Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part III: Protozoa (2): Plasmodium spp.

Genomic analysis of P. falciparum revealed more than thirty cysteine proteases (CPs). However, the most studied CPs are four falcipains (FPs), three dipeptidyl peptidases, a calpain and a metacaspase. Beside the main function of hemoglobin degradation, CPs are not only essential for protein trafficking, but they are also involved in egress cascade, i.e. rupture of infected erythrocytes as well as de novo RBCs invasion. In addition, studies showed their essential role in exo-erythrocytic hepatic stages, as well as oocyst production and gamete egress in mosquitoes. Accordingly, CPs inhibitors (CPIs) are of great interest in development of novel anti-malarial drugs as well as a new strategy to eliminate malaria transmission. Several compounds were investigated as CPIs including herbal extracts, known proteases with reported inhibitory potency against papain-like family, chemical compounds and synthesized derivatives as well as commercially available drugs approved for human use for other diseases. However, no commercial drug-targeting FPs has been developed yet. On the other hand, endogenous parasites cystatins (CYSs) regulate CPs and prevent inappropriate effects of host enzymes. The present review will discuss the role of essential plasmodial CPs and the importance of search for or development of potent specific selective CPI as a novel anti-marital drug. Hopefully the rapid development of highly efficient technology promises advances of expression systems using genetic tools for metabolic regulation of protein expression. This is in addition to recent technology for advanced screening directed with molecular modeling using three-dimensional construction of the target CP. Abbreviations: ADMET: Computational tool to evaluate drug absorption, distribution, metabolism, excretion and toxicity; CALP: Calpain; CP: Cysteine proteinase; CPI: Cysteine proteinase inhibitor; CSP: Circumsporozoite protein; CYS: Cystatin; DPAP: Dipeptidyl aminopeptidase; E-64: A broad spectrum CPI; FP: Falcipain; HTS: High throughput screening; MCA: Metacaspase; MSP: Merozoite surface protein; PV: Parasitophorus vacuole; SAR: Structure activity relationship; SERA: Serine-repeat antigen; VP: Vivapain; VS: Virtual screening. CPs, CYSs, CPIs and Plasmodium spp. Abaza 73 [I] Cysteine proteinases (CPs) In three review articles published by Rosenthal[1-3], the important roles of malarial proteases in the erythrocytic life cycle stages were designated. These stages account for malarial clinical manifestations, passing from merozoites invasion to mature schizonts, rupture of infected RBCs and release of numerous invasive merozoites. The reviewer discussed all types of proteases, including CPs, required for hemoglobin degradation in the trophozoite stag and for synthesis in subsequent stages, as well as their roles in rupture and subsequent reinvasion of new RBCs. He also claimed that knockout gene encoding falcipain 2 (FP-2) led to a transient block in hemoglobin breakdown with significant increase in parasite sensitivity to CPIs. In contrast, disruption of the gene encoding FP-1 showed its essential role for oocysts production in mosquitoes. Other than FPs, P. falciparum genome revealed possession of three dipeptidyl aminopeptidases (DPAPs), previously recognized as calpain (CALP) homologs, and three serine-repeat antigens (SERAs) that have a cysteine motif. As it was only localized in the food vacuole, the reviewer discussed the role of DPAP1 in hemoglobin breakdown, in addition to the roles of DPAP-3 and SERA-5 in egress cascade. Accordingly, he suggested FP-2, DPAPs 1 and 3, and SERA-5 as potential anti-malarial drug targets[3]. In an attempt to characterize putative proteases in P. falciparum, a group of American investigators[4] predicted 92 proteinases using comparative genomic analysis. Their prediction was confirmed by further phylogenetic analysis. Among them, 88 proteases were identified with their assigned transcribed proteins using microarray analysis, and reverse transcript PCR. The transcript proteinases were classified and the highest (36%) were found to belong to CPs. Beside the identified and characterized CPs at that time, the investigators identified only two new potentially essential CPs; CALP and metacaspase (MCA). The first is a calcium-activated CP and was suggested as an essential catalytic enzyme in merozoite invasion. Its usefulness as anti-malarial drug target was suggested for two reasons. One, was the discovery of a typical endogenous CALP substrate (protein kinase C, PKC) in P. falciparum genome that is crucial for signal transduction pathways affecting biology and host-parasite interactions. Two, it is not similar to host CALPs, therefore use of inhibitors would have minimal effect on the host. Although previous studies neither reported apoptosis in P. falciparum, nor presence of MCA in protozoa, the investigators recommended further studies to investigate the role of plasmodial MCA as potential anti-malarial drug target[4]. In 2011, a fourth review article was published and the reviewer tabulated the stages of gene expression of CPs in P. falciparum and P. vivax using two methods; immunoblotting and proteomic screening[5]. The trophozoite is the only stage that showed gene expression of all FPs and vivapains (VPs) which are the FP homologs in P. vivax. Gene expression of FP-1 and VP-4 was detected in ring and schizont stages. Added to the previous two CPs, FP-3 was also detected in schizont stage. While FP-1 gene expression was detected only in sporozoites, VP-4 was only detected by immunoblotting in gametocytes. The reviewer suggested that the transient accumulation of undegraded hemoglobin in the food vacuole in response to knockout gene encoding FP-2 was compensated with FP-3 gene expression in late trophozoite (12 h later after FP-2 expression). For P. vivax, the reviewer attributed the fewer studies working on P. vivax CPs to two reasons; unavailability of continuous in vitro P. vivax cultures and limited animal models (only primates). The reviewer observed that FPs and VPs have a specific unique conserved role of their motif and time of expression to facilitate their main function. In other words, they might have developed gradually to efficiently degrade host hemoglobin. Accordingly, he recommended further studies to design potent inhibitors for FPs and VPs assisted by three-dimensional structure guided technology[5]. Beside the above mentioned functions, Plasmodium CPs proved to catalyze circumsporozoite protein (CSP), the major surface protein of Plasmodium sporozoites. It was found that CSP facilitates sporozoite adhesion to the host hepatocytes and subsequent invasion, and it should be cleaved prior to adhesion. A group of American scientists succeeded to identify its cleavage site by plasmodial CPs[6]. Using pulse-chase metabolic labeling experiments in absence and presence of different protease inhibitors, they found that more than 80% of the labeled CSP was cleaved after 2 h in absence of inhibitors. In contrast, E-64, a well-known broad spectrum CPI, inhibited CSP cleavage and subsequently inhibited both in vitro and in vivo sporozoites ability to adhere and invade their target cells. Using inhibitors of serine, metalloor aspartyl-proteases were not effective to inhibit CSP functions[6]. A] Falcipains (FPs) 1. FP-1: Rosenthal and Nelson[7] identified the first CP in the trophozoite of P. falciparum that has an essential role as potent hemoglobinase. The American investigators named the 28-kDa protein “trophozoite CP”, termed latter as falcipain-1 (FP-1). They succeeded to synthesize its encoding gene and primed it with P. falciparum genomic DNA using PCR. Although FP-1 is expressed by trophozoites, the investigators did not observe its essentiality for development of erythrocytic stages. This was confirmed by two American studies[8,9]. In the first study, the investigators succeeded to identify gene expression of all P. falciparum FPs in malarial stages of gametocytogenesis. Results revealed upregulation of FP-1 transcripts in gametocytes. Knockout gene encoding FP-1 neither affected the morphological features of gametocytes or gametes, nor parasite sensitivity to CPIs. However, it significantly reduced oocyst production when fed to a mosquito. Accordingly, the investigators concluded the potential use of FP-1 as new strategy in malaria transmission[8]. PARASITOLOGISTS UNITED JOURNAL 74 Meanwhile, CPIs did not inhibit erythrocyte invasion by merozoites in both wild type and knockout parasites[9]. It was found that FP-1 shares only ~40% of sequence identity to the other FPs. In addition, it was detected in the transcriptome and proteomes of Plasmodium asexual and sexual erythrocytic stages[10] as well as in sporozoites[11]. 2. FP-2: In 1995, the second trophozoite ‘P. falciparum CP’ (FP-2) was recognized in hemoglobin degradation in an acidic environment of trophozoite food vacuole[12]. It is well known that hemoglobin degradation into heme and globin is an essential process in Plasmodium life cycle, as heme is processed into malarial pigment, and globin is hydrolyzed to free amino acids essential for parasitic stages synthesis. To characterize the catalytic role of P. falciparum FP-2 in the initial step for globulin breakdown, Salas et al.[12] succeeded to express its functional proteolytic activity, i.e. as a recombinant form, in globulin hydrolysis of denatured and native hemoglobin. Results revealed that FP-2 is responsible for multiple cleavages resulting in small peptides shown in SDS–PAGE gel. Moreover, quantitative assays showed that FP-2 was capable of degrading hemoglobin at the rate of 1-3 pg/parasite/h, i.e. 25-75% of RBC hemoglobin/parasite/h. Another American group of investigators demonstrated that, similar to all CPs, FP-2 required activation prior to its proteolytic activity. They also observed high transcriptomics of the gene encoding FP-2 in the early intra-erythrocytic stages[13].