Keywords

1 Introduction

Malaria is an ancient disease caused by protozoan parasite of the genus Plasmodium. Till date malaria remains a major health burden for impoverished regions of the world with limited sanitation and healthcare facilities. In humans, malaria is caused by five Plasmodium species, namely, P. falciparum, P. vivax, P. ovale, P. malariae, and P. cynomolgi. Of the five species, P. falciparum is the causative agent of severe malaria, responsible for most of the malaria-related deaths. Due to the combined global efforts to curb the menace of malaria, disease cases have come down by 37% globally and by 42% in Africa between 2000 and 2015. The more encouraging outcome of the efforts has been the drastic reduction in malaria-related deaths that have come down by 60% globally and 66% in the African regions in the same time span [1].

Despite these successes in malaria control, complete malaria eradication remains an ambitious goal for the global scientific community and healthcare providers. A completely protective malaria vaccine is still a dream for the scientists and the available arsenals of antimalarial drugs are limited. Till date major antimalarials are natural compounds or their derivatives and the knowledge about their mechanisms of action are limited. In addition, the ability of the parasite to develop resistance against antimalarials is common. The parasite quickly develops resistance against the extensively used antimalarials, especially when they are used as a monotherapy, although successes have been achieved to counter drug resistance using combination therapy [2,3,4,5,6,7,8,9,10,11]. Due to above-mentioned reasons, development of novel antimalarial drugs remains a high priority.

For development of new drugs, identification of crucial parasite-specific molecules is required, which could be used for specific targeting. One group of such molecules is parasite proteases. They constitute the major virulence factors in various parasitic diseases and are crucial for the pathogens for their survival and ability to cause the diseases [12]. Malarial proteases are crucial molecules for parasite development and virulence. Some of these proteases possess parasite-specific functions and structural features that could be specifically targeted by drugs. Potential of these molecules as antimalarial drug targets has been substantiated by the use of protease inhibitors as drugs against a number of diseases. Protease inhibitors are already in use as drugs against human immunodeficiency virus (HIV) [13], hepatitis C virus (HCV) [14], and in the treatment of hypertension [15] and coagulopathies [16].

These groups of molecules are widespread in organisms and carry out a variety of biological processes by regulating the fate, localization, and activity of the target proteins [17]. Through evolution they have adapted to a variety of physiological conditions found in complex organisms and employ different mechanisms for substrate hydrolysis. Depending on the mechanisms of action, proteases have been classified into different classes (serine, cysteine, metallo, aspartic, threonine, and glutamic proteases). Serine, threonine, and cysteine proteases rely upon the nucleophilic character of the side chains of serine, threonine, and cysteine, respectively, at the active site. These side chains directly attack the peptide bond to form a transient, covalent enzyme–substrate intermediate, which breaks to give rise to the product and the enzyme. Aspartic and metalloproteases employ water molecule as nucleophile and do not form a covalent intermediates [18]. A number of proteases of various classes are found in Plasmodium. This chapter describes the available information on P. falciparum proteases crucial for parasite development in the human host and the studies related to drug development efforts to target these molecules.

2 Proteases as Potential Antimalarial Drug Targets

Malaria parasite completes its life cycle inside the vertebrate and invertebrate host cells. The host cell provides a rich source of nutrients for the parasite and a safe niche where the parasite is protected from the host immune responses. Asexual erythrocytic life cycle is responsible for the clinical manifestations of malaria, which starts when the merozoites released from liver infect the erythrocytes. The intracellular parasite feeds on erythrocyte hemoglobin and develops into more metabolically active trophozoite, which then undergoes nuclear division to transform into multinucleated schizont. After maturation, schizonts rupture to release the merozoites, which start a new wave of infection by invading new erythrocytes.

Completion of parasite life cycle in vertebrate and invertebrate hosts is dependent on a number of proteolytic activities. Inhibitor studies have shown that cysteine, aspartic, metallo, and serine proteases play critical roles in parasite-specific processes during the erythrocytic cycle such as hemoglobin digestion, merozoite release from infected erythrocytes, and invasion of fresh erythrocytes by the released merozoites. In addition a number of proteases are involved in metabolic processes necessary for parasite survival (Fig. 1). These processes have been the focus of drug development efforts [19]. In addition to erythrocytic stages, liver stages of the parasite are also attractive drug targets due to their low numbers and distinct metabolism [20, 21]. Liver stage parasites also employ proteolytic activities for their maturation and subsequent dispersal in the blood stream [22, 23]. Aspartate, cysteine, and metalloproteases are involved in hemoglobin degradation, a pathway necessary for parasite growth in the blood stages, whereas serine and cysteine proteases are crucial for the parasite egress and invasion at blood and liver stages. Table 1 summarizes the role of parasite proteases in P. falciparum development and propagation. Data mining revealed the presence of 92 putative proteases in P. falciparum genome [24]. Further analysis of P. falciparum genome database revealed the presence of 124 predicted proteases [25]. The roles of proteases in various parasite-specific processes and their potential as drug targets are described below.

Fig. 1
figure 1

Role of proteases in asexual erythrocytic life cycle of malaria parasite

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Table 1 Role of proteases in development of P. falciparum parasitic stages
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3 Proteases Involved in Hemoglobin Digestion

P. falciparum has a limited capacity to synthesize amino acids [26]. In the blood stages, it relies on degradation of abundant host cell molecule hemoglobin for its utilization [27, 28]. During the trophozoite stage, parasite proteases degrade most of the hemoglobin [29] and supply the parasite with amino acids for protein synthesis and metabolism [30]. Plasmodium ingests hemoglobin by an invagination spanning from parasite plasma membrane to parasitophorous vascuolar membrane known as cytostome. The cytostome takes host cell cytoplasm and pinches off to form double-membrane transport vesicles loaded with hemoglobin, which fuse with food vacuoles to empty their contents there. Food vacuoles are acidic compartments with a pH between 5.0 and 5.4 [31, 32] and provide the site for hemoglobin degradation and heme detoxification [33].

Hydrolysis of hemoglobin is a semi-ordered process mediated by the action of a series of proteases. Aspartic [34,35,36] and metalloproteases [37] are involved in hemoglobin degradation in the food vacuole. Four proteases predominantly carry out hemoglobin degradation (aspartic proteases plasmepsin 1 and 2, cysteine protease falcipain 2, and metalloprotease falcilysin) [38]. Plasmepsin 1 and 2 possess the ability to cleave hemoglobin under non-denaturing conditions between Phe33 and Leu34 in the hinge region of the alpha globin chain, which probably allows the globin subunits to unwind that facilitates further proteolysis [33, 39]. After the initial cleavage by plasmepsins 1 and 2, falcipain 2 cleaves the globin fragments [38]. It has been demonstrated that plasmepsin 1 can degrade native hemoglobin efficiently under nonreducing conditions while plasmepsin 2 has a preference for denatured hemoglobin. Although falcipains cleave denatured globin but not native hemoglobin [39], under mild reducing conditions falcipain 2 cleaves both native and denatured globin [40]. Falcilysin further cleaves the globin fragments into smaller peptides [41]. The digested peptides are then transported to parasite cytosol, where cytosolic aminopeptidases hydrolyse these peptides to free amino acids [42].

Human hemoglobin lacks a single amino acid isoleucine. Liu et al. [43] demonstrated that P. falciparum could grow in a medium containing only a single amino acid isoleucine. This finding indicated that host hemoglobin is sufficient to provide the rest of the amino acid requirements of the parasite. Knockout parasite lines of the hemoglobin-degrading enzymes (falcipain-2, plasmepsins, alone or in combination) grown in this medium showed reduced growth. A potent inhibitor of plasmepsin pepstatin A did not significantly affect the growth of wild-type parasites but was able to kill the parasites lacking falcipain-2 at low concentrations. It was even more effective in killing the parasites triple knockout parasites lacking falcipain-2, plasmepsin 1, and 4. This study provided evidence that both plasmepsins and falcipain-2 are hemoglobinases with overlapping functions. While falcipain-2 was essential for parasite survival plasmepsins provided subsidiary roles and were dispensable for the parasite [43], different classes of hemoglobin-degrading proteases and their role in the parasite are summarized below.

3.1 Aspartic Proteases (Plasmepsins)

P. falciparum genome data predicted the presence of 10 plasmepsin genes. Expression, localization, and enzymatic data suggested that four of the plasmepsins (plasmepsin 1, plasmepsin 2, plasmepsin 4, and a histo-aspartic protease (HAP)) were found in the food vacuole and were capable to degrade hemoglobin [41, 44]. Gene knockout of P. falciparum food vacuole plasmepsins revealed that growth rates were reduced by 30–35% upon plasmepsin 1 and plasmepsin 4 disruption, whereas disruption of plasmepsin 2 did not cause any growth defect. Disruption of HAP caused a slight drop in growth rates that was not statistically significant [45]. In another similar study, targeted gene disruption by double crossover homologous recombination was carried out for plasmepsin 1, plasmepsin 2, HAP, and plasmepsin 4/plasmepsin 1. All the four knockout parasites were morphologically normal. In amino acid-limited medium, they exhibited slower growth rate as compared to the parental strain. Sensitivity of the knockout parasites to aspartic and cysteine protease inhibitors changed minimally, compared to wild-type 3D7 parasite, suggesting the functional redundancy of these proteases [46].

Among the two major hemoglobin-degrading plasmepsins, 1 and 2, plasmepsin 2 was found to be 5–10-fold more efficient on peptide substrates and also had 80-fold higher inhibition constant compared to plasmepsin 1 [47]. Because of their higher hemoglobin-degrading activities, a number of studies on drug screening have been focused on plasmepsins 1 and 2 [48,49,50,51,52,53,54]. Due to their redundant function, it is suggested that in order to achieve higher antiparasite activity, as many as of these enzymes have to been targeted by the inhibitors [55] and inhibitors targeting multiple plasmepsins could be used as antimalarials [56].

3.2 Cysteine Proteases (Falcipains)

P. falciparum genome encodes four clan CA cysteine proteases known as falcipains (falcipain 1, falcipain 2, falcipain 2′, and falcipain 3). These proteases have been biochemical and genetically characterized [57,58,59]. Genetic disruption of falcipain 2 caused block in hemoglobin degradation resulting in accumulation of undegraded hemoglobin in the food vacuole, similar to that caused by cysteine protease inhibitors [58]. Falcipain 3 was refractory to genetic deletion, although a tagged copy of the gene could readily be produced. Falcipain 1 and falcipain 2′ were dispensable for the parasite as their disruption did not cause any defect in parasite phenotype [59]. Based on these studies, falcipains 2 and 3 appear to be crucial hemoglobinases with redundant functions [57,58,59].

Activities of falcipain 2 and 3 have been assessed on peptide substrates in vitro. Falcipain 2 accounted for 90% of the cysteine protease activity on peptide substrates in trophozoite lysates [40], while falcipain 3 showed relatively lesser activity on peptide substrates [57]. Differential activities of these enzymes correlated with their timings of expression. Falcipain 2 was predominantly expressed in early trophozoites when parasite growth is maximum and hence maximum hemoglobin degradation is required while falcipain 3 showed maximum expressions in late trophozoites when the nutrient requirement is minimal and parasite prepares for division [58]. Falcipain 2′ did not exhibit any biochemical differences with falcipain 2. Gene knockout of falcipain 2′ did not cause any phenotypic defect in the parasite and the biological role of this enzyme remains unknown [60,61,62].

Due to the crucial role of falcipains 2 and 3, numerous studies have been focused on designing of their inhibitors [38, 63]. These inhibitors caused accumulation of undegraded hemoglobin in the food vacuole resulting in swollen morphology. They have also inhibited parasite development and have been effective in cure of experimental malaria [28, 40, 64,65,66,67].

3.3 Metalloproteases

Following degradation by aspartic and cysteine proteases, peptide fragments generated from hemoglobin are further hydrolyzed by metalloprotease falcilysin [37]. Specificity analysis using hydrolysis of a series of random peptide substrates revealed that the enzyme was highly active at acidic pH, consistent with its role in hemoglobin degradation. Surprisingly, the enzyme also showed robust activity at neutral pH and with slightly different substrate specificities [68]. Further studies revealed that in addition to food vacuole falcilysin was also translocated to the apicoplast. Based on in vitro studies, it was suggested that this enzyme might have a role in transit peptide hydrolysis [69].

3.4 Aminopeptidases

Nine aminopeptidases are annotated in P. falciparum genome. These enzymes catalyze the hydrolysis of amino acids from the N-terminus. Five aminopeptidases are involved in hydrolysis of peptides generated by hemoglobin digestion [70]. Two of these enzymes M1 alanyl aminopeptidase (PfM1AAP) and M17 leucine aminopeptidase (PfM17LAP) are essential for P. falciparum growth and development as inhibitors of these peptidases were found to be lethal to P. falciparum culture in vitro [71].

4 Proteases Involved in Parasite Egress from Erythrocytes

Erythrocyte rupture by malaria parasites is a temporally regulated process. Different models of erythrocyte rupture have been proposed based on live video microscopic and electron microscopic studies [72,73,74,75,76]. Although the exact mechanism of egress is obscure, the most widely experimentally supported model of erythrocyte rupture suggests that egress is a two-step process, in which the degradation of parasitophorous vacuole membrane takes place prior to RBC membrane rupture [74]. This model was supported by electron microscopy (EM)-based evidences demonstrating that highly mature schizonts occasionally lack a discrete parasitophorous vacuole membrane (PVM) [77, 78]. Similar results were obtained by immune electron microscopy of mature schizonts with antibody against parasitophorous vacuole (PV) resident protein S-antigen [74].

Mechanistically egress consists of destabilization of erythrocyte cytoskeleton, PVM, and the erythrocyte membrane. Studies based on serine, cysteine, and aspartic protease inhibitors suggest that egress is dependent on proteolytic activities [79,80,81,82,83]. Analysis on unruptured schizonts in the protease inhibitor-treated parasites revealed the defects in processing of merozoite surface protein (MSP1) [82] and PV protein p126 that is now known as SERA5 [83].

In vitro studies have suggested that hemoglobin-degrading proteases plasmepsin 2 and falcipain 2 could also have role in egress. Plasmepsin 2 digested erythrocyte cytoskeleton proteins spectrin, protein 4.1, and actin at neutral pH in vitro. Detection of this protease in the cytoplasm of schizont-infected erythrocytes was also suggestive of its role in destabilization of host cell membrane skeleton [84]. Falcipain 2 digested cytoskeleton proteins ankyrin and protein 4.1 at neutral pH in vitro [85, 86]. A peptide based on ankyrin cleavage site was found to be inhibitory to falcipain 2 activity and merozoite egress when added to schizont-stage parasites. Localization of falcipain 2 in PV and subcellular structures extending into the erythrocyte cytosol also provide strength to its possible involvement in erythrocyte cytoskeleton destabilization [87]. Surprisingly, plasmepsin 2 and falcipain 2 were dispensable for the parasite as shown by gene disruption studies and these knockout lines showed no defect in egress [43, 45, 46, 57, 58]. Although the role of these proteases in egress has not been conclusively ascertained, they seem to be redundant in function [88].

The role of a protease in egress was demonstrated in an exquisite study by Aly and Matuschewski [89] using reverse genetics approach. They showed that disruption of a P. berghei gene termed as “egress cysteine protease 1” (ecp1) inhibited the release of sporozoites from mosquito oocyst [89]. ecp1 belongs a family of cysteine proteases known as serine repeat antigens (SERAs). There are nine members of SERA proteases found in P. falciparum genome. Eight of them are arranged on chromosome 2 in a head-to-tail tandem orientation, whereas the ninth member SERA9 is located on chromosome 9 [90]. P. berghei ecp1 was found to be ortholog of P.falciparum SERA8.

The central regions of SERA proteins show homology to papain family cysteine proteases. However, in some SERAs active site cysteine is substituted with a serine residue [91,92,93]. In P. falciparum, SERAs 1–5 and SERA9 possess active site serine and SERAs 6–8 possess active site cysteine [93]. Proteolytic activity of P. falciparum SERA5, the most highly expressed member, has been demonstrated in vitro on peptide substrates [94]. To study the essentiality of P. falciparum SERAs, gene knockout studies were conducted. All but SERA5 and SERA6 could not be disrupted suggesting the essential function of these proteins for the parasite [90, 95].

SERA5, also known as SERA, is the most highly expressed member and a number of studies have suggested its involvement in merozoite egress from erythrocytes. It is synthesized as a 126 kDa precursor (p126) [96] and secreted into the lumen of the parasitophorous vacuole. Around the time of schizont rupture, it undergoes proteolytic processing into multiple fragments. These processing seem to be essential for its role in egress. The full-length p126 is processed into a 47-kDa N-terminal and a 73-kDa fragment (P73) C-terminal fragments. P73 is processed into 56-kDa and 18-kDa fragments and the 56-kDa fragment is further processed to form a 50-kDa central domain and a 6-kDa fragment [97]. The complex of N-terminal 47-kDa and C-terminal 18-kDa fragments is found associated with the merozoite surface and the central 50-kDa fragment is detected in the culture medium after schizont rupture [98, 99]. The cleavage that produces p47 and p73 from p126 and cleavage of p73 to p56 and p18 is carried out by PfSUB1. p56 is cleaved by an unknown protease to produce p50 and a 6-kDa fragment [88].

Evidence for involvement of SERA5 and other SERA family members came from inhibitor studies. A specific inhibitor of PfSUB1 was found to block the rupture of schizonts. Further biochemical analysis revealed that PfSUB1 mediated rupture of schizonts through the processing of SERA5 and other SERA family members including SERA4 and SERA6 [100]. In a reciprocal approach to identify the mediators of merozoite egress, using a chemical screen of 1200 covalent serine and cysteine protease inhibitors demonstrated that PfSUB1 and a cysteine protease DPAP3 as the regulators of this process. This study demonstrated that chemical blocking of PfSUB1 and DPAP3 resulted in blockade of SERA5 processing, correlating with the blockade in schizont rupture. Inhibition of DPAP3 resulted in reduced level of mature PfSUB1 suggesting that both the proteases regulate the efficient release of merozoites from the infected red blood cells by SERA5 processing [101]. Attempts to target the enzyme domain of SERA5 using peptides resulted in the block of merozoite egress [102]. In another study, incubation of P. falciparum culture with the SERA5 prodomain and a peptide derived from it resulted in block in schizont rupture [103]. These studies also suggested the role of SERA5 in merozoite egress and the possibility of targeting the protein for parasite growth inhibition. In addition to SERA5, SERA6 is also supposed to have role in merozoite egress. It is cleaved in the PV by PfSUB1 just before egress. Mutation of active site cysteine or the blockade in processing by PfSUB1 was lethal for the parasite. Processing of P. berghei ortholog of SERA6 (PbSERA3) by PfSUB1 converted it into an active cysteine protease. Taken together, these findings suggest the role of SERA6 in merozoite egress [104].

Based on these studies, PfSUB1 appears to be involved in maturation of multiple SERA proteases and hence is an important regulator of merozoite egress. Its role in egress at blood stages and expression in liver stages raised the possibility of similar role in liver stages. A conditional mutagenesis-based invalidation of P. berghei SUB1 in liver stages revealed that SUB1-deficient parasites failed to egress from hepatocytes [23]. Based on its role in both blood and liver stages, PfSUB1 qualifies an attractive multistage target against malaria. Molecular modeling, substrate specificity, and characterization of PfSUB1 and its homologs from Plasmodium vivax, Plasmodium knowlesi, and P. berghei revealed many unusual features in SUB1 substrate-binding cleft, although SUB1 from all the species cleaved the conserved parasite substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibited SUB1 from all the species [105]. In another study, high throughput screening of a proprietary library of compounds against PfSUB1 identified hydrazine 2 as an inhibitor of PfSUB1 [106]. Due to its potential as an attractive multistage druggable target, identification of potent inhibitors against PfSUB1 could pave the way for the development antimalarials acting at schizont rupture.

5 Proteases Involved in Erythrocyte Invasion by Merozoites

Malaria parasites efficiently invade their host cells to survive inside the host and protect themselves from the host immune responses. Apicomplexan parasites utilize their specialized invasion apparatuses to identify, penetrate, and establish themselves within the host cells. Host cell entry is a crucial checkpoint where parasite development can be blocked, and hence an important target for antimalarial drug development [107]. Evidences for the involvement of parasite proteases in invasion came from inhibitor studies. Protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and chymostatin blocked merozoite invasion in P. falciparum and a number of other Plasmodium species [108, 109]. The chymostatin-sensitive effect was reversed by pretreatment of RBCs with chymotrypsin, suggesting that parasite-induced proteolytic modification of RBCs was involved in chymostatin-sensitive step [108, 110, 111]. Inhibitor studies on simian malaria parasite P. knowlesi revealed that treatment of isolated, invasive merozoites with N-tosyl phenylalanyl chloromethyl ketone, or N-tosyl lysyl chloromethyl ketone prevented primary attachment of parasites to host cells, whereas chymostatin blocked invasion at a later stage suggesting the involvement of multiple proteolytic activities in the pathway [81]. Consistent with this, a P. falciparum serine protease activity that mediated an essential processing and shedding of a major merozoite surface protein (merozoite surface protein 1; MSP1) at invasion was identified that was highly sensitive to inhibition by PMSF but not by chymostatin [112].

Invasion is a rapid process, taking about 60 s for the egressed merozoites to invade new erythrocytes. These merozoites distinguish between erythrocytes and other cells by initial low-affinity reversible contacts between merozoite and erythrocyte surface molecules [113]. The initial attachment occurs anywhere between the merozoite surface and erythrocyte; hence, the apical end of the merozoite orientates toward the erythrocyte surface in the subsequent steps. This orientation results in the formation of a tight junction between the merozoite apical end and the erythrocyte surface. This junction moves from anterior to the posterior end of the merozoite with the help of actin-myosin motor as it invades the erythrocytes [114]. The interaction between the merozoite and the erythrocyte molecules during the movement of the tight junction involves activity of parasite proteases [116,117,118]. Studies in Toxoplasma gondii and P. falciparum have revealed that micronemal secretion is induced upon initial contact. These secretory proteins are exposed at the parasite surface and mediate parasite orientation and formation of tight junction. Soon after microneme secretion, another apical organelles known as rhoptries discharge their contents, which are also supposed to have role in tight junction formation, although their precise role is not known [119, 120].

The initial interaction between the merozoite and the host cell is mediated by a protein complex on the merozoite surface known as merozoite surface protein (MSP1) complex MSP1 complex is composed of MSP1 and its associated proteins MSP6 and MSP7 [121,122,123]. This complex is processed in the parasitophorous vacuole by the serine protease PfSUB1. This processing termed as “primary processing” makes the merozoite competent for the initial interaction [124]. After initial interaction, micronemal secretions mediate the high affinity interaction resulting in the formation of tight junction. This tight junction moves from anterior to posterior pole of the parasite in actomyosin motor-dependent manner. During this movement, shedding of MSP1 complexes and another micronemal protein apical membrane antigen (PfAMA1) takes place that is essential for invasion. Cleavage of both these proteins takes place at the juxtamembrane site. MSP1 is cleaved at the site distal to the epidermal growth factor-like domain at its C-terminal, shedding the MSP1 complex except the C-terminal region known as MSP119 that enters into the host cell following invasion [125]. Similarly, cleavage of AMA1 29 residues away from the transmembrane domain releases the bulk of the ectodomain. In this way the juxtamembrane “stub” along with the transmembrane and cytoplasmic domains enters into the host cell after invasion [126]. In vitro studies on the shedding of these parasite proteins strongly suggest that micronemal subtilisin-like serine protease PfSUB2 is the most likely candidate for these processing events, and hence termed as “merozoite surface sheddase” (MESH) [116].

By reverse genetics approaches both PfSUB1 and PfSUB2 appear to be essential for the parasite [100, 105]. Based on the numerous studies on their role in merozoite egress and invasion, these two proteases appear to be promising drug targets against malaria. Recently, efforts have been made to identify PfSUB1 and PfSUB2 inhibitors. Characterization of PfSUB1 and its orthologs in P. knowlesi and P. berghei revealed that cleavage sites in the parasite substrates of these proteases in conserved despite the differences in the enzyme substrate-binding sites. Consistent with these features, two peptidyl alpha-ketoamide inhibitors of PfSUB1 also inhibited its orthologs [127]. A low toxic natural pentacyclic triterpene maslinic acid (MA) was inhibitory to P. falciparum transition from ring to schizont stage. MA also inhibited the processing of MSP1 complex and hence supposed to target PfSUB1 [128]. In an in vitro study, PfSUB2 prodomain selectively inhibited the shedding of MSP1 and AMA1, it could be used an attractive design for PfSUB2 inhibitors. Structural study of PfSUB2 prodomain using nuclear magnetic resonance (NMR) identified a likely catalytic domain-binding interface region in it, which could act as a design for peptidomimetic inhibitor against the enzyme [129].

In addition to subtilisin-like proteases, intramembrane rhomboid proteases also have role during parasite invasion by cleaving the adhesins inside the parasite membrane. Rhomboid proteases carry out intramembrane proteolysis, and hence have their catalytic triad embedded within the membrane bilayer that is surrounded by a hydrophilic cavity [130]. P. falciparum rhomboid proteases are largely uncharacterized till date with two members PfROM1 and PfPROM4 partially characterized. PfROM1 andPfROM4 carried out the intramembrane cleavage of the adhesins AMA1 [126, 131] and erythrocyte-binding antigen 175 (EBA-175), respectively [132]. In addition, they were able to cleave a variety of adhesins involved in host–parasite interaction within the transmembrane domains [131]. Although this group of proteases seems to be crucial for the invasion process, they need to be extensively characterized and evaluated for druggability.

Overall, proteolytic enzymes involved in invasion are considered to be attractive targets of antimalarial drug development. Invasion is a very rapid process, taking place within a minute of merozoite release; the access of parasite molecules involved in invasion is debatable. Dowse et al. [132] suggested that proteases involved in invasion have the potential to serve as drug targets as their biosynthesis and maturation start much earlier in the life cycle, making them available for targeting by drugs [132].

6 Other Cellular Proteases

In addition to the above-described proteases, a number of parasitic proteases are involved in regular metabolic processes and cell cycle regulation in the parasite, with unique parasite-specific features. The parasite possesses a single mitochondrion and a relict plant plastid-like organelle called apicoplast [133,134,135,136,137]. The mitochondrion possesses some unique biochemical features. It is defective in tricarboxylic acid cycle and does not appear to oxidize glucose to produce ATP in the blood stages of the parasite [138], although it seems to be involved in pyrimidine biosynthesis [139]. An intriguing P. falciparum protease is ClpQ threonine protease (heat shock loci V or HslV) is localized in the mitochondrion [140, 141]. Its ATPase partner is ClpY (heat shock loci U or HslU) [142]. These two proteins interact to form ClpQY machinery, a prokaryotic predecessor of the eukaryotic proteosomal machinery [142]. ClpQ appears to be critical for parasite survival as the disruption of the interaction between ClpQ and ClpY using peptide inhibitors resulted in the death of asexual blood stages in a phenotype resembling apoptosis [143].

The apicoplast is crucial for the biosynthesis of parasite heme, isopentenyl diphosphate, fatty acids, and isoprenoid precursors [144, 145]. The apicoplast possesses its own genome but 95% of its proteins are nuclear-encoded and transported to the organelle. Targeting of proteins to apicoplast through the secretory pathway is mediated by bipartite signal and transit peptide sequences. Two parasite proteases are involved in the targeting process; the stromal processing protease (SPP) cleaves the transit peptide to release the mature protein [146] and the metalloprotease falcilysin carries out the degradation of the transit peptide [69]. Another serine protease ClpP dependent on ATPase for functioning has been localized in the apicoplast and its inhibitor was found to significantly inhibit parasite growth in vitro [147].

Parasite signal peptide peptidases mediate protein trafficking to their destinations within the parasite and host cytosol [24, 147–149]. The P. falciparum signal peptide peptidase (PfSPP) is essential for parasite growth inside the erythrocytes and could be targeted by small molecule inhibitors [150]. Gamma secretase and signal peptide peptidase inhibitor LY411,575 was evaluated for the targeting Plasmodium berghei liver stages in human hepatoma cell lines and in mouse primary hepatocytes. LY411,575 was found to be inhibitory for normal liver stage development at nanomolar concentration (IC50 value of 80 nM). This inhibitor also decreased the liver stage parasite load in vivo and also conferred 55% resistance to cerebral malaria in mice [151].

The malaria parasite contains a minimal endoplasmic reticulum-associated degradation (ERAD) network relative to higher eukaryotes. P. falciparum is highly sensitive to the inhibition of protease component of this system (PfSSP). The compounds inhibiting PfSSP also showed low nanomolar activity against liver stage malaria parasites [152]. Many nuclear-encoded proteins are targeted to subcellular organelles like apicoplast and mitochondria using the signal and transit peptides. Upon reaching the target, the transit peptides are cleaved to release the proteins. Metalloprotease falcilysin is known to cleave the transit peptide in malaria parasite [68, 69]. Parasite exports a number of proteins to modify the host erythrocytes. These proteins contain a pentameric (RxLxE/Q/D) motif required for export into the erythrocytes known as PEXEL (Plasmodium EXport ELement) motif, which is responsible for trafficking of these proteins into the host cytosol [153, 154]. An ER-resident aspartic protease plasmepsin V is responsible for cleavage of PEXEL and facilitating trafficking of the proteins [155].

7 Conclusion

Despite the availability of drugs and significant successes in reducing malaria-related illnesses and deaths globally, malaria still poses a serious threat to human health. Due to the ability of the parasite to quickly acquire resistance against the drugs, development of novel drugs against parasite-specific molecules remains a priority. The parasite genome encodes proteases of many different classes, many of which carry out processes crucial for parasite survival and propagation. These molecules have been extensively studied and many of these have been shown promises as potential drug targets because they posses unique parasite-specific features that could be specifically targeted. The success of protease inhibitors as drugs has already been shown against other infectious agents like HIV and HCV, invoking interest in the use of malarial protease inhibitors as drugs. Overall, malarial proteases represent an intriguing group of molecules that could be utilized for specific targeting by the novel drugs.