Malaria is widespread in tropical and subtropical regions, including parts of the Americas, Asia and Africa. An estimated three billion people were at the risk of malaria and half to one million deaths were reported in 2010
. Most deaths by malaria are caused by Plasmodium falciparum, one of the five species of human infectious malaria parasites, and malaria is related to the distribution of Anopheles mosquitoes
. Unfortunately, the increasing resistance of malaria parasites to available drugs has being reported
[3, 4]. Therefore, there is a need to develop new anti-malarial drugs.
Plasmodium falciparum invades erythrocytes and consumes the available haemoglobin as a means to obtain nutrients during growth and maturation
. Many Plasmodium proteases appear to play key roles during the life cycle of malaria, including: 1) invasion of an erythrocyte, 2) degradation of haemoglobin, and 3) rupture of erythrocytes. The degradation of haemoglobin occurs in the acidic food vacuole (FV) formed by the parasite in an erythrocyte, and up to 80% of haemoglobin is consumed by malarial parasites
[2, 6]. In P. falciparum, three different classes of proteases are mainly responsible for the haemoglobin degradation; they include aspartic proteases (plasmepsin I, II, IV and HAP), cysteine proteases (falcipain-1, -2 and −3) and the metalloprotease (falcilysin)
[6–9]. Several exopeptidases such as dipeptidyl aminopeptidase 1 (DPAP1) and three metallo-aminopeptidases (A-M1, APP and LAP) have also essential roles in haemoglobin degradation
[10–12]. Plasmepsin is known to be synthesized in an inactive precursor form (membrane-bound proplasmepsin), and to be processed to mature form by mature plasmepsin and falcipain, a cysteine protease
[13, 14]. Thus, an aspartic protease inhibitor, Pepstatin A has been known to block the processing of plasmepsin
[5, 6, 14, 15]. Since Plasmodium plasmepsin and falcipain are involved in haemoglobin degradation, which is necessary for parasite proliferation in the host, they have been targeted for development of anti-malarial drugs for decades
[5, 16–19]. However, plasmepsin activation does not seem to be completely blocked by inhibitors of aspartic proteases and/or cysteine proteases
[5, 20]. Recently, ALLN, a calpain inhibitor has been proposed to have the inhibitory effect of plasmepsin and falcipain
[14, 15]. Although its antimalarial activity is likely due primarily to the inhibition of falcipain, it still opens the possibility that calpain could be the one of the mediators for haemoglobin degradation and, thereby, a potential anti-malarial drug target.
Calpain is a cytoplasmic Ca2+-dependent, non-lysosomal cysteine protease that is ubiquitously expressed in mammals and many other organisms
. The P. falciparum genome encodes a single calpain homologue, although no biochemical data are available and it is not clear whether the calpain is expressed or catalytically active in any parasitic stage
. The P. falciparum calpain (Pf-calpain) gene differs significantly from those found in vertebrates to date
. A putative calpain (MAL13P1.310) in P. falciparum has high sequence similarity to Caenorhabditis elegans calpain-7
[22–24]. They belong to a monophyletic group of calpain-7, which might have contributed to an alternative Ca2+-independent calpain activity
. Pf-calpain consists of a central catalytic domain II (subdomain IIa and IIb) and a C-terminal catalytic domain III. This domain composition is a distinct type that is not common to any other types of calpain classes
. Pf-calpain was believed to be an essential mediator of merozoite invasion, based on the observation that a calpain inhibitor blocked invasion
. As the parasite progresses from trophozoite to schizont stage, there is a 30-fold increase in the level of calpain transcription
Based on reports, Pf-calpain seems to play a role in haemoglobin degradation along with plasmepsin and falcipain
[14, 15, 26]. However, no direct evidence was proposed that Pf-calpain participates in haemoglobin degradation and ALLN indeed acts against this enzyme. Thus, in this study, the active form of Pf-calpain was investigated in the first place to check its enzymatic activity and inhibition by ALLN. This active Pf-calpain was further utilized to establish the high-throughput screening system for Pf-calpain inhibitors. Results suggest that Pf-calpain is active only with catalytic subdomain IIa, resulting in a monomeric form of enzyme. In addition, the enzymatic activity of Pf-calapin was efficiently inhibited by ALLN treatment. The monomeric structure of Pf-calpain is considerably distinct from mammalian typical calpains and, thereby, Pf-calpain could serve as a target to develop parasite specific anti-malarial drugs. In addition, the monomeric structure might accelerate drug development by simplifying the synthesis steps of Pf-calpain selective inhibitors.