysteine proteases confirmed the

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essential role that these enzymes have during L2 to L3 and the L3 to L4 molting [23, 25]. Ancylostoma caninum L3 infective larvae were shown to secrete a metalloprotease capable of degrading gelatin, collagen, laminin, and fibronectin [26]. The metalloprotease inhibitors EDTA and 1,10phenanthroline reduced the penetration of A. caninum L3 larvae in dog skin in vitro [26]. The infective larvae of the human hookworm, Necator americanus, also secrete metalloproteases capable of degrading collagen and elastin [27]. 1,10-phenanthroline and EDTA inhibited hamster skin penetration to some degree, but pepstatin A (an aspartic protease inhibitor) had ~54.8% inhibition compared to the control untreated larvae [27]. This study showed the importance of aspartyl proteases in the skin penetration process, but also verified the presence of other proteolytic enzymes, especially metalloproteases and serine proteases [27]. A metalloprotease from the infective filariform (L3) larvae of Strongyloides stercoralis has been shown to aid with skin penetration and tissue migration [28] in a similar fashion to the cercarial larvae of Schistosoma mansoni (discussed below). Upon contact with human skin, the S. stercoralis larvae rapidly penetrate the dermal extracellular matrix by secreting a neutral metalloprotease [29]. This metalloprotease (Ss40 [30] or strongylastacin [28]) was shown to cleave components of the extracellular matrix, elastin, and glycoproteins, but not collagen. Ss40 activity and larval skin invasion was inhibited by 1,10-phenanthroline, the metal chelator EDTA, but not by TIMP-proteins, the tissue inhibitor of metalloprotease [28, 29]. Ss40 was also shown to be immunogenic, stimulating IgG antibodies during infection of humans [30]. These results, coupled with the identification of other proteases involved in molting (i.e. serine proteases [18, 31]), led to the hypothesis that proteases are potential targets for anti-nematode drugs [18, 19, 24, 31]. 2.2. Trematodes 2.2.1. Schistosoma Mansoni Trematodes (flatworms) depend on a cercarial larval stage for infection of the mammalian host [32]. For schistosomes (blood flukes), the cercariae escape the intermediate host (typically a snail) and use different environmental cues (light-dark contrast, thermal gradients, and/or motion) to find the definitive host [33]. The cercariae then penetrate the host skin leading to infection [32-34]. Cercariae transform into schistosomula (young worms), which migrate through the lungs to the liver via the bloodstream, where they eventually mature into adult worms [33, 34]. To complete the lifecycle, the female adult worms lay eggs that pass through the wall of the intestine or bladder to exit the host in the stool or urine. Proteases are involved in each of the developmental stages of S. mansoni [5, 35]. With the annotation of the S. mansoni genome, a total protease profile has been described [36]. Proteases comprise ~2.5% of the proteome in S. mansoni, with the major difference between the parasite and human proteases being in the scarcity of chymotrypsin-like S1 family of enzymes (22 versus 135 sequences in human) [36]. Vertebrates and some invertebrates utilize this class of enzymes in highly complex and regulated proteolysis cascades, such as nutrition acquisition, development, and blood coagulation [36]. However, the reduced complexity in S. mansoni

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proteases translates to fewer proteolytic enzymes available to perform essential life functions, which make them attractive targets for drug discovery [36]. Nonetheless, a serine chymotrypsin-like protease (cercarial elastase) has been implicated in tissue invasion and migration of the cercarial stage [3740]. This protease facilitates degradation of elastin and related fibrillar macromolecules found in human skin [39]. It may also be involved in host immune evasion [41, 42]. S. mansoni cercarial elastase was shown to cleave human complement proteins (C3, C3b, iC3b, and C9, responsible for oponization of the parasite by macrophages) in vitro [41]. In addition, cercarial elastase can cleave human IgE, an antibody that plays a pivotal role in parasite killing [42]. PMSF, elastatinal, a chloromethyl ketone derivatized peptide, and a soybean trypsin inhibitor prevented cleavage of the complement proteins and IgE. Furthermore, in vivo and in vitro inhibition studies with the Suc-Gly-Ala-Leu-chloromethyl ketone [35] and other serine specific tetrapeptide reversible and irreversible chloromethyl ketones [39, 43] were shown to prevent cercariae from invading human skin. Research on the mechanism by which cercarial larvae invade human skin was initially based on studies using nonhuman animal models, artificial skin systems, or in the presence of skin-derived chemicals [44]. Studies using the hamster cheek pouch found that cercariae can penetrate the epidermal basement within 30 min, but are arrested in the epidermis for about 40 h before entering the dermis (mean time of 52.5 h) [45-47]. However, if human skin rather than the hamster cheek pouch is used, cercariae penetrate skin as far as the dermal-epidermal basement membrane within 30 min and enter the dermis within one hour [48]. Using viable human skin supported in an air/fluid phase static diffusion cell (a Franz cell), Bartlett et al. [44] showed that cercariae could attach to the skin within 1 min of exposure. The skin of model mammals, such as rat, mouse, guinea pig, and hamster differ from human skin in the amount of hair, chemical nature of the skin surface secretions, and the thickness of the epidermis and dermal layer [44, 49]. Therefore, it is important to use human skin in experiments when unambiguous results from human infection are sought [49]. A cathepsin B-like cysteine protease and a leucine aminopeptidase have been identified in S. mansoni eggs and are believed to help with migration and egg hatching, respectively [5, 35, 50]. Studies using RNAi against the leucine aminopeptidase, SmLAP1 transcript (and a closely related transcript, SmLAP2) inhibited the hatching of S. mansoni eggs by ~80% [51]. The gene transcripts are expressed at the egg stage, with SmLAP2 more highly expressed. These results indicate a role of leucine aminopeptidases in the hatching of S. mansoni miracidia [51]. 2.2.2. Fasciola Hepatica Fasciola hepatica is a food-borne parasite that causes the liver fluke disease, fasciolosis [52]. Infection by F. hepatica begins with ingestion of dormant metacercarial cysts found on contaminated vegetation [53]. After ingestion, the larvae excyst in the duodenum where they penetrate the intestinal wall, travel through the peritoneal cavity, enter the liver capsule, and eventually migrate to the common and hepatic bile ducts where they mature into adult flukes [53, 54]. The para-

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site secretes a large family of cysteine proteases [52, 55, 56]. Proteomic analysis of F. hepatica secretions identified cathepsin L enzymes as the major components involved in virulence [52]. McVeigh et al. [52] reviewed proteomic studies demonstrating the differential expression of cathepsin L proteases, as well as the identity and function of the individual cathepsin L members. Adult F. hepatica flatworms are found within bile ducts and primarily rely on cathepsin L proteases for hemoglobin digestion [52, 56]. Newly excysted juvenile larvae secrete cathepsin L, cathepsin B, and asparaginyl endopeptidases, but at 21 days of development, when the flukes are migrating through host tissue, the protease expression pattern changes to enhanced production of cathepsin L proteases [52]. Using general cysteine protease and cathepsin-specific inhibitors, Fasciola larvae were shown to depend on cathepsin L (FhCL3) and cathepsin B (FhCB) proteases for traversal of the gut wall [52 and references within]. The motility and viability of F. hepatica newly excysted juveniles were significantly reduced by the cathepsin B-selective inhibitor CA-074, indicating a key role of these proteases during development and excystment [52, 57]. RNAi against cathepsin B (FhCB2) and cathepsin L (FhCL1) proteases prevented newly excysted juveniles from invading and migrating through rat gut sacs [52, 58]. Cathepsin L proteases have been shown to be localized to the specialized gastrodermal cells that line the F. hepatica gut [52, 59]. Inactive pro-cathepsin L is secreted from the gastrodermal cells and activated in the parasite gut either autocatalytically or through pathways that lead to proteolytic cleavage of the propeptide [60]. Once activated, the proteases facilitate hemoglobin digestion in the fluke gut, and induce potent Th2 immune responses when secreted into host tissues [52]. Modulation of the host response by trematode cathepsins (including F. hepatica) has been reviewed, see [61]. See also [55, 59, 62, 63]. 2.2.3. Paragonimus Westermani Newly excysted larvae of the lung fluke Paragonimus westermani cause pulmonary or extrapulmonary paragonimiasis in humans [64, 65]. Humans become infected with P. westermani after consuming undercooked freshwater crayfish, crabs, or raw boar meat containing metacercariae (the infective larval stage of the mammalian host) [64]. After ingestion, the metacercariae invade the duodenum and migrate through several intra-abdominal organs before reaching the lungs where they mature into adult worms [64]. The lung fluke larvae release excretory/secretory products (ESP), including two abundant cysteine proteases [65]. Other proteins are found in the ESP of the lung fluke, but the origin, function, and biochemical nature of these proteins remains undetermined [66]. The two ESP cysteine proteases are implicated in excystment of the metacercariae [67], larval tissue invasion [64], and evasion of the host immune response [68]. They have also been reported to regulate interleukin-8 (IL-8) (an important protein involved in the human inflammatory response) and the lifespan of human eosinophils (white blood cells directed against multicellular parasites), resulting in eosinophil-mediated tissue inflammation [65]. These two cysteine proteases (one with a MW of 27 kDa and the other of 28 kDa) were purified from P. westermani newly excysted larvae using an anion exchange column [65]. Of the two iso-

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lated cysteine proteases, only the 27 kDa cysteine protease induced eosinophil superoxide anion production and degranulation in vitro indicating a possible role in eosinophilmediated tissue inflammation response during larval migration of P. westermani [65]. The 28 kDa cysteine protease species did not have an effect on eosinophil superoxide anion production and degranulation, but it has been shown to play an important role in modulating P. westermani metacercariae excystment [67]. In addition, other cysteine proteases have been detected at different developmental stages of P. westermani, including the egg and adult stages [66]. Proteomic analysis of P. westermani adult worm ESP revealed at least 15 different cysteine proteases [65, 66]. Due to the abundance of cysteine proteases in the different developmental stages of P. westermani, especially in the adult fluke ESP, these enzymes have been used in the immunodiagnosis of fluke infections [69, 70]. 2.2.4. Clonorchis Sinensis Clonorchis sinensis, the oriental liver fluke, causes clonorchiasis, a disease that can lead to cholecystitis, cholangiectasis, cholelithiasis, hepatic fibrosis, liver cancer and cholangiocarcinoma [71, 72]. Humans become infected with C. sinensis when raw or undercooked freshwater fish of the family Cyprinidae or shrimp containing C. sinensis metacercariae are ingested [72]. After ingestion, the metacercariae excyst in the duodenum and migrate to the liver bile ducts where they mature into adult worms [73]. Humans are the only definitive hosts. Regardless of the life stage, cysteine proteases in C. sinensis are thought to be essential in host nutrient uptake, immune evasion, and stage transition [74, 75]. A recent study using real-time PCR showed that an mRNA cysteine protease transcript of C. sinensis is present in the egg, metacercaria, excysted juvenile, and adult worm stages [72]. The cysteine protease is localized to the oral sucker, excretory bladder and tegument of both the cercariae and metacercaria stages, as well as in the intestine of the adult worm [72]. In addition, a cathepsin L-like cysteine protease was localized to the adult intestine and tegument of both the cercariae and metacercariae stages [76]. The excretory/secretory products, especially the cysteine proteases in the ESP of the C. sinensis adult worm, have been used in the serodiagnosis of clonorchiasis [72, 77, 78]. Enzymatic and proteomic analysis of C. sinensis ESP identified a legumain enzyme [79] and revealed multiple isoforms of cathepsin F-like cysteine proteases [80]. However, the main proteolytic activity of the ESP has been mainly attributed to these cathepsin F-like enzymes, which are localized in the intestine and the intestinal contents of C. sinensis [80]. Proteolytic activity was measured in in vitro assays using artificial fluorogenic cysteine protease substrates and defined by cysteine protease specific inhibitors (E-64, E-64d (a cell permeable form of E-64), and Z-FA-FMK). Since these cathepsin F-like cysteine proteases are synthesized and localized in the parasite intestine, the authors concluded that they might have a cooperative role in nutrient acquisition and in host-parasite interactions [80]. 2.3. Nutrition Acquisition: Nematodes and Trematodes Some nematodes and trematodes require the digestion of hemoglobin as a nutritional source for viability and egg pro-

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duction [16, 81-83]. However, unlike biting insects like the Aedes aegypti mosquito [84, 85] and other exoparasites that utilize serine proteases as the main digestive enzymes [82], helminths use cysteine and aspartic proteases for blood meal digestion [16, 35, 81, 83, 86]. This is consistent with the acidic (low pH) environment of the trematode gut [81] and (presumably) the nematode gut [10]. In schistosomes, cysteine and aspartic proteases are present in the surrounding gastrodermis and gut lumen of the adult worm [81]. No serine proteases have been localized to these compartments in schistosomes [87] or other parasitic worms. In vitro activity assays of recombinant cysteine proteases from the schistosome gut [16, 81] and from the nematode canine hookworm A. caninum gut [82], using both synthetic peptidyl and protein substrates, demonstrated optimal activity in the acidic range (pH 5.0 – 6.0 for synthetic substrates and pH 2.0 – 4.0 for hemoglobin). A recombinant hemoglobinase (a cysteine protease) from P. westermani was also shown to degrade hemoglobin at acidic pH values (3.0 – 5.0) [88]. Incubation of the recombinant cysteine protease (CP-2) from A. caninum with E-64 inhibited hemoglobin digestion in in vitro assays [82]. As for schistosomes, protease-specific irreversible inhibitors (diazomethane and fluoromethyl ketonederivatized peptides [16, 89]) have been shown to interrupt hemoglobin digestion and interfere with schistosome worm development [10, 16, 81]. Although fluoromethyl ketone peptide inhibitors were successful in reducing egg production and total worm burden in schistosome-infected mice [89], at the doses needed to cure schistosomiasis the fluoromethyl ketone moiety was toxic [86, 90]. These inhibitors could enter the Krebs cycle as fluoromethyl ketonederivatized amino acids and shut down cellular ATP production [90]. With the development of vinyl sulfone inhibitors the toxicity of this compound was eliminated in animal models [86, 90]. A 2007 study demonstrated the efficacy of a vinyl sulfone cysteine protease inhibitor (K11777, Fig. 2) in a murine model of schistosomiasis [91]. If given early in infection, K11777 reduced the number of mature worms recovered and significantly reduced egg production when given after worms matured and mated [91]. Inhibitors and RNAi applied against schistosome aspartic proteases also block hemoglobin degradation, affect egg production, and arrest worm development [92, 93]. To date, the only known aspartic protease from schistosomes is a cathepsin D-like enzyme [94]. The enzyme is localized to the gastrodermis and gut lumen [81, 95], but a recent study using RNAi against the S. mansoni cathepsin D transcript showed that schistosomules were not able to mature, reproduce, or survive in mice, indicating possible roles in growth, development, and maturation of schistosome larvae [93]. In S. japonicum, transcripts encoding cathepsin D were also found in eggs, miracidia, and adult male and female worms, indicating functional roles other than digestion [81, 96]. The recombinant enzymes from S. mansoni and S. japonicum have an acidic pH optima and digested human hemoglobin in vitro [95]. Two other helminth aspartic proteases, one from the canine hookworm A. caninum (Ac-APR1) and the second from the human hookworm N. americanus (Na-APR-1), were also studied in vitro using recombinant enzymes [97, 98]. These proteases are localized to the intestinal brush border membrane and cleaved hemoglobin in

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vitro. Notably, specific proteases were more efficient at digesting hemoglobin if the substrate was from specific permissive hosts [97, 98]. Hookworm proteases may also be involved in tissue migration of L3 infective larvae, since they degrade human collagen, albumin, and fibrinogen [98]. A second aspartic protease from N. americanus (Na-APR-2) is more similar to the family of nematode-specific aspartic proteases than to vertebrate cathepsin D [99]. Na-APR-2 is expressed by immature and mature adult hookworms and is able to digest hemoglobin and serum proteins in vitro. No aspartic protease activity was detected in the excretory/secretory products of adult hookworms [97, 99]. After initial cleavage of hemoglobin and serum proteins by the cysteine and aspartic proteases of parasitic worms, the newly digested fragments are further hydrolyzed by exopeptidases into smaller peptides or amino acids [81, 86, 100]. Dipeptidyl peptidase I (cathepsin C) secreted by the S. mansoni and S. japonicum gastrodermis (into the trematode gut) has been identified as the enzyme that cleaves dipeptides from larger protein fragments [81 and references within, 101]. In addition to cathepsin C, metallo-aminopeptidases are also believed to be crucial for the final stage of liberating free amino acids from fragments and peptides initially digested by the parasite gut cysteine and aspartic proteases. Only three parasitic worm metalloproteases have been recombinantly expressed and studied in vitro. A leucine aminopeptidase from S. mansoni was isolated and expressed as a recombinant protein. The recombinant leucine aminopeptidase was localized to the gastrodermal cells surrounding the gut lumen and had activity against fluorogenic peptidyl substrates [100]. The other two recombinantly expressed metalloproteases are Ac-MEP-1from A. caninum [82] and NaMEP-1 from N. americanus [102]. Both metallo-enzymes are localized to the brush border of the adult worm intestine and were not able to digest hemoglobin or heat-denatured globin [82, 102]. However, both Ac-MEP-1 and Na-MEP-1 did digest protein fragments generated by the cleavage of substrates by recombinant cysteine and aspartic proteases. A protease network (or cascade) involving aspartic, cysteine, and metalloproteases was initially described in the blood-feeding nematode, A. caninum, and was suggested to follow a semi-ordered pathway similar to that of the Plasmodium falciparum food vacuole [82]. In P. falciparum, aspartic and cysteine proteases make initial cuts in hemoglobin, which are further processed by metalloproteases [82, 103] (P. falciparum hemoglobin digestion is discussed further in the Protozoan Section). To show that this process happens in A. caninum, recombinant aspartic (APR-1), cysteine (CP-2), and metalloproteases (MEP-1) were cloned, expressed, purified, and tested against hemoglobin [82]. Results from hemoglobin digestion gel analysis and mass spectroscopy demonstrated that only APR-1 and CP-2 were able to digest native hemoglobin or denatured globin in vitro. MEP-1 could only cleave globin fragments after initial cleavage with the aspartic and cysteine proteases. Using class-specific protease inhibitors (pepstatin for APR-1, E-64 for CP-2, and 1,10phenanthroline for MEP-1) abolished hemoglobinolytic digestion in vitro. In S. mansoni, using a combination of protease class-specific inhibitors and RNAi, the role of each major endopeptidase was determined [87]. The proteases SmCB1, SmCL1, SmAE, and SmCD were shown to function

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Fig. (2). Structures of WRR483 and WRR605, analogs of K11777.

in a cooperative network for the degradation of hemoglobin and other host serum proteins. SmCB1, which is found in the schistosome gut, was directly implicated in hemoglobin degradation [50, 81]. However, studies using RNAi demonstrated that this enzyme was not essential for this process, but rather necessary for normal parasite growth [104]. Double-stranded RNA (dsRNA) against the SmCB1 transcript was electroporated into larval schistosomes, and although the newly treated schistosomes were viable, they could not be maintained in culture or survive when introduced into the host mouse. The SmCB1-dsRNA larvae digested hemoglobin, but compared to the control parasites, significant growth retardation was observed [104]. The cathepsin D-like cysteine protease (SmCD) was shown to have the greatest role in the primary cleavage of hemoglobin (based on inhibition studies and RNAi gene suppression), with fragment degradation by cathepsin B and L [87]. The cysteine proteases (cathepsins B and L) initiated cleavage of host serum albumin. SmAE may produce site-specific cleavages in albumin [87]. From these studies, the authors proposed a substratespecific cascade, in which cathepsin D makes the initial cleavage in hemoglobin followed by further cleavage into fragments and peptides by the cysteine proteases (cathepsins B, L, and C) and a metallo-aminopeptidase. The cysteine proteases (cathepsin B and L) are the primary enzymes responsible for albumin cleavage [87]. A proteolytic cascade for hemoglobin digestion was also found in the hookworm N. americanus. An aspartic protease (Na-APR-1), a cysteine protease (Na-CP-3), and a metalloprotease (Na-MEP-1) were recombinantly expressed and tested in vitro with human hemoglobin [102]. Only Na-APR-1 was shown to cleave intact hemoglobin, while Na-CP-3 and Na-MEP-1 could cleave globin fragments that had been initially digested by NaAPR-1 [102]. An alternative approach to drug inhibition against parasite digestive proteases is the development of a vaccine targeting gut-associated enzymes [10, 81, 83, 86, 102]. Cys-

teine proteases released into the host bloodstream elicit a strong immune response an effect that has been used as a serodiagnostic test for schistosomiasis [5, 16, 81]. Herd animal vaccines were produced that target gut proteases from the trematode F. hepatica [105-107] and the nematodes H. contortus, Ancylostoma spp. and N. americanus [83]. In H. contortus (the most pathogenic nematode of ruminants), cathepsin B-like cysteine proteases were found to be protective antigens [108]. The H. contortus cathepsin B-like enzymes are encoded by at least 23 cathepsin B genes [109]. The biological and biochemical roles for only a few of these proteases have been partially characterized [109-111]. These cysteine proteases are found in the nematode intestinal microvilli [111, 112] and studies using excretory/secretory products (ESP), crude extracts of whole worms, and gut tissue have demonstrated a possible role in blood meal protein digestion [111-115]. A cysteine protease (AC-5) from the excretory/secretory product of H. contortus was purified, enriched, and evaluated for its protective effect against H. contortus infection in lambs [108]. Vaccination with AC-5 reduced cumulative egg output and worm burden. This cysteine protease had not been previously identified in the ESP of H. contortus [108]. Other studies using purified protein extracts from adult worms (enriched for cysteine protease activity) [114] and recombinant inactive cysteine proteases [116, 117] have also been shown to confer significant levels of protection and reduce fecal egg outputs and final worm burden. However, the protection levels using the inactive recombinant enzyme were much lower than using cysteine protease-enriched fractions [117]. To overcome this problem, and the difficulties of expressing proteases in standard recombinant expression systems, Caenorhabditis elegans was used to express an active H. contortus cathepsin L cysteine protease (Hc-CPL-1) [118, 119]. Initial studies with a C. elegans cathepsin L mutant, rescued by transgenically expressing the Hc-cpl-1 gene, demonstrated that the enzyme was properly expressed in its active (correctly folded) form [118] and produced sufficient protein [119]. Unfortunately, vaccination of sheep with the purified recombinant Hc-CPL-

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1 did not provide any protection against infection or have any effects on embryonic development, egg hatching, or adult worms of H. contortus [119]. Vaccination of dogs with recombinant Ac-CP-2 (cysteine protease) from the canine nematode A. caninum did result in a decrease in parasite fecal counts and a decrease in the number of female adult hookworms in the intestine [120, 121]. In addition to vaccines against cysteine proteases, there are also effective vaccines against aspartic proteases and a leucine aminopeptidase. Vaccination studies with canines using recombinant Na-APR-1 from the human hookworm N. americanus and Ac-APR-1 from A. caninum demonstrated protective immunity against infection, significantly lowered fecal egg counts, and reduced hookworm burden [121-123]. Vaccination studies with sheep using recombinant leucine aminopeptidase from F. hepatica led to a significant reduction in fluke burdens compared to the adjuvant-alone control group [107]. 2.4. Cestodes 2.4.1. Echinococcus Echinococcosis is a zoonotic infection caused by Echinococcus spp. There are six known species, but only four have been shown to cause human disease: E. granulosus, E. multilocularis, E. vogeli, and E. oligarthus [124]. Of the four, E. granulosus and E. multilocularis are of greater concern to human health due to their potential to be lethal and because the diseases caused by these cestodes are emerging or re-emerging in endemic areas around the world [124]. Infection by E. granulosus metacestodes (an intermediate larval stage) (known as cystic echinococcosis or hydatid disease) leads to formation of cysts in the liver and lungs, but may also affect the kidneys, spleen, brain, and heart. If left untreated, cysts grow to a large size affecting the function of the organ causing rupture, which leads to anaphylaxis [125, 126]. Infection with E. multilocularis (or alveolar echinococcosis) also leads to formation of cysts in the liver. However, infection is characterized by multiple smaller cysts with an alveolar structure. Proliferation may lead to the destruction of host tissue and dissemination [127]. Although rarely diagnosed, alveolar echinococcosis is more fatal than cystic echinococcosis if left untreated [127]. Infection with E. vogeli and E. oligarthus (polycystic echinococcosis) also leads to cysts in the liver and abdomen, but is rarely diagnosed and is not as widespread in humans as E. granulosus or E. multilocularis [128, 129]. The lifecycle of Echinococcus spp. is very complex, involving a definitive carnivorous host (dogs and other canids) and an herbivore intermediate host (sheep, cattle, horses, pigs) [126]. Humans, however, serve as “dead end” intermediate hosts for all Echinococcus spp. due to accidental infection from the natural transmission cycles in domesticated or wild mammals [125, 130]. In the definitive host, the adult worms of Echinococcus are firmly attached to the mucosa of the small intestine. After reaching maturity they shed gravid proglottids (tapeworm segments with a mature sexual reproductive system) containing hundreds of eggs [126, 130-132]. The proglottids and/or Echinococcus eggs are released in the feces of the definitive host and are then ingested by the natural intermediate herbivore hosts. Human hosts may accidentally ingest proglottids or eggs. Upon ingestion, eggs release oncospheres (embryos) that penetrate the intestinal wall of both herbivore and human hosts and travel through the blood

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or lymphatic system. Upon reaching the liver, lungs, or other sites cyst formation begins [132]. After organ localization, the oncospheres develop into metacestodes (larval echinococcal cyst or hydatid cyst), which enlarge over time and produce protoscoleces (eventual tapeworm heads) [125, 131, 132]. To complete the lifecycle, the carnivore definitive host must ingest the protoscoleces found in the organs of the intermediate herbivore host or from those excreted in human feces. After ingestion, the protoscoleces evaginate in the small intestine, attach to the intestinal mucosa, and develop to adult worms [125]. Proteases from Echinococcus spp. are less well characterized than proteases from nematodes and trematodes. This is due to the difficulty of obtaining cestode-derived materials from animal model systems. Echinococcus spp. (and other cestodes) infiltrate and proliferate by exogenous budding of the cellular germinal layer making it difficult to separate contaminated host cells from parasite materials [133]. Although tapeworms are found worldwide, the most affected people are in developing countries where severe poverty is prevalent [132, 134]. The World Health Organization has recently included echinococcosis in its neglected tropical diseases (2008-2015) strategic plan [132]. Cystic echinococcosis (hydatid disease), alveolar echinococcosis, and taeniasis (beef or pork tapeworm infections, discussed later) have become increasingly common in the United States, with immigrants from countries endemic with these diseases being the most frequently diagnosed [132, 134]. Early work with total crude (tegument) homogenates of E. granulosus protoscoleces (the tissue-dwelling larval stage enclosed within hydatid cysts) demonstrated leucine aminopeptidase activity in vitro [135]. In 1991, researchers from Uruguay observed metalloprotease activity in the hydatid cyst fluid, cyst membranes, and protoscoleces of E. granulosus [136]. To measure proteolytic activity, each isolated preparation was run on polyacrylamide gels containing gelatin as a substrate. Each preparation was incubated in the presence or absence of different protease inhibitors. Serine, cysteine, and aspartyl protease inhibitors did not have an effect on proteolytic activity, but the metalloprotease inhibitors EDTA and 1,10-phenanthroline inhibited proteolytic activity. The highest gelatinase activity was observed in the cyst membrane samples indicating that the major metalloproteases originate from the cyst membrane [136]. The cyst membrane is the boundary between the host and the parasite, and the metalloproteases might play an important role in host-parasite interactions [136]. To date, only two cathepsin L-like [137] and two cathepsin B-like [133] cysteine proteases from E. multilocularis metacestodes have been characterized. The recombinant enzymes were shown to degrade IgG, albumin, type I and IV collagen, and fibronectin in vitro. The authors suggested these proteases might be involved in the pathogenesis of E. multilocularis and in protein degradation for parasite nutrition acquisition [133, 137]. However, unlike nematodes and trematodes, the cestodes lack a definitive gut and so must depend on the tegument for nutritional absorption [138]. The tegument is likened to the intestinal mucosa in humans, which performs similar absorptive functions [138]. Degradation of macromolecules by cysteine proteases would facilitate nutrient absorption by the parasite tegument [133, 137].

Inhibition of Parasitic Proteases

Unfortunately, no inhibition studies or gene knockouts have been done to elucidate the true function of these E. multilocularis cysteine proteases in vivo [133]. With limited studies on proteases from Echinococcus spp., the main focus has been on antigenic stimulation by the hydatid cyst. The Echinococcus hydatid cyst cavity is filled with a clear liquid (called hydatid cyst fluid), which contains proteins that stimulate an antigenic response [132]. Two major components of this cyst fluid are antigen 5 (Ag5) and antigen B (AgB), with Ag5 being the predominant protein species expressed and secreted in all lifecycle stages [139]. Ag5 is also used as a serodiagnostic test against echinococcosis [132]. Very little is known about the biological role of Ag5, but initial characterization revealed a 38 kDa subunit, which seems to be closely related to the serine protease trypsin family [140]. The protein was shown to have the critical cysteine residues for disulfide bond formation and part of the catalytic triad: the active site histidine and aspartic acid residues. However, the catalytic serine residue was replaced by threonine [140]. This mutation may not preclude catalytic activity [141]. However, native Ag5 from E. granulosus did not show activity in vitro with a variety of substrates [140]. Bacterially expressed and purified Ag5 protein from E. granulosus also failed to show protease activity in in vitro assays. An E. granulosus Ag5 homolog from Taenia solium (TsAg5) was recombinantly expressed in bacteria and purified. In contrast to the inactive E. granulosus Ag5 fusion protein, the trypsinlike domain of TsAg5 showed marginal activity in in vitro assays using an artificial trypsin substrate [142]. TsAg5 has 82% protein sequence identity with E. granulosus Ag5, including all the conserved critical amino acids at the active site, especially the catalytic serine to threonine mutation [142]. Like E. granulosus Ag5, TsAg5 was detected in the cyst fluid and in the excretory/secretory antigens of the cysticercus (metacestode larval stage) of T. solium [142], but whether or not TsAg5 (or E. granulosus Ag5 for that matter) is directly involved in tissue invasion is still unknown. 2.4.2. Taenia (Pork and Beef Tapeworms) T. solium (pork tapeworm) and Taenia saginata (beef tapeworm) are cestodes that can infect humans. Both species require a definitive host (humans) and an animal intermediate host, pigs (T. solium) and cattle (T. saginata) [143]. The lifecycle of these parasites is as complex as the Echinococcus spp. Upon ingestion of tainted meat that contains cysticerci (Taenia larval stage metacestodes), the metacestode evaginates in the small intestine and attaches to the intestinal wall, where it matures to an adult worm [134, 144]. The adult tapeworms shed proglottids (containing hundreds of eggs) daily, which are released in the feces. The proglottids and/or eggs are ingested by the natural intermediate hosts, and upon ingestion, the eggs lose their coats and free the oncospheres, or invasive larva, which can cross the intestinal wall reaching the brain or muscle to form a cysticercus [134, 144]. Humans can also be intermediate hosts of cysticerci upon ingestion of T. solium eggs. This leads to neurocysticercosis, a severe infection of the nervous system and the leading cause of acquired epilepsy worldwide [134]. Ingestion of T. saginata eggs does not cause cysticercosis in humans, only in cattle [143].

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Host tissue invasion by Taenia oncospheres in the intermediate hosts is a key step in the pathogenesis of these cestodes. Proteases have long been suspected as key factors in the pathogenesis of Taenia oncospheres, but no direct evidence is available. Nonetheless, proteolytic activity has been observed in Taenia oncosphere cultures. Studies using spent media from cultured T. saginata oncospheres identified excretory/secretory peptidases including serine and cysteine endopeptidases and an aminopeptidase [145]. Only PMSF and E-64 had an appreciable effect on activity [145]. The authors suggested that since cestode oncospheres must invade the host intestinal mucosa, secreted proteases might play an important role in invasion. A similar study compared the protease activities of the excretory/secretory antigens of T. solium and T. saginata oncospheres to determine if the profile of the proteolytic activities correlated with a difference in pathogenicity [143]. Oncospheres were cultured from both T. solium and T. saginata spent media and proteolytic activity measured using serine, cysteine, and aminopeptidase B fluorescent substrates. A variety of serine and cysteine protease inhibitors were used. Proteolytic activities attributed to serine proteases (trypsin-, chymotrypsin-, and elastaselike), cysteine proteases (cathepsin B, cathepsin L, and calpain-like), and aminopeptidase (B-like peptidases) were observed in both species of Taenia oncospheres [143]. The dominant protease activity was from the chymotrypsin-like serine proteases, consistent with the pH of the small intestine [143]. T. solium had 10-fold higher chymotrypsin-like activity than T. saginata at a 24 hr time point. The level of cathepsin B-like activity observed in both Taenia species was fairly low. T. solium had higher cathepsin B-like protease activity, while T. saginata had higher cathepsin L-like protease activity [143]. Cysteine proteases from T. solium were hypothesized to be involved in immune evasion since they cleave human immunoglobulins. They can also induce CD4+ lymphocytes apoptosis in vitro [146]. Lymphocytes co-incubated with living T. solium metacestodes (or metacestode excretory/secretory products) and E-64 maintained their structural integrity, while those not incubated with E-64 showed signs of apoptosis. Cysteine protease(s) in metacestodes might therefore down-modulate the host cell immune response by inducing apoptosis in CD4+ T cells. Cell death by CD4+ lymphocytes may serve as a mechanism to evade the host immune response, but the exact mechanism is unknown [146]. A cysteine protease from T. solium cysts was isolated from cysticercotic pigs and shown to cleave IgG in vitro at an acidic pH and reducing environment, and more importantly was inhibited with E-64 [147]. Assays with a recombinant cathepsin L-like cysteine protease from T. solium metacestodes confirmed this class of enzymes can cleave IgG [148]. Purified recombinant protease cleaved IgG and bovine serum albumin, but not collagen, with a pH optimum at 6.5. The addition of DTT increased activity in vitro and the enzyme was inhibited by E-64 [148]. 3. PROTOZOAN PARASITES 3.1. Hemoglobin Degradation, Cell Invasion and Cell Exit (Egress): Plasmodium Falciparum Malaria is estimated to cause 800,000 deaths and several hundred million clinical cases per year [11, 149]. Four dif-

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ferent species of Plasmodium protozoa cause most of the malaria in humans (P. falciparum, P. vivax, P. ovale, and P. malariae). P. falciparum is the deadliest form [150]. There have also been observations of transmission of the primate malarial parasite, P. knowlesi, harbored in monkeys, to humans, raising concern that this species might be easily spread to humans, making it that much more difficult to control [149]. Transmission of the parasite occurs via the bite of female anopheline mosquitoes [11, 149]. Upon ingestion of a blood meal, the Plasmodium sporozoite (the human-infective stage of the parasite) is injected from the salivary glands of the mosquito into the bloodstream of the human host. Sporozoites first develop in oocysts found within the extracellular environment of the mosquito epithelium. Sporozoites emerge, or “egress” from the oocyst compartment [151]. Egress of sporozoites depends on a cysteine protease called ECP1 [152]. In P. berghei, ECP1 gene deletion resulted in mature oocysts packed with sporozoites incapable of escaping [151, 152]. To regulate the number of sporozoites in the oocysts, programmed cell death (apoptosis) may be initiated by metacaspases, cysteine proteases structurally related to metazoan caspases [153, 154]. Knockout of P. berghei metacaspase 1 did not have an effect on any stage of parasite development, but metacaspase 1 may be compensated by two other metacaspases [153]. Caspase-specific inhibitors inhibited apoptosis in vivo [154]. Newly emerged sporozoites are carried through the mosquito hemolymph into the salivary glands where they differentiate into mature sporozoites capable of infecting vertebrate hosts [151]. Following a mosquito bite, sporozoites leave the salivary glands and travel to the liver and invade hepatocytes, where they undergo a transformation from sporozoites to exoerythrocytic forms [11, 149, 151]. Direct injection of sporozoites into the lumen of the blood capillary is not necessary to cause infection. Aly et al. [151] reviewed studies showing that blood-stage infection in mice is initiated after sporozoites are directly injected into the skin, muscle, peritoneum, and tail. Further, fluorescent labeling and intravital microscopy studies showed that sporozoites remain in the skin at the bite site, eventually entering the bloodstream [151]. The authors discuss the roles played by sporozoite proteins in cell traversal, but there is no mention of proteases involved in this process [151]. Proteolytic cleavage of a multifunctional protein, circumsporozoite protein (CSP), by a cysteine protease of parasite origin has been shown to be required for hepatocyte cell invasion [151, 155]. CSP is the major surface protein of the sporozoite and has been shown to mediate sporozoite adhesion and entry into hepatocytes [155, 156]. Treating sporozoites with E-64 prior to infection of hepatocyte cells in vitro inhibited 90% of infectivity [155]. However, proteolytic processing of CSP was not required for cell traversal. After sporozoite infection and transformation in hepatocytes, the hepatocytes eventually rupture releasing the merozoites into the bloodstream (the pre-erythrocytic stages) [11, 149]. The newly released merozoites then invade red blood cells in the bloodstream and the erythrocytic stage is initiated [11]. This stage is similar to the liver stage and involves the multiplication and asexual reproduction of merozoites. In disease pathogenesis, the erythrocytic stage is the key stage in the lifecycle because destruction of red blood cells is associated

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with morbidity and death in humans [149, 157]. Degradation of host hemoglobin at this stage provides a rich source of amino acids and metabolic energy needed by the parasite for survival [149]. A group of cysteine proteases (falcipains), aspartic proteases (plasmepsins), a metalloprotease (falcilysin), and aminopeptidases have been implicated in the degradation of hemoglobin in the parasitic food vacuole [158]. During the erythrocytic stage, the cytostome, an invagination in the food vacuole membrane required for phagocytosis, takes up hemoglobin from red blood cells and degradation ensues. Proteases work cooperatively to degrade hemoglobin, a process necessary to produce amino acids for protein biosynthesis, prevent erythrocyte cell lysis by maintaining osmotic stability, provide space needed for the asexual production of merozoites, and for metabolic energy [150, 158, 159]. Hemoglobin cleavage follows a semi-ordered process where either plasmepsins or falcipains initiate hemoglobin digestion, followed by cleavage of the smaller peptides by falcilysin and dipeptidyl aminopeptidase 1, and final cleavage into amino acids by aminopeptidases [11, 158, 159]. Inhibition by E-64 (cysteine proteases) and leupeptin (an inhibitor of serine, cysteine, and threonine peptidases) inhibited hemoglobin degradation and produced food vacuole swelling, while inhibition of aspartic proteases did not [159 and references within]. Four falcipains (clan CA cysteine proteases) have been identified and knockout of falcipain-2 resulted in arrest of hemoglobin degradation and production of dark stained swollen food vacuoles, consistent with hemoglobin accumulation [159, 160]. Knockout of both falcipains and aspartic protease genes led to phenotypes similar to wildtype, but with 50-fold more sensitivity to pepstatin (an aspartic protease inhibitor) [159, 160]. Knockout of falcipain-3 was lethal, but replacement with a tagged functional copy was achieved, indicating that falcipain-3 is an essential enzyme [159, 161]. Knockout of falcipain-1 and falcipain-2’ did not yield a phenotype [159, 161]. Knockout of plasmepsins PM-I, PM-II, PM-III (also known as histo-aspartic protease (HAP)), and/or PM-IV in the presence of aspartic protease inhibitors had no effect on hemoglobin degradation, but did inhibit parasite development at an earlier stage [162]. PM gene disruptions did enhance parasite susceptibility to cysteine protease inhibitors, which in turn inhibited hemoglobin degradation and noticeably affected food vacuole morphology [162]. These results suggest that both plasmepsins and cysteine proteases (falcipains) play a complementary or redundant role in hemoglobin degradation. Falcipains were also shown to activate pro-plasmepsins to the active mature form [163] indirectly contributing to hemoglobin degradation [159]. E-64d or a combination of E-64d and pepstatin inhibited plasmepsin processing, and purified falcipain-2 and falcipain-3 were shown to cleave plasmepsin-II at the natural cleavage site [159, 163]. Genome analysis has revealed the presence of ten aspartic protease plasmepsins in P. falciparum, but only four of these are found in the acidic food vacuole [150, 158]. PM-V resides in the endoplasmic reticulum of the parasite and is responsible for erythrocyte remodeling by exporting effector proteins beyond the parasitophorous vacuole membrane [164, 165]. PM-IX and PM-X have potential roles in the early developmental stages of P. falciparum [162].

Inhibition of Parasitic Proteases

After plasmepsin or falcipain processing, falcilysin (FLN) [103, 166], dipeptidyl aminopeptidase 1 (DPAP1) [167], and aminopeptidases [168] cleave the remaining oligopeptides of hemoglobin into smaller peptides and amino acids. Metal chelators have a generalized inhibitory effect on FLN activity [103], but selective inhibitors have not as yet been identified [158]. The FLN metalloprotease has been shown to have dual functions, one as a food vacuole protease at acidic pH (5.2) and as a protease for degradation of transiting peptides in the apicoplast and mitochondria of the parasite (at neutral pH) [169]. More studies are needed to determine the transcriptional regulation of this protease [169] and to determine the effects on Plasmodium survival by FLN-specific inhibitors. DPAP1 semicarbizide- and nitrilepeptide analogs inhibit recombinant DPAP1 activity in vitro, inhibiting dipeptide formation in vivo and further processing into individual amino acids [167]. Potent nonpeptidic DPAP1 inhibitors also result in P. falciparum death in vitro [170]. DPAP1 is the only dipeptidyl aminopeptidase found in the hemoglobin degradation pathway [170]. Nine metallo-aminopeptidases have been identified in the P. falciparum genome, with five thought to act in concert in the final stages of hemoglobin degradation [171]. These include an alanine aminopeptidase (PfA-M1), a leucine aminopeptidase (PfA-M17), an aspartyl aminopeptidase (PfM18 AAP), a prolyl aminopeptidase, and a post-prolyl aminopeptidase (or prolyl iminopeptidase), which cleaves amino acids from the N-terminus of peptides with a prolyl group in the P1’ position [168, 171]. The other four peptidases are methionine aminopeptidases believed to play a housekeeping role by removing the initiator methionine from synthesized polypeptides [168, 171]. The use of activity-based probes against PfA-M1 and PfA-M17, based on the general scaffold of bestatin (a metallo-aminopeptidase inhibitor), lead to inhibition of both enzymes in cultured parasites [172]. Specific inhibition of PfA-M1 prevented hemoglobin degradation and caused swelling of the parasitic food vacuole and death. Inhibition of PfA-M17 led to death prior to hemoglobin degradation [172]. PfM18APP gene disruption suggested that the enzyme was nonessential for blood-stage replication, but did result in a loss of fitness to the parasite [171, 173]. To date, no potent PfM18APP inhibitors have been identified. The structures of PfA-M1 [174], PfA-M17 [175], and PfM18 AAP [171] have been solved, but structures of other aminopeptidases are not available. Very little biochemical information is available for the remaining aminopeptidases [168]. The processes that lead to hepatocyte and red blood cell invasion and cell exit (egress) share some common mechanistic aspects involving proteases. Some consider invasion to begin at the moment the parasite escapes the cell, but in contrast to hemoglobin degradation, cell invasion involves serine proteases, especially those of the subtilisin family [176]. The first indication that serine proteases were involved in red blood cell invasion by malaria parasites came from results with chymostatin, a serine protease inhibitor. In experiments with P. knowlesi and P. chabaudi merozoites, chymostatin, but not leupeptin or E-64, inhibited cell invasion; the compound inhibited a subtilisin-like protease [159 and references within]. Three subtilisin-like proteases were identified (PfSUB1, PfSUB2, and PfSUB3) in the Plasmodium genome. Only PfSUB2 is directly implicated in cleaving merozoite surface proteins required for erythrocytic invasion

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[176-178]. PfSUB1 is involved in the rupture and release of Plasmodium merozoites from erythrocytes [179]. PfSUB1 was shown to activate a group of proteins, the serine rich antigen (SERA) proteases, so named because of the presence of more than 30 serines on the N-terminus [159, 180]. These enzymes have modest homology to the papain-family clan CA proteases, but some SERA homologues have a Ser rather than a Cys catalytic residue [159, 180]. Of the nine SERA proteases identified, SERA1-5 and SERA9 have the catalytic serine residue [180] and SERA6-8 have the catalytic cysteine [159]. Only SERA5 and SERA6 are localized in the parasitophorous vacuole, the membrane that surrounds the intracellular parasite, indicating their possible roles in egress [159, 176]. An ortholog of SERA8, ECP1 (described earlier), was shown to block sporozoite egress from oocysts in P. berghei [152, 159]. The precise roles of these SERA proteases are still unknown, but prior to parasitic egress PfSUB1 is transported to the parasitophorous vacuole space where it proteolytically cleaves SERA5 [159], which may facilitate degradation of the merozoite surface proteins required for exit [179]. PfSUB1is the best studied of the three subtilisins and has been shown to be essential in the asexual blood-stage of the P. falciparum lifecycle [179]. Selective inhibitors against PfSUB1 were shown to inhibit egress and reduce the invasiveness of newly released merozoites [179, 181, 182]. Activation of the PfSUB1 precursor (inactive) protease depends on the proteolytic activity of DPAP3 (dipeptidyl aminopeptidase 3) [182]. E-64 prevented invasive merozoite egress in P. falciparum cultures, possibly due to the inhibition of DPAP3 [179]. Unpublished gene disruption experiments have shown that PfSUB3 is not required for the asexual blood-stage of P. falciparum in vitro [179, 181]. The specific role of this subtilisin is not known and has yet to be functionally characterized [177, 183]. It is important to note that parasite egress from erythrocytes depends on a tightly regulated series of events involving all of the aforementioned proteases. Initially, the process was thought to be a single rapid explosive event happening at the end of the 48-hour lifecycle, but recent work has shown that the process takes place in a more systematic way over a 15-20 h period, and possibly involving the SERA family of proteases [184]. 3.2. Virulence and Pathogenesis of Protozoan Kinetoplastids: Leishmania and Trypanosoma Kinetoplastids are single-cell flagellated protozoa. Within this class, the trypanosomatids, especially Leishmania and Trypanosoma spp., are of great medical and economic importance [185, 186]. Leishmania spp. invade vertebrate macrophages causing the disease, human leishmaniasis, in more than 2 million people a year in 88 countries from North and South America to Asia, Africa, and Europe [5, 14, 186, 187]. Trypanosoma cruzi causes Chagas’ disease, the leading cause of dilated cardiomyopathy in Central and South America [187, 188]. The common pathway of infection by trypanosomatids is via an insect vector. Leishmania spp. requires a sand fly for transmission, T. brucei the Tsetse fly, while T. cruzi is transmitted by the Reduviidae family of true bugs [5, 186, 188]. 3.2.1. Leishmania Spp The lifecycle of Leishmania spp. shuttles between a motile flagellated promastigote stage that asexually reproduces

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in the gut of the sand fly, and a non-motile amastigote stage [5, 186, 187]. Upon ingestion of a blood meal, the sand fly transmits the infective metacyclic promastigotes into the host dermis where they are phagocytosed by mammalian macrophages [186]. Within the macrophages, the promastigotes transform into amastigotes, replicate within parasitophorous vacuoles (eventually rupturing the host macrophage) and are primed for uptake by another sand fly. Replication and macrophage activation leads to the development of immune response-dependent lesions [5]. In the mid-1980s, a zinc metalloprotease was discovered on the surface of Leishmania spp. promastigotes that was shown to play a role in pathogenesis [186]. This major surface protease, MSP (also called GP63 or leishmanolysin), accounts for about 1% of the total promastigote proteins in L. major and L. mexicana [186]. MSP belongs to the M8 endopeptidase family and is homologous to the mammalian matrix metalloproteases. It is anchored to the plasma membrane by glycosyl phosphatidyl inositol (GPI) [186]. GP63 was implicated in the evasion of complement-mediated cell lysis, phagocytosis of promastigotes into macrophages, and survival of intracellular amastigotes [186]. Leishmania spp. have multiple MSP genes that are differentially expressed at different lifecycle stages [186]. Nonetheless, the enzymatic domains within the different MSP genes are conserved, which could indicate stagespecific regulation [186]. Peptidomimetic metalloprotease inhibitors against purified GP63 from L. major promastigotes inhibited cleavage of a synthetic peptide in vitro, but no effect was observed in the growth culture of L. major promastigotes [189]. However, injecting susceptible BALB/c mice with liposome-encapsulated recombinant GP63 conferred significant protection against L. major [186, 190]. Promastigote GP63 may facilitate phagocytosis of parasites by host macrophages by acting as a parasite receptor. When recombinant encapsulated GP63 was injected into the mice, prior to L. major infection, it stimulated host immunity, presumably protecting the mice from promastigote invasion [190]. Cysteine (papain-like) class proteases of Leishmania are involved in virulence, cell differentiation, and immune evasion [185, 191]. The role of cysteine proteases in Leishmania infection has been extensively reviewed, see [5, 16, 86, 185 187, 191]. Cathepsin L (CPA and CPB) and cathepsin B-like (CPC) proteases are found in all Leishmania species and are predominantly expressed in amastigotes [5, 187, 191]. In L. chagasi and L. donovani, cathepsin B-like proteases are expressed in all lifecycle stages [185, 191]. Cysteine protease specific inhibitors have inhibited growth/replication of L. major and L. mexicana [187, 191]. Functional information on CPA, CPB, and CPC was obtained through gene disruption experiments in L. mexicana. CPA [192] and CPC [193] deletion mutants were shown to have wild-type phenotype, producing lesions on BALB/c mice, whereas CPB mutants [192, 194] were much less infective and produced small, slow-growing lesions [191]. Double CPA and CPB deletion mutants produced a similar phenotype to the CPB deletion mutant [192]. CPB is encoded by a tandem array of 19 genes [191, 194, 195]. To restore sustained virulence in CPB null mutants, multiple CPB genes had to be reinserted [196]. Single gene reinsertions did not restore virulence [191, 196]. Inhibitors developed against CPB had some efficacy in vivo

Rascón and McKerrow

and in vitro against Leishmania, but only at higher concentrations [197]. The lack of efficacy of cysteine protease inhibitors in models of Leishmania infections may reflect the redundancy of these proteases in Leishmania spp., in contrast to those in the related T. cruzi and T. brucei parasites. Two serine-like proteases, a subtilisin and an oligopeptidase, have been identified and characterized in L. donovani. As mentioned, the lifecycle of Leishmania spp. involves a promastigote stage in the midgut of the sand fly vector and an amastigote stage in the mammalian host [5, 186, 187]. During its lifecycle, the protozoan is exposed to reactive oxygen species, and the ability of these protozoa to withstand oxidant damage is essential for their survival, and particularly for amastigotes, which must be capable of overcoming the oxidative burst generated by activated host macrophages [198]. Leishmania and other kinetoplastids possess a unique redox metabolic system based on trypanothione, an antioxidant thiol, as the main transporter of electrons [198200]. This complex system involves the reduction of a thioredoxin-related protein by trypanothione, which in turn reduces a peroxiredoxin-type peroxidase that ultimately reduces the highly oxidative species [200]. The levels of these key peroxidases are regulated by a subtilisin-like serine protease (SUB) [201]. Gene deletions in L. donovani and proteomic analysis of wild-type and SUB knockouts revealed that subtilisin is responsible for processing the C-termini of tryparedoxin peroxidases, as indicated by an increase in the higher molecular weight isoforms of these enzymes [201]. In addition, L. donovani SUB knockouts were inefficient at undergoing promastigote to amastigote differentiation in vitro, and SUB-knockout amastigotes had abnormal membrane structures, retained flagella, and had increased binucleation [201]. In hamster and murine infection models, SUB knockout Leishmania displayed reduced virulence and the absence of lesions in the spleen. Similar approaches using gene disruption and proteomic analysis identified oligopeptidase B (OPB) as the enzyme responsible for the high level of serine protease activity expressed by L. donovani [202]. Knockout of the OPB gene resulted in the disappearance of serine protease activity in L. donovani extracts, and more importantly, decreased virulence in a murine footpad infection model. The specific role of OPB was determined by proteomic analysis of the wildtype versus OPB null. There was a significant increase in four enolase protein species [202]. Apart from its role in carbohydrate metabolism, enolase has also been shown to be an essential virulence factor in both bacterial pathogens and protozoan parasites, acting as a cell surface ligand binding host plasminogen [202, 203]. Enolase-bound plasminogen could facilitate macrophage entry by the parasite or prevent fibrin deposition at the site of infection if activated to plasmin. At the same time, enolase retention could activate host macrophages and negatively affect parasite survival, as was observed with OPB null parasites [202]. Wild-type L. donovani did not cause a change in macrophage gene expression, but OPB knockouts did induce a key increase in macrophage gene transcription. This study demonstrated a key role of OPB in Leishmania virulence, and with the determination of the OPB crystal structure, specific OPB inhibitors with therapeutic potential could be developed [204].

Inhibition of Parasitic Proteases

Current Medicinal Chemistry, 2013, Vol. 20, No. 25 3089

3.2.2. Trypanosoma cruzi

teine protease inhibitors was shown to prevent autocatalytic processing and cruzain trafficking causing accumulation of cruzain in Golgi-specific vesicles [209]. Validation of T. cruzi cysteine proteases by gene deletion has not been possible due to their essential roles in the parasite lifecycle [208]. However, the expression of cruzain does result in enhanced metacyclogenesis [210]. Peptide-fluoromethyl ketones arrested intracellular replication and intercellular transmission of T. cruzi in mammalian cells in vitro [211] and rescued mice from lethal T. cruzi infection [212]. The fluoromethyl ketone moiety proved to be too toxic as a drug candidate; however, vinyl sulfones were equally effective [86, 185, 188] (as mentioned earlier). K11777, a vinyl sulfone inhibitor that blocks the lifecycle of T. cruzi both in vitro and in animals [5], is the only parasite protease inhibitor awaiting final FDA approval for use in Phase 1 clinical trials [188] (see Table 1). Preclinical studies that have been completed for this inhibitor include a 7-day dosing of monkeys at 200 mg/Kg, a dose escalation study in rodents, dogs, and monkeys, and a 14-day chronic dosing study in rodents. The key remaining study before a “first in man” exposure is a 28-day chronic dosing of monkeys. This latter study is funded by the NIAID and should occur in 2012.

In T. cruzi infection, the parasite is not transmitted directly from the vector to the host during blood feeding. Instead, the insect vector deposits parasite-infested feces onto the skin of the vertebrate host. Fecal contamination at the wound site or mucous membranes (the eyes, nose, or mouth) allows the transmission of the infective trypomastigote stage [187, 188]. Upon entry into the host cell the bloodstream trypomastigote transforms into the non-motile replicative amastigote [186, 188]. The amastigotes transform back to trypomastigotes just before host cell rupture and then reinvade host cells or are taken up by a Reduviid insect vector during a subsequent infection event [188]. In the insect, the trypomastigotes transform to replicative epimastigotes, which are not infective to humans [187, 188]. MSP (GP63) Leishmania homologues have been found on the surface of T. cruzi. Approximately 425 MSP gene homologues are present in the T. cruzi genome and are differentially expressed at different lifecycle stages [186]. T. cruzi GPI-anchored MSP homologues do show zinc metalloprotease activity [186, 205]. In addition, affinity purified antibodies raised against these MSPs showed 50% inhibition of mammalian cell invasion by trypomastigotes in vitro [186, 205]. Other proteases implicated in T. cruzi cell invasion include two members of the serine protease prolyl oligopeptidase (POP) family, POP Tc80 and oligopeptidase B [13, 205]. POP Tc80 has been implicated in adhesion to host cells and cell invasion, and was shown to have specificity for fibronectin and human collagen (types I and IV) [13, 205]. POP Tc80 was identified in cell-free extracts of all T. cruzi stages (trypomastigotes, amastigotes, and the insect stage epimastigotes) [205]. Oligopeptidase B, on the other hand, is a cytosolic enzyme believed to generate a calcium (Ca2+) signaling agonist, which facilitates entry into nonphagocytic cells [13]. Epoxy--lap (a napthoquinone compound) and PMSF against T. cruzi whole extracts, inhibited TAME (4toluenesulfonyl-L-arginine-methyl ester) activity in vitro [206]. Although the authors do not attribute the activity solely on the POP family serine proteases (both POP Tc80 and oligopeptidase B), it is possible that both proteases contributed to TAME activity and both were inhibited. Cysteine proteases play an important role in the lifecycle and pathogenicity of T. cruzi. Of the seventy-cysteine protease genes predicted from the T. cruzi genome [207], one, cruzain (cruzipain), is the major protease essential for survival and proliferation of the parasite. It has been extensively studied for more than two decades and many reviews are available, see [5, 90, 185, 188, 207, 208]. Cruzain is expressed in all stages of the T. cruzi lifecycle, with higher levels in the epimastigote stage [188]. In the insect vector stage, cruzain aids with the degradation of endocytosed nutrients and is localized in lysosomal-like organelles [5]. In the human stages, it is localized in the flagellar pocket of trypomastigotes, and to the cell surface in the amastigote stage [5, 188]. Inhibitor studies with peptidyl diazomethane derivatives (a class of cysteine protease-specific inhibitors) demonstrated the role of cruzain in host cell invasion and in differentiation of trypomastigotes to amastigotes, and amastigote to trypomastigote differentiation [188, 205]. In addition, pre-treating T. cruzi epimastigotes with different cys-

3.2.3. Trypanosoma brucei The lifecycle of T. brucei and some aspects of T. brucei disease progression are also dependent on cysteine proteases [213]. Rhodesain (or brucipain, trypanopain) (cathepsin L) and TbCatB (cathepsin B) are the two major cysteine proteases expressed by bloodstream T. brucei parasites [214-216]. Rhodesain and TbCatB have been identified in all lifecycle stages of the parasite, especially the infective stage [215, 217]. Rhodesain is likely required to cross the blood-brain barrier [218-220]. In murine models of infection, trypanosomes with cathepsin L (rhodesain) expression reduced by RNAi showed no effect on parasitemia or splenomegaly, but a prolonged survival rate of infected mice was observed [218] coupled with in vitro data from Grab et al. [220]. This suggested rhodesain might be required for parasites to cross the blood-brain barrier and produce fatal encephalitis. RNAi knockdown of TbCatB also enhanced mouse survival [218] and eliminated T. brucei parasites in vitro [216]. RNAi knockdown of TbCatB in cultured T. brucei resulted in defective cytokinesis and parasites with a “tadpole” morphology due to an enlarged endosome [216]. In addition, single allele tbcatb deletion studies resulted in T. brucei mutants exhibiting decreased growth rates, accumulation of host and parasite proteins, and morphological abnormalities [221]. The diazomethane inhibitor Z-Phe-Ala-CHN2 [222, 223], K11777 [216, 224, 225], and cathepsin L-specific inhibitors [226] led to parasite death both in vivo and in vitro [217]. Other cysteine protease inhibitors are lethal to T. brucei parasites in vitro and have been shown to cure T. brucei infection in animal models [215, 216, 222-224, 227, 228]. Oligopeptidase B (TbOPB) is a serine protease proposed to play a role in crossing of endothelial cell blood barriers by trypanosomes and possibly also in host hormone deregulation [219, 229]. TbOPB null mutants in T. brucei did not have an affect on trypanosome parasite survival in vitro or in

3090 Current Medicinal Chemistry, 2013, Vol. 20, No. 25

Table 1.

Rascón and McKerrow

Parasite Protease Targets and Selected General Or Validated Inhibitors (In Order of Appearance Within Text)

Organism

Target Protease

Inhibitor

HTLa/LOb

Animal Model

Preclinical

Phase 1

Reference

Validation B. pahangi

Aminopeptidase

Phosphoroamidon and H-boro-

No/No

No

No

No

[21]

Phe-(pinacol) O. volvulus

Ov-CPL (cathepsin L)

Fluoromethyl ketones

No/No

No

No

No

[22, 23]

A. suum

Aminopeptidase

Amastatin

No/No

No

No

No

[24]

A. suum

Cysteine Protease

Fluoromethyl ketones

No/No

No

No

No

[24]

Ac-MTP-1

1,10-phenanthroline

No/No

No

No

No

[26]

1,10-phenanthroline, EDTA

No/No

No

No

No

[28, 29]

Fluoromethyl ketones, E-64,

Yes/Yes

Yes

No

No

[35, 50, 89, 91, 279, 280]

Bestatin

No/No

No

No

No

[35, 50, 51, 100, 281]

Various serine protease inhibi-

No/No

No

No

No

[35, 39, 41-43]

No/No

No

No

No

[52 and references within]

A. caninum

(metalloprotease) S. stercoralis

Strongylastacin (metalloprotease)

S. mansoni

Cathepsin B

K11777, CA-074, and other inhibitors S. mansoni

Leucine aminopeptidase

S. mansoni

Cercarial elastase

tors (i.e. PMSF, chloromethyl ketones) F. hepatica

Cathepsin L

General cysteine and cathepsinspecific inhibitors

F. hepatica

Cathepsin B

CA-074

No/No

No

No

No

[57]

P. westermani

Cysteine proteases

E-64

No/No

No

No

No

[65]

C. sinensis

Cathepsin F

E-64, fluoromethyl ketones

No/No

No

No

No

[80]

A. caninum

Cysteine protease

E-64

No/No

No

No

No

[82]

No/No

No

No

No

[87, 95, 282]

(CP-2) S. mansoni and

Cathepsin D

Pepstatin A and other statin

S. japonicum

(aspartic protease)

inhibitors

Ac-APR-1

Pepstatin A

No/No

No

No

No

[82, 97, 98]

Pepstatin A

No/No

No

No

No

[97]

Pepstatin A

No/No

No

No

No

[99]

E-64, Iodoacetamide

No/No

No

No

No

[101]

1,10-Phenanthroline

No/No

No

No

No

[82]

1,10-Phenanthroline

No/No

No

No

No

[27, 102]

A. caninum

(aspartic protease) N. americanus

Na-APR-1 (aspartic protease)

N. americanus

Na-APR-2 (aspartic protease)

S. mansoni and

Cathepsin C

S. japonicum

(Dipeptidyl peptidase I)

A. caninum

Ac-MEP-1 (metalloprotease)

N. americanus

Na-MEP-1 (metalloprotease)

S. mansoni

Cathepsin L

E-64, K11777

No/No

No

No

No

[87]

S. mansoni

SmAE (asparaginyl endopeptidase)

Iodoacetic acid, Aza-peptidyl Michael acceptors, and peptidyl allyl sulfones

Yes/No

No

No

No

[87, 283, 284, 285]

H. contortus

AC-5 (cysteine protease)

E-64

No/No

No

No

No

[108]

Inhibition of Parasitic Proteases

Current Medicinal Chemistry, 2013, Vol. 20, No. 25 3091 (Table 1) contd….

Organism

Target Protease

Inhibitor

HTLa/LOb

Animal Model

Preclinical

Phase 1

Reference

Validation H. contortus

Hc-CPL-1

E-64

No/No

No

No

No

[119]

(cysteine protease) E. granulosus

Metalloproteases

1,10-phenanthroline

No/No

No

No

No

[136]

E. multilocularis

Cathepsin L

E-64

No/No

No

No

No

[137]

E. multilocularis

Cathepsin B

E-64

No/No

No

No

No

[133]

TsAg5

Chymostatin

No/No

No

No

No

[142]

T. solium

(serine-like protease) T. solium

Cysteine proteases

E-64

No/No

No

No

No

[146, 147]

T. solium

Cathepsin L-like

E-64

No/No

No

No

No

[148]

ECP1

E-64

No/No

No

No

No

[152]

Falcipains

Leupeptin, E-64, peptidyl

Yes/Yes

No

No

No

[159 and references within, 286]

(cathepsin L-like)

fluoromethyl ketones, vinyl

Yes/Yes

No

No

No

[287]

Yes/Yes

No

No

No

[162]

P. falciparum

(cysteine protease) P. falciparum

sulfone, and aldehyde inhibitors P. falciparum

P. falciparum

Falcipains

2-Pyrimidinecarbonitrile

(cathepsin L-like)

inhibitors

Plasmepsins I-IV

Pepstatin A and other aspartic

(aspartic proteases)

protease inhibitors

P. falciparum

Plasmepsin V

Pepstatin A

No/No

No

No

No

[164]

P. falciparum

Falcilysin

EDTA, 1,10-phenantrholine

No/No

No

No

No

[103]

P. falciparum

DPAP1 (aminopeptidase)

Semicarbizide-, nitrile-peptide, and nonpeptidic analogs

No/No

No

No

No

[167, 170]

P. falciparum

DPA1 (aminopeptidase)

JCP405, JCP410, SAK2

Yes/Yes

No

No

No

[182]

P. falciparum

PfA-M1 and PfAM17 (metalloaminopeptidases)

Bestatin-based analogs

No/No

No

No

No

[172]

P. falciparum

Subtilisin family

Chymostatin

No/No

No

No

No

[159 and references within]

P. falciparum

PfSUB1 (subtilisin)

Peptidyl alpha-ketoamides

No/No

No

No

No

[179]

P. falciparum

PfSUB1

MRT12113

Yes/No

No

No

No

[181]

P. falciparum

DPAP3 (aminopeptidase)

JCP405, JCP410, SAK1

Yes/Yes

No

No

No

[182]

Leishmania spp.

GP63 (leishmanolysin) (metalloprotease)

Peptidomimetic metalloprotease inhibitors

No/No

No

No

No

[189]

Leishmania spp.

Cysteine proteases

K11002 (Peptidomimetic vinyl sulfone cysteine protease inhibitor)

No/No

Yes

No

No

[197]

L. donovani

OPB (oligopeptidase B, serine protease)

Various serine protease inhibitors (i.e. antipain, PEFABLOC, and others)

No/No

No

No

No

[202]

T. cruzi

Serine protease prolyl oligopeptidase (POP Tc80 and oligopeptidase B)

Epoxy-alpha-lap (napthoquinone compound) and PMSF

No/No

No

No

No

[206]

3092 Current Medicinal Chemistry, 2013, Vol. 20, No. 25

Rascón and McKerrow

(Table 1) contd….

Organism

Target Protease

Inhibitor

HTLa/LOb

Animal Model

Preclinical

Phase 1

Reference

Validation T. cruzi

Cruzain

K11777

Yes/Yes

Yes

Yes

Yes

[188, 288]

T. brucei

Rhodesain (brucipain,

Aryl Nitrile inhibitors, K11777,

Yes/Yes

No

No

No

[215, 227, 288, 289, 290, 291, 292]

trypanopain) (cathep-

K11017, K11002, Azadipeptide

sin L)

Nitrile Inhibitors, Thiosemicar-

Yes/Yes

No

No

No

[289, 290, 215, 291, 293]

bazone inhibitors T. brucei

TbCatB (cathepsin B)

K11777, Azadipeptide Nitrile inhibitors, CA-074, Thiosemicarbazone inhibitors, Purine Nitrile Inhibitors

E. histolytica

EhCP1

WRR483

Yes/Yes

Yes

No

No

[241, 288]

E. histolytica

EhCP4

WRR605

Yes/Yes

Yes

No

No

[244]

G. muris

Cysteine proteases

E-64, fluoromethyl ketones

No/No

No

No

No

[250]

GlCP2

Leupeptin and E-64

No/No

No

No

No

[255]

Bestatin

No/No

No

No

No

[258]

AEBSF

No/No

No

No

No

[257]

G. lamblia

(cysteine protease) G. lamblia

DPP IV (dipeptidyl peptidase)

G. lamblia

gSPC (subtilisin-like proprotein convertase)

a

HTL = Hit to Lead Chemistry

b

LO = Lead Optimization Chemistry.

mouse infectivity [229]. However, activity of other serine oligopeptidases was up-regulated and is believed to take over function in the absence of TbOPB [229]. 3.3. Virulence and Pathogenesis: Entamoeba histolytica Entamoeba histolytica is an intestinal protozoan parasite and the causative agent of human amoebiasis (amebic colitis). Infection by E. histolytica can lead to colonic inflammation and ulcers. “Metastatic” invasive amoebae may form abscesses in the liver, lung, and brain [230, 231]. Amoebiasis causes ~70,000 deaths per year and is the fourth leading cause of death by a protozoan infection [232, 233]. Human infection begins with the ingestion of cysts (the dormant stage of E. histolytica) found in contaminated food or water. After ingestion, the cysts undergo excystation in the terminal ileum (the most distal part of the small intestine) releasing eight E. histolytica trophozoites per cyst [231, 233-235]. The trophozoites migrate to the large intestine where they attach to the intestinal mucus layer and epithelial cells, colonizing the colon [231, 233-235]. Once infection is established, the trophozoites may spread to other parts of the body, through the bloodstream, leading to abscesses in the liver, brain, and lungs [230, 231, 233]. To complete the lifecycle, trophozoites undergo encystation (cyst formation) and are excreted in the stool. Cysteine proteases have been implicated in the virulence of E. histolytica [230-235]. A key aspect of this parasitic disease is tissue destruction [234]. E. histolytica cysteine proteases have been shown to degrade components of the extracellular matrix of the colon, including fibronectin, laminin, and collagen. Cysteine proteases of E.

histolytica also degrade components of the complement system (anaphylatoxins C3a and C5a), as well as IgA and IgG [230, 231, 233, 235]. With the completion of the E. histolytica genome, 50 cysteine protease genes have been identified [236]. However, only three of these genes (ehcp1, ehcp2, and ehcp5) were expressed at high levels in cultured trophozoites. They account for 90% of the total cysteine protease gene transcripts [233, 237, 238]. The expression of these transcripts correlates with cysteine protease activity [237, 238]. Cultured E. histolytica trophozoites express and secrete abundant cysteine proteases both intracellularly and extracellularly [235]. E. histolytica cysteine proteases have been observed extracellularly in amoebic liver abscesses [230]. E. histolytica cysteine proteases therefore represent exploitable targets for new chemotherapy [235]. E. histolytica trophozoites pre-incubated with E-64 showed reduced liver abscess formation when introduced into severe combined immunodeficient (SCID) mice [239]. E-64 did not affect the growth or viability of the cultured trophozoites because E-64 does not permeate cell membranes [240]. The availability of other cysteine protease specific inhibitors (peptidyl chloromethyl ketones, diazomethyl ketones, and fluoromethyl ketones) [235] prompted inhibitor studies with both E. histolytica trophozoites and recombinant E. histolytica cysteine proteases. Cultured E. histolytica trophozoites were pre-incubated with K11777 and then injected into a human colon xenograft mouse model. Amoeba invasion was quantified 24 hours post-injection [241]. Compared to a no inhibitor control, there was >80% reduction in detectable E. histolytica trophozoites in human colon tissue

Inhibition of Parasitic Proteases

[241]. EhCP1, one of the three highly expressed and released cysteine proteases, was successfully expressed and purified. By mapping the specificity at the P2 position (Arg), a new vinyl sulfone inhibitor based on the K11777 scaffold was synthesized, WRR483 [241] (Fig. 2). Amoebic invasion of the colon tissue was >95% blocked by WRR483. This inhibitor may also target the other two highly expressed and released cysteine proteases, EhCP2 and EhCP5, which have similar catalytic activities [242, 243]. The EhCP4 gene was shown to be up-regulated during trophozoite invasion and colonization of murine cecal tissue [244]. Unlike EhCP1, EhCP2, and EhCP5, EhCP4 has a substrate preference for small hydrophobic groups at the P2 position [244]. Therefore, a new inhibitor, WRR605 (with a valine at the P2 position) was synthesized [244] (Fig. 2). Incubation of trophozoites with WRR605 followed by introduction into mouse colon models resulted in both depletion of trophozoites and a decrease in inflammation of the infected cecum [244]. However, pre-incubation of WRR605 with cultured trophozoites did not block invasion. The role of proteases in the encystation and excystation process in E. histolytica is not known. Although a reliable, reproducible in vitro encystation model for E. histolytica is not available, researchers have found an alternative model in the reptilian parasite E. invadens [245]. Trophozoites from E. invadens encyst in hypoosomolar conditions; in the presence of cysteine protease inhibitors the efficiency of this process was significantly reduced [245, 246]. To date, only one other protease, the metallosurface protease 1 (EhMSP-1) has been studied [247]. This metalloprotease belongs to the M8 zinc metalloprotease family and is homologous to the Leishmania GP63 metalloprotease (see Kinetoplastids, Leishmania section). Gene silencing of EhMSP-1 in E. histolytica trophozoites reduced amebic adherence, cell motility, cell monolayer destruction, and phagocytosis [247]. In addition, gene sequence analysis and phylogenetic studies showed that the EhMSP-1 gene is not found in E. dispar, the non-pathogenic human parasite. 3.4. Excystation and Encystation: Giardia lamblia Giardia lamblia (syn. G. intestinalis, G. duodenalis) is a protozoan parasite that causes a severe diarrheal pathology known as giardiasis. Giardiasis is found worldwide, particularly affecting immune-compromised individuals, pregnant women, children, and people living in impoverished, crowded areas where sanitation is inadequate [248, 249]. Giardiasis is the most common cause of waterborne, foodborne, and restaurant associated outbreaks of diarrhea, diarrhea in childcare facilities, and travelers’ diarrhea [248, 250]. Giardia infection begins with the ingestion of cysts found in contaminated water or food, through direct fecal-oral contact or through direct contact with an infected person [248, 249]. After ingestion and exposure to the acidic environment in the stomach, trophozoites begin to excyst, colonize the upper small intestine, and establish infection [248]. The vegetative replicating trophozoites asexually divide by binary fission in the small intestine, and after exposure to biliary secretions, some trophozoites encyst in the jejunum [248]. The infective cysts are then secreted in the feces to be ingested by another host, completing the lifecycle. Cysts can survive diverse en-

Current Medicinal Chemistry, 2013, Vol. 20, No. 25 3093

vironmental conditions [248]. It takes only a few cysts to establish infection [249]. Protease activity from cultured Giardia trophozoites includes cysteine proteases [251-254], serine proteases [251254], aspartic proteases [251, 253], metalloproteases [252], and aminopeptidases [251, 253]. Protease activity in cultured trophozoites is dominated by cysteine proteases. In Giardia, cysteine proteases are localized in endosome-lysosome vacuoles where they function to degrade endocytosed proteins [248]. The best-characterized roles of cysteine proteases are in the encystation and excystation processes in Giardia. Ward et al. [250] inhibited the excystation process in cultured Giardia muris trophozoites with E-64 and three different fluoromethyl ketone-derivatized dipeptides. They demonstrated that cysteine protease activity was localized in cytoplasmic vacuoles, which upon excystation, release their contents into the space between the trophozoite and cyst wall for cyst wall protein degradation [250]. The cysteine protease inhibitors did not affect trophozoite growth, motility, or cyst viability. A specific cysteine protease, GlCP2, was implicated in the degradation of cyst wall proteins [250]. The identification of GlCP2 led to further studies on encystation. In 2008, Dubois et al. [255] showed that the GlCP2 gene transcript was also highly expressed in the vegetative and encysting stages of G. lamblia trophozoites, and co-localized to encystation-specific vesicles. These vesicles contain the precursor materials needed for cyst wall formation, including cyst wall proteins that need to be proteolytically processed before being incorporated into the cyst wall [256]. To confirm these findings, recombinant GlCP2 (a clan CA cysteine protease) was purified and assayed against recombinant cyst wall protein 2 (rCWP2). Recombinant GlCP2 processed the protein in vitro [255]. Y01, a Giardia cathepsin-C specific inhibitor, did not inhibit rCWP2 cleavage by rGlCP2. Activity attributed to other proteases has been observed in cultures of G. lamblia trophozoites [251-254] and independent genome analyses [257] have confirmed the presence of serine, threonine, metallo-, aspartic, and aminopeptidases. However, only two other proteases have been partially characterized in Giardia. One is the membraneassociated dipeptidyl peptidase (DPP) IV [258] and the other is the subtilisin-like proprotein convertase (gSPC) [257], both serine proteases. Each may play a role in encystation (DPP IV and gSPC) and excystation (gSPC). Giardia DPP IV was identified as a potential target in cultured trophozoites with bestatin. The inhibitor blocked cyst formation and abolished the expression of cyst wall proteins without having an effect on trophozoite growth [258]. The protease of interest was isolated from encysting and non-encysting trophozoites using a bestatin-affinity matrix, sequenced, and shown to degrade a DPP IV fluorogenic substrate [258]. The exact function of this protease in trophozoite differentiation into cysts is still unknown, but the authors hypothesized that changes in the fluidity of the membrane, stimulated by environmental conditions, exposes the active site to proteolytically initiate an unknown transduction process leading to the expression of encystation-specific genes [258]. gSPC was identified by bioinformatic analysis and expressed as an epitope-tagged gSPC protein in G. lamblia cultures [257]. Endogenous gSPC activity was inhibited using AEBSF (a spe-

3094 Current Medicinal Chemistry, 2013, Vol. 20, No. 25

cific serine protease inhibitor), resulting in arrest of encystation and excystation. 4. NATURALLY DERIVED PARASITE PROTEASE INHIBITORS: CYSTATINS AND SERPINS Parasite-derived cystatins and serpins have been previously reviewed [5, 259-262]. However, they merit a brief discussion because these naturally derived inhibitors play important roles in parasite development, virulence, and pathogenesis. Cystatins are cysteine protease inhibitors of the papain-like family that function to regulate oogenesis, molting during larval stages, and migration through host tissues [259, 261]. The best-characterized parasitic cystatin is onchocystatin from the nematode Onchocerca volvulus. This inhibitor is expressed in the eggshell of microfilariae, the cuticle of L3, L4 larvae, and in both male and female adult worms [259]. Expression of the inhibitor in adult and female worms was shown to inhibit the mammalian lysosomal cysteine proteases (cathepsins L and S) involved in host immunity [5]. Cystatins have also been identified in H. contortus and are thought to play similar roles by inhibiting host cathepsin enzymes [259]. In B. malayi, cysteine protease inhibitors are expressed at different stages of the lifecycle, with expression in the mosquito vector, but not in the mammalian host [259]. A cysteine protease inhibitor from Ascaris lumbricoides may be involved in host immune suppression [263]. A novel cysteine protease inhibitor from the trematode C. sinensis has also been described. This inhibitor was shown to inhibit endogenous cathepsin F, human cathepsin B, human cathepsin L, and papain [264]. Immunolocalization studies showed that the C. sinensis cysteine protease inhibitor was localized to the intestinal epithelium where it plays a role in modulating cathepsin F activity and processing, and may also protect the parasite from host proteases [264]. Plasmodium also expresses cysteine protease inhibitors, falstatin in P. falciparum [265] and an inhibitor of cysteine proteases (PbICP) in P. berghei [266]. These inhibitors are suggested to play a role in sporozoite invasion and parasite survival by limiting proteolysis from host or parasite proteases [265, 266]. Falstatin and PbICP are known as chagasin-like inhibitors. When first discovered, chagasin from T. cruzi showed no sequence similarity to other cysteine protease inhibitors and so was proposed to represent a novel family [267]. All of these chagasin-like cysteine protease inhibitors have high affinity for papain-like enzymes and their respective endogenous cysteine proteases [267]. Both NMR and crystal structures of chagasin are published and validate this family of inhibitors as novel [268-270]. Serpins function as serine protease inhibitors, inhibiting host blood coagulation and activation of host complement to evade the host immune system [260]. Although mammalian serpins have been reported as inhibitors of cathepsins and caspases [271], no data exists as of yet for the target diversity of parasite serpins. Gene sequence analysis identified two distinct families of nematode serpin families: one is structurally homologous to the mammalian serpins, and the other belongs to a novel group of small serine protease inhibitors (smapins) [260, 262]. Smapins are a small group of proteins composed of less than 100 amino acids with 10 cysteine groups that form five disulfide linkages [5, 260]. Smapins are unique to parasitic nematodes and are found in

Rascón and McKerrow

A. suum, Anisakis simplex, O. volvulus, and A. caninum [5, 260]. Gene expression studies of two O. volvulus smapins found that both genes were expressed in all life stages of the parasite lifecycle, with highest expression observed during the molting larval stages and reproducing adults [260]. An A. simplex serpin isolated from adult worms was shown to inhibit bovine trypsin, human leukocyte elastase, and chymotrypsin, suggesting a protective role against host intestinal proteases [272]. Three other A. simplex elastase isoinhibitors were isolated and believed to function in reproduction [273], but the target serine proteases have not been identified [260]. Only two serpins from B. malayi have been characterized to date [260]. Recent studies identified a serpin from H. contortus, which is believed to target host proteases rather than endogenous proteases [274]. Serpins from trematodes have also been identified. In C. sinensis, a serpin was indentified in the infective metacercaria (possibly playing a role in excystation) [275] and another serpin was identified in the adult worms and eggs [276]. Serpins from P. westermani and from the cestode species Echinococcus have also been identified [260]. A schistosome serpin (S. mansoni protease inhibitor 56) was shown to inhibit neutrophil elastase, suggesting a protective role against host proteolytic attack [260, 277]. More recently, an S. mansoni larval secreted serpin was shown to regulate the activity of cercarial elastase on skin degradation [278]. Molehin et al. [260] reviews, in detail, parasitic helminth serpins and smapins, along with information regarding the structure, function, and stoichiometry of inhibition of these naturally derived protease inhibitors. 5. CONCLUSION Proteases play important roles in the development, virulence, and pathogenesis of parasitic organisms. Protease inhibitors have served as tools for “chemical knockout” to help identify the function of major proteases in parasite lifecycles. Examples include the identification of schistosome gut proteases involved in the degradation of host hemoglobin and other serum proteins, of proteases involved in development and excystment of newly excysted F. hepatica juveniles, and of Plasmodium proteases involved in hemoglobin degradation, cell invasion, and cell exit. RNA interference technology also has some success in validating the role of proteases in parasitic infections. Further biochemical and cell biological analysis coupled with structural studies of parasitic proteases, will provide a better foundation for the development of parasite-specific protease inhibitors. CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest. ACKNOWLEDGEMENTS We would like to thank Drs. Paul J. Brindley (George Washington University, Washington D.C., USA), Alex Loukas (James Cook University, Australia), John P. Dalton (McGill University, Quebec, Canada), Sara Lustigman (New York Blood Center, USA), Phil Rosenthal (UCSF, USA), Sharon L. Reed (UCSD, USA) and Conor Caffrey (CDIPD, UCSF, USA) for their helpful suggestions. We would also

Inhibition of Parasitic Proteases

Current Medicinal Chemistry, 2013, Vol. 20, No. 25 3095

like to thank Potter Wickware (CDIPD, UCSF, USA) for his editorial comments. A.A.R., Jr. is supported by the NIH Institutional Research and Academic Career Development Award (IRCADA) Post-Doctoral fellowship through the IRACDA Scholars in Science (ISIS) program at UCSF (National Institute of General Medical Sciences/National Institutes of Health Award Number K12GM081266). ABBREVIATIONS AEBSF

=

[13]

[14] [15]

[16]

4-(2-aminoethyl)-benzene-sulfonyl ride Hydrochloride

Fluo[17]

ATP

=

Adenosine-5’-triphosphate

CP

=

Cysteine Protease

E-64

=

trans-epoxy-succinyl-L-leucyl-amido-4guanidino butane

[18]

DTT

=

Dithiothreitol

[19]

EhCP (ehcp) =

Entamoeba histolytica Cysteine Protease

EhMSP-1

Entamoeba histolytica Metallosurface Protease 1

=

GlCP2

=

Giardia lamblia Cysteine Protease 2

gSPC

=

Giardial Subtilisin-Like Proprotein Convertase

Ig

=

Immunoglobulin

pfSUB

=

Plasmodium Protease

POP Tc80

=

Prolyl Oligopeptidase from Trypanosoma cruzi

PMSF

=

falciparum

Subtilisin-Like

[2] [3] [4] [5] [6] [7] [8]

[9] [10] [11]

[12]

[22]

[23]

[24]

Phenyl Methyl Sulfonyl Fluoride

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Received: July 21, 2012

Revised: October 01, 2012

Accepted: October 16, 2012

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