Cysteine proteases of malaria parasites

A number of cysteine proteases of malaria parasites have been described, and many more putative cysteine proteases are suggested by analysis of the Plasmodium falciparum genome sequence. Studies with protease inhibitors have suggested roles for cysteine proteases in hemoglobin hydrolysis, erythrocyte rupture, and erythrocyte invasion by erythrocytic malaria parasites. The best characterised Plasmodium cysteine proteases are the falcipains, a family of papain-family (clan CA) enzymes. Falcipain-2 and falcipain-3 are hemoglobinases that appear to hydrolyse host erythrocyte hemoglobin in the parasite food vacuole. This function was recently confirmed for falcipain-2, with the demonstration that disruption of the falcipain-2 gene led to a transient block in hemoglobin hydrolysis. A role for falcipain-1 in erythrocyte invasion was recently suggested, but disruption of the falcipain-1 gene did not alter parasite development. Other papain-family proteases predicted by the genome sequence include dipeptidyl peptidases, a calpain homolog, and serine-repeat antigens. The serine-repeat antigens have cysteine protease motifs, but in some the active site Cys is replaced by a Ser. One of these proteins, SERA-5, was recently shown to have serine protease activity. As SERA-5 and some other serine-repeat antigens localise to the parasitophorous vacuole in mature parasites, they may play a role in erythrocyte rupture. The P. falciparum genome sequence also predicts more distantly related (clan CD and CE) cysteine proteases, but biochemical characterisation of these proteins has not been done. New drugs for malaria are greatly needed, and cysteine proteases may provide useful new drug targets. Cysteine protease inhibitors have demonstrated potent antimalarial effects, and the optimisation and testing of falcipain inhibitor antimalarials is underway.     

Blocking effect of a biotinylated protease inhibitor on the egress of Plasmodium falciparum merozoites from infected red blood cells

The malaria parasite Plasmodium falciparum invades human red blood cells. Before infecting new erythrocytes, the merozoites have to exit their host cell to get into the blood plasma. Knowledge about the mechanism of egress is scarce, but it is thought that proteases are basically involved in this step. We have introduced a biotinylated dibenzyl aziridine-2,3-dicarboxylate (bADA) as an irreversible cysteine protease inhibitor to study the mechanism of merozoite release and to identify the proteases involved. The compound acts on parasite proteins in the digestive vacuole and in the host cell cytosol, as judged by fluorescence microscopy. The inhibitor blocks rupture of the host cell membrane, leading to clustered merozoite structures, as evidenced by immunoelectron microscopy. Interestingly, bADA did not prevent rupture of the parasitophorous vacuole membrane (PVM) that surrounds the parasite during the period of intraerythrocytic maturation. The compound appears to be a valuable template for the development of inhibitors specific for individual plasmodial proteases, which would be useful tools to dissect the molecular mechanisms underlying the process of merozoite release and consequently to develop potent antimalarial drugs.     

Antimalarial activity of thiosemicarbazones and purine derived nitriles

Malaria is a devastating illness caused by multiple species of the Plasmodium genus. The parasite’s falcipain proteases have been extensively studied as potential drug targets. Here we report the testing of two established cysteine protease inhibitor scaffolds against both chloroquine sensitive and chloroquine resistant parasites. A subset of purine derived nitriles killed the parasite with moderate potency, and these inhibitors do not seem to exert their antiproliferative effects as cysteine protease inhibitors. Compound potency was determined to be similar against both parasite strains, indicating a low probability of cross resistance with chloroquine. These compounds represent a novel antimalarial scaffold, and a potential starting point for the development new inhibitors.     

Double-stranded RNA-mediated gene silencing of cysteine proteases (falcipain-1 and -2) of Plasmodium falciparum

Malaria remains a public health problem of enormous magnitude, affecting over 500 million people every year. Lack of success in the past in the development of new drug/vaccines has mainly been attributed to poor understanding of the functions of different parasite proteins. Recently, RNA interference (RNAi) has emerged as a simple and incisive technique to study gene functions in a variety of organisms. In this study, we report the results of RNAi by double-stranded RNA of cysteine protease genes (falcipain-1 and -2) in the malaria parasite, Plasmodium falciparum. Using RNAi directed towards falcipain genes, we demonstrate that blocking the expression of these genes results in severe morphological abnormalities in parasites, inhibition of parasite growth in vitro and substantial accumulation of haemoglobin in the parasite. The inhibitory effects produced by falcipain double-stranded (ds)RNAs are reminiscent of the effects observed upon administering E-64, a cysteine protease inhibitor. The parasites treated with falcipain's dsRNAs also show marked reduction in the levels of corresponding endogenous falcipain mRNAs. We also demonstrate that dsRNAs of falcipains are broken into short interference RNAs approximately 25 nucleotides in size, a characteristic of RNAi, which in turn activates sequence-specific nuclease activity in the malaria parasites. These results thus provide more evidence for the existence of RNAi in P. falciparum and also suggest possibilities for using RNAi as an effective tool to determine the functions of the genes identified from the P. falciparum genome sequencing project.     

Falcipain cysteine proteases of malaria parasites: An update

BackgroundThe malaria parasite Plasmodium falciparum expresses four related papain-family cysteine proteases known as falcipains. These proteases play critical roles in the parasite life cycle, and as such are potential targets for new modes of antimalarial chemotherapy, as discussed in this review.Scope of reviewThis review summarizes available knowledge describing falcipain cysteine proteases of malaria parasites.Major conclusionsBased on available data the falcipains can be broken into two sub-families, the falcipain-1 and the falcipain-2/3 sub-families. Falcipain-1 has been difficult to study; it appears to play its most important roles in nonerythrocytic parasites, but not the erythrocytic stage responsible for human disease. Falcipain-2 and falcipain-3 have similar biochemical features, and are expressed sequentially during the erythrocytic cycle. Inhibition of either of these enzymes blocks hemoglobin hydrolysis and completion of the parasite developmental cycle. Knockout of falcipain-2 blocks hemoglobin hydrolysis, but parasites recover, presumably due to subsequent expression of falcipain-3. Knockout of falcipain-3 has not been possible, suggesting that the protease is essential for erythrocytic parasites. Determination of structures of falcipains and extensive chemistry efforts have facilitated identification of numerous small molecule falcipain inhibitors as potential new antimalarial agents. Other malaria parasites express close homologs of falcipain-1 and falcipain-2/3 proteases, suggesting that agents that target the falcipains will also be active against other human malaria parasites.General Significance. Falcipain-2 and falcipain-3 play vital roles during the erythrocytic stage of infection with P. falciparum and thus are promising targets for new agents to treat malaria.     

A role of falcipain-2, principal cysteine proteases of Plasmodium falciparum in merozoite egression

The process of merozoite release in Plasmodium falciparum involves rupture of the parasitophorous vacuole membrane and erythrocyte plasma membrane. Through the use of protease inhibitors that halt the merozoite release, a number of parasite proteases, especially serine, aspartic, and cysteine proteases, have been implicated in the schizont rupture. To understand the precise role of cysteine proteases in the merozoite release, in the present study, we treated P. falciparum cultures with siRNAs corresponding to falcipain-1, falcipain-2, and falcipain-3, the three papain-family proteases of the parasite. Treatment of malaria parasites with either of the falcipain siRNAs considerably reduced parasite growth. Morphological examination of the siRNA treated parasite cultures revealed that most of the parasites in falcipain-2 siRNA treated cultures were arrested at schizont stage. Analysis of a transgenic P. falciparum line expressing chimeric-GFP upon treatment with falcipain-2 siRNA revealed block in the rupture of erythrocyte membrane at the time of merozoite egression. These results suggest that falcipain-2 is an important parasitic protease that participates in hemoglobin degradation and in the merozoite release.     

Irreversible effect of cysteine protease inhibitors on the release of malaria parasites from infected erythrocytes

By studying the inactivation of malaria parasite culture by cysteine protease inhibition using confocal microscopy of living cells and electron microscopy of high-pressure frozen and freeze-substituted cells, we report the precise step in the release of malaria parasites from erythrocytes that is likely regulated by cysteine proteases: the opening of the erythrocyte membrane, liberating parasites for the next round of infection. Inhibition of cysteine proteases within the last few minutes of cycle does not affect rupture of the parasitophorus vacuole but irreversibly blocks the subsequent rupture of the host cell membrane, locking in resident parasites, which die within a few hours of captivity. This irreversible inactivation of mature parasites inside host cells makes plasmodial cysteine proteases attractive targets for antimalarials, as parasite-specific cysteine protease inhibitors may significantly augment multi-target drug cocktails.     

The impact of whole genome sequence data on drug discovery--a malaria case study.

BACKGROUND: Identification and validation of a drug discovery target is a prominent step in drug development. In the post-genomic era it is possible to reevaluate the association of a gene with a specific biological function to see if a homologous gene can subsume this role. This concept has special relevance to drug discovery in human infectious diseases, like malaria. A trophozoite cysteine protease (falcipain-1) from the papain family, thought to be responsible for the degradation of erythrocyte hemoglobin, has been considered a promising target for drug discovery efforts owing to the antimalarial activity of peptide based covalent cysteine protease inhibitors. This led to the development of non-peptidic non-covalent inhibitors of falcipain-1 and their characterization as antimalarials. It is now clear from sequencing efforts that the malaria genome contains more than one cysteine protease and that falcipain-1 is not the most important contributor to hemoglobin degradation. Rather, falcipain-2 and falcipain-3 appear to account for the majority of cysteine hemoglobinase activity in the plasmodium trophozoite. MATERIALS AND METHODS: We have modeled the falcipain-2 cysteine protease from one of the major human malaria species, Plasmodium falciparum and compared it to our original work on falcipain-1. As with falcipain-1, computa-tional screening of the falcipain-2 active site was conducted using DOCK. Using structural superpositions within the protease family and evolutionary analysis of substrate specificity sites, we focused on the commonalities and the protein specific features to direct our drug discovery effort. RESULTS: Since 1993, the size of the Available Chemicals Directory had increased from 55313 to 195419 unique chemical structures. For falcipain-2, eight inhibitors were identified with IC50's against the enzyme between 1 and 7 microM. Application of three of these inhibitors to infected erythrocytes cured malaria in culture, but parasite death did not correlate with food vacuole abnormalities associated with the activity of mechanistic inhibitors of cysteine proteases like the epoxide E64. CONCLUSIONS: Using plasmodial falcipain proteases, we show how a protein family perspective can influence target discovery and inhibitor design. We suspect that parallel drug discovery programs where a family of targets is considered, rather than serial programs built on a single therapeutic focus, will become the dominant industrial paradigm. Economies of scale in assay development and in compound synthesis are expected owing to the functional and structural features of individual family members. One of the remaining challenges in post-genomic drug discovery is that inhibitors of one target are likely to show some activity against other family members. This lack of specificity may lead to difficulties in functional assignments and target validation as well as a complex side effect profile.     

Antimalarial effects in mice of orally administered peptidyl cysteine protease inhibitors.

The Plasmodium falciparum cysteine protease falcipain is required for the degradation of hemoglobin by erythrocytic malaria parasites. In prior studies, peptidyl inhibitors of falcipain blocked hemoglobin degradation and development by cultured parasites and one of these compounds, when administered parenterally, cured Plasmodium vinckei-infected mice. We now report an evaluation of orally administered peptidyl inhibitors of falcipain in a mouse malaria model. In studies with a fluoromethyl ketone, orally administered morpholine urea-phenylalanine-homophenylalanine-fluoromethyl ketone delayed the progression of murine malaria. In studies of a new series of vinyl sulfones, a set of related compounds demonstrated marked inhibition of falcipain and of parasite biological activities in vitro. One of these compounds, N-methyl piperazine urea-leucine-homophenylalanine-2-naphthalene vinyl sulfone, cured about 40% of mice when administered orally twice-a-day for four days. Our results suggest that peptidyl inhibitors of falcipain have promise as antimalarial chemotherapeutic agents.     

Stage-specific antimalarial activity of cysteine protease inhibitors

Cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are promising targets for antimalarial chemotherapy. We evaluated cultured parasites for the stage-specific expression of cysteine proteases and sensitivity to cysteine protease inhibitors. Protease activity and inhibitor sensitivity varied markedly over time. Cysteine protease activity was greatest in early trophozoites, while sensitivity to cysteine protease inhibitors was greatest in mature trophozoites. Our results indicate the importance of considering the stage-specific effects of antimalarials and are consistent with the conclusion that the principal antimalarial activity of cysteine protease inhibitors is due to a block in hemoglobin hydrolysis.     

Falstatin, a cysteine protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.     

Expression,Characterization, and Cellular Localization of Knowpains,Papain-Like Cysteine Proteases of the Plasmodium knowlesi Malaria Parasite

Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4). Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5), suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3) to moderate (KP4) preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease inhibitor libraries against knowpains for developing broadly effective compounds active against multiple human malaria parasites.     

Mining a cathepsin inhibitor library for new antiparasitic drug leads

The targeting of parasite cysteine proteases with small molecules is emerging as a possible approach to treat tropical parasitic diseases such as sleeping sickness, Chagas' disease, and malaria. The homology of parasite cysteine proteases to the human cathepsins suggests that inhibitors originally developed for the latter may be a source of promising lead compounds for the former. We describe here the screening of a unique ～ 2,100-member cathepsin inhibitor library against five parasite cysteine proteases thought to be relevant in tropical parasitic diseases. Compounds active against parasite enzymes were subsequently screened against cultured Plasmodium falciparum, Trypanosoma brucei brucei and/or Trypanosoma cruzi parasites and evaluated for cytotoxicity to mammalian cells. The end products of this effort include the identification of sub-micromolar cell-active leads as well as the elucidation of structure-activity trends that can guide further optimization efforts.     

A cathepsin B-like protease is required for host protein degradation in Trypanosoma brucei

Identification and analysis of Clan CA (papain) cysteine proteases in primitive protozoa and metazoa have suggested that this enzyme family is more diverse and biologically important than originally thought. The protozoan parasite Trypanosoma brucei is the etiological agent of African sleeping sickness. The cysteine protease activity of this organism is a validated drug target as first recognized by the killing of the parasite with the diazomethane inhibitor Z-Phe-Ala-CHN(2) (where Z is benzyloxycarbonyl). Whereas the presumed target of this inhibitor was rhodesain (also brucipain, trypanopain), the major cathepsin L-like cysteine protease of T. brucei, genomic analysis has now identified tbcatB, a cathepsin B-like cysteine protease as a possible inhibitor target. The mRNA of tbcatB is more abundantly expressed in the bloodstream versus the procyclic form of the parasite. Induction of RNA interference against rhodesain did not result in an abnormal phenotype in cultured T. brucei. However, induction of RNA interference against tbcatB led to enlargement of the endosome, accumulation of fluorescein isothiocyanate-transferrin, defective cytokinesis after completion of mitosis, and ultimately the death of cultured parasites. Therefore, tbcatB, but not rhodesain, is essential for T. brucei survival in culture and is the most likely target of the diazomethane protease inhibitor Z-Phe-Ala-CHN(2) in T. brucei.     

Exoerythrocytic Plasmodium Parasites Secrete a Cysteine Protease Inhibitor Involved in Sporozoite Invasion and Capable of Blocking Cell Death of Host Hepatocytes

Plasmodium parasites must control cysteine protease activity that is critical for hepatocyte invasion by sporozoites, liver stage development, host cell survival and merozoite liberation. Here we show that exoerythrocytic P. berghei parasites express a potent cysteine protease inhibitor (PbICP, P. berghei inhibitor of cysteine proteases). We provide evidence that it has an important function in sporozoite invasion and is capable of blocking hepatocyte cell death. Pre-incubation with specific anti-PbICP antiserum significantly decreased the ability of sporozoites to infect hepatocytes and expression of PbICP in mammalian cells protects them against peroxide- and camptothecin-induced cell death. PbICP is secreted by sporozoites prior to and after hepatocyte invasion, localizes to the parasitophorous vacuole as well as to the parasite cytoplasm in the schizont stage and is released into the host cell cytoplasm at the end of the liver stage. Like its homolog falstatin/PfICP in P. falciparum, PbICP consists of a classical N-terminal signal peptide, a long N-terminal extension region and a chagasin-like C-terminal domain. In exoerythrocytic parasites, PbICP is posttranslationally processed, leading to liberation of the C-terminal chagasin-like domain. Biochemical analysis has revealed that both full-length PbICP and the truncated C-terminal domain are very potent inhibitors of cathepsin L-like host and parasite cysteine proteases. The results presented in this study suggest that the inhibitor plays an important role in sporozoite invasion of host cells and in parasite survival during liver stage development by inhibiting host cell proteases involved in programmed cell death.     

Plasmodium food vacuole plasmepsins are activated by falcipains

Intraerythrocytic malaria parasites use host hemoglobin as a major nutrient source. Aspartic proteases (plasmepsins) and cysteine proteases (falcipains) function in the early steps of the hemoglobin degradation pathway. There is extensive functional redundancy within and between these protease families. Plasmepsins are synthesized as integral membrane proenzymes that are activated by cleavage from the membrane. This cleavage is mediated by a maturase activity whose identity has been elusive. We have used a combination of cell biology, chemical biology, and enzymology approaches to analyze this processing event. These studies reveal that plasmepsin processing occurs primarily via the falcipains; however, if falcipain activity is blocked, autoprocessing can take place, serving as an alternate activation system. These results establish a further level of redundancy between the protease families involved in Plasmodium hemoglobin degradation.     

Homology modeling of falcipain-2: validation, de novo ligand design and synthesis of novel inhibitors

Increasing resistance of malaria parasites, in particular Plasmodium falciparum, demands a serious search for novel targets. Cysteine protease in P. falciparum, encoded by a previously unidentified gene falcipain 2, provides one such target to design chemotherapeutic agents for treatment of malaria. In fact, a few cysteine protease inhibitors have been shown to inhibit growth of cultured malarial parasites. In absence of a crystal structure for this enzyme, homology modeling proved to be a reasonable alternative to study binding requirements of the enzyme. A homology model for falcipain 2 was developed and validated by docking of known vinyl sulfone inhibitors. Further, based on the observations of these studies, novel isoquinoline inhibitors were designed and synthesized, which exhibited in vitro enzyme inhibition at micromolar concentrations.     

Antimalarial activity of azadipeptide nitriles

Azadipeptide nitriles—novel cysteine protease inhibitors—display structure-dependent antimalarial activity against both chloroquine-sensitive and chloroquine-resistant lines of cultured Plasmodium falciparum malaria parasites. Inhibition of parasite’s hemoglobin-degrading cysteine proteases was also investigated, revealing the azadipeptide nitriles as potent inhibitors of falcipain-2 and -3. A correlation between the cysteine protease-inhibiting activity and the antimalarial potential of the compounds was observed. These first generation azadipeptide nitriles represent a promising new class of compounds for antimalarial drug development.