Puran Singh Sijwali

Expression and characterization of the Plasmodium falciparum haemoglobinase falcipain-3.

In the malaria parasite Plasmodium falciparum, erythrocytic trophozoites hydrolyse haemoglobin to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block parasite haemoglobin hydrolysis and development, indicating that cysteine proteases are required for these processes. Three papain-family cysteine protease sequences have been identified in the P. falciparum genome, but the specific roles of their gene products and other plasmodial proteases in haemoglobin hydrolysis are uncertain. Falcipain-2 was recently identified as a principal trophozoite cysteine protease and potential drug target. The present study characterizes the related P. falciparum cysteine protease falcipain-3. As is the case with falcipain-2, falcipain-3 is expressed by trophozoites and appears to be located within the food vacuole, the site of haemoglobin hydrolysis. Both proteases require a reducing environment and acidic pH for optimal activity, and both prefer peptide substrates with leucine at the P(2) position. The proteases differ, however, in that falcipain-3 undergoes efficient processing to an active form only at acidic pH, is more active and stable at acidic pH, and has much lower specific activity against typical papain-family peptide substrates, but has greater activity against native haemoglobin. Thus falcipain-3 is a second P. falciparum haemoglobinase that is particularly suited for the hydrolysis of native haemoglobin in the acidic food vacuole. The redundancy of cysteine proteases may offer optimized hydrolysis of both native haemoglobin and globin peptides. Consideration of both proteases will be necessary to evaluate cysteine protease inhibitors as antimalarial drugs.

Cysteine proteases of malaria parasites: Targets for chemotherapy

New drugs to treat malaria are urgently needed. Cysteine proteases of malaria parasites offer potential new chemotherapeutic targets. Cysteine protease inhibitors block parasite hemoglobin hydrolysis and development, indicating that cysteine proteases play a key role in hemoglobin degradation, a necessary function of erythrocytic trophozoites. These inhibitors also block the rupture of erythrocytes by mature parasites, suggesting an additional role for cysteine proteases in the hydrolysis of erythrocyte cytoskeletal proteins. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites is larger than was previously realized. Plasmodium falciparum, the most virulent human malaria parasite, expresses three papain-family cysteine proteases, known as falcipains. All three proteases are expressed by trophozoites and hydrolyze hemoglobin at acidic pH, suggesting roles in this process. Falcipain-2 also hydrolyzes ankyrin at neutral pH, suggesting additional activity against erythrocyte cytoskeletal targets. Multiple orthologs of the falcipains have been identified in other plasmodial species. Analysis of orthologs from animal model rodent parasites identified similar features, but some noteworthy biochemical differences between the cysteine proteases. These differences must be taken into account in interpreting in vivo experiments. A number of small molecule cysteine protease inhibitors blocked parasite hemoglobin hydrolysis and development, and inhibitory effects against parasites generally correlated with inhibition of falcipain-2. Some compounds also cured mice infected with otherwise lethal malaria infections. Current research priorities are to better characterize the biological roles and biochemical features of the falcipains. In addition, efforts to identify optimal falcipain inhibitors as antimalarials are underway.

Folding of the Plasmodium falciparum Cysteine Protease Falcipain-2 Is Mediated by a Chaperone-like Peptide and Not the Prodomain

Papain-family cysteine proteases of the malaria parasite Plasmodium falciparum, known as falcipains, are hemoglobinases and potential drug targets. Available data suggest that papain-family proteases require prodomains for correct folding into functional conformations. However, in prior studies of falcipain-2, anEscherichia coli-expressed construct containing only a small portion of the prodomain refolded efficiently, suggesting that this enzyme differs in this regard from other papain-family enzymes. To better characterize the determinants of folding for falcipain-2, we expressed multiple pro- and mature constructs of the enzyme in E. coli and assessed their abilities to refold. Mature falcipain-2 refolded into active protease with very similar properties to those of proteins resulting from the refolding of proenzyme constructs. Deletion of a 17-amino acid amino-terminal segment of the mature protease yielded a construct incapable of correct folding, but inclusion of this segment in trans allowed folding to active falcipain-2. The prodomain was a potent, competitive, and reversible inhibitor of mature falcipain-2 (K i 10−10 m). Our results identify a chaperone-like function of an amino-terminal segment of mature falcipain-2 and suggest that protease inhibition, but not the mediation of folding, is a principal function of the falcipain-2 prodomain.

Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax

Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.

Synthesis and insight into the structure-activity relationships of chalcones as antimalarial agents.

Licochalcone A (I), isolated from the roots of Chinese licorice, is the most promising antimalarial compound reported so far. In continuation of our drug discovery program, we isolated two similar chalcones, medicagenin (II) and munchiwarin (III), from Crotalaria medicagenia , which exhibited antimalarial activity against Plasmodium falciparum . A library of 88 chalcones were synthesized and evaluated for their in vitro antimalarial activity. Among these, 67, 68, 74, 77, and 78 exhibited good in vitro antimalarial activity against P. falciparum strains 3D7 and K1 with low cytotoxicity. These chalcones also showed reduction in parasitemia and increased survival time of Swiss mice infected with Plasmodium yoelii (strain N-67). Pharmacokinetic studies indicated that low oral bioavailability due to poor ADME properties. Molecular docking studies revealed the binding orientation of these inhibitors in active sites of falcipain-2 (FP-2) enzyme. Compounds 67, 68, and 78 showed modest inhibitory activity against the major hemoglobin degrading cysteine protease FP-2.

Falcipain Cysteine Proteases Require Bipartite Motifs for Trafficking to the Plasmodium falciparum Food Vacuole

The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 hydrolyze hemoglobin in an acidic food vacuole to provide amino acids for erythrocytic malaria parasites. Trafficking to the food vacuole has not been well characterized. To study trafficking of falcipains, which include large membrane-spanning prodomains, we utilized chimeras with portions of the proteases fused to green fluorescent protein. The prodomains of falcipain-2 and falcipain-3 were sufficient to target green fluorescent protein to the food vacuole. Using serial truncations, deletions, and point mutations, we showed that both a 20-amino acid stretch of the lumenal portion and a 10-amino acid stretch of the cytoplasmic portion of the falcipain-2 prodomain were required for efficient food vacuolar trafficking. Mutants with altered trafficking were arrested at the plasma membrane, implicating trafficking via this structure. Our results indicate that falcipains utilize a previously undescribed bipartite motif-dependent mechanism for targeting to a hydrolytic organelle, suggesting inhibition of this unique mechanism as a new means of antimalarial chemotherapy.

Critical role of amino acid 23 in mediating activity and specificity of vinckepain-2, a papain-family cysteine protease of rodent malaria parasites

Cysteine proteases of Plasmodium falciparum, known as falcipains, have been identified as haemoglobinases and potential drug targets. As anti-malarial drug discovery requires the analysis of non-primate malaria, genes encoding related cysteine proteases of the rodent malaria parasites P. vinckei (vinckepain-2) and P. berghei (berghepain-2) were characterized. These genes encoded fairly typical papain-family proteases, but they contained an unusual substitution of Gly23 with Ala (papain numbering system). Vinckepain-2 was expressed in Escherichia coli, solubilized, refolded and autoprocessed to an active enzyme. The protease shared important features with the falcipains, including an acidic pH optimum, preference for reducing conditions, optimal cleavage of peptide substrates with P2 Leu and ready hydrolysis of haemoglobin. However, key differences between the plasmodial proteases were identified. In particular, vinckepain-2 showed very different kinetics against many substrates and an unusual preference for peptide substrates with P1 Gly. Replacement of Ala23 with Gly remarkably altered vinckepain-2, including loss of the P1 Gly substrate preference, markedly increased catalytic activity ( k cat/ K m increased approx. 100-fold) and more rapid autohydrolysis. The present study identifies key animal-model parasite targets. It indicates that drug discovery studies must take into account important differences between plasmodial proteases and sheds light on the critical role of amino acid 23 in catalysis by papain-family proteases.

Antimalarial effects of human immunodeficiency virus type 1 protease inhibitors differ from those of the aspartic protease inhibitor pepstatin

ABSTRACT Human immunodeficiency virus type 1 protease inhibitors (HIVPIs) and pepstatin are aspartic protease inhibitors with antimalarial activity. In contrast to pepstatin, HIVPIs were not synergistic with a cysteine protease inhibitor or more active against parasites with the cysteine protease falcipain-2 knocked out than against wild-type parasites. As with pepstatin, HIVPIs were equally active against wild-type parasites and against parasites with the food vacuole plasmepsin aspartic proteases knocked out. The antimalarial mechanism of HIVPIs differs from that of pepstatin.

Blocking Plasmodium falciparum Development via Dual Inhibition of Hemoglobin Degradation and the Ubiquitin Proteasome System by MG132

Among key potential drug target proteolytic systems in the malaria parasite Plasmodium falciparum are falcipains, a family of hemoglobin-degrading cysteine proteases, and the ubiquitin proteasomal system (UPS), which has fundamental importance in cellular protein turnover. Inhibition of falcipains blocks parasite development, primarily due to inhibition of hemoglobin degradation that serves as a source of amino acids for parasite growth. Falcipains prefer P2 leucine in substrates and peptides, and their peptidyl inhibitors with leucine at the P2 position show potent antimalarial activity. The peptidyl inhibitor MG132 (Z-Leu-Leu-Leu-CHO) is a widely used proteasome inhibitor, which also has P2 leucine, and has also been shown to inhibit parasite development. However, the antimalarial targets of MG132 are unclear. We investigated whether MG132 blocks malaria parasite development by inhibiting hemoglobin degradation and/or by targeting the UPS. P. falciparum was cultured with inhibitors of the UPS (MG132, epoxomicin, and lactacystin) or falcipains (E64), and parasites were assessed for morphologies, extent of hemoglobin degradation, and accumulation of ubiquitinated proteins. MG132, like E64 and unlike epoxomicin or lactacystin, blocked parasite development, with enlargement of the food vacuole and accumulation of undegraded hemoglobin, indicating inhibition of hemoglobin degradation by MG132, most likely due to inhibition of hemoglobin-degrading falcipain cysteine proteases. Parasites cultured with epoxomicin or MG132 accumulated ubiquitinated proteins to a significantly greater extent than untreated or E64-treated parasites, indicating that MG132 inhibits the parasite UPS as well. Consistent with these findings, MG132 inhibited both cysteine protease and UPS activities present in soluble parasite extracts, and it strongly inhibited recombinant falcipains. MG132 was highly selective for inhibition of P. falciparum (IC50 0.0476 µM) compared to human peripheral blood mononuclear cells (IC50 10.8 µM). Thus, MG132 inhibits two distinct proteolytic systems in P. falciparum, and it may serve as a lead molecule for development of dual-target inhibitors of malaria parasites.

Functional Evaluation of Plasmodium Export Signals in Plasmodium berghei Suggests Multiple Modes of Protein Export

The erythrocytic stage development of malaria parasites occurs within the parasitophorous vacuole inside the infected-erythrocytes, and requires transport of several parasite-encoded proteins across the parasitophorous vacuole to several locations, including the cytosol and membrane of the infected cell. These proteins are called exported proteins; and a large number of such proteins have been predicted for Plasmodium falciparum based on the presence of an N-terminal motif known as the Plasmodium export element (PEXEL) or vacuolar transport signal (VTS), which has been shown to mediate export. The majority of exported proteins contain one or more transmembrane domains at the C-terminus and one of three types of N-terminus domain architectures. (1) The majority, including the knob-associated histidine rich protein (KAHRP), contain a signal/hydrophobic sequence preceding the PEXEL/VTS motif. (2) Other exported proteins, including the P. berghei variant antigen family bir and the P. falciparum skeleton binding protein-1, do not appear to contain a PEXEL/VTS motif. (3) The P. falciparum erythrocyte membrane protein-1 (PfEMP1) family lacks a signal/hydrophobic sequence before the motif. These different domain architectures suggest the presence of multiple export pathways in malaria parasites. To determine if export pathways are conserved in plasmodia and to develop an experimental system for studying these processes, we investigated export of GFP fused with N- and C-terminus putative export domains in the rodent malaria parasite P. berghei. Export was dependent on specific N- and C-terminal domains. Constructs with a KAHRP-like or bir N-terminus, but not the PfEMP1 N-terminus, exported GFP into the erythrocyte. The C-terminus of a P. falciparum variant antigen rifin prevented GFP export by the KAHRP-like N-terminus. In contrast, GFP chimeras containing KAHRP-like N-termini and the PfEMP1 C-terminus were exported to the surface of erythrocytes. Taken together, these results suggest that proteins with KAHRP-like architecture follow a common export pathway, but that PfEMP1s utilize an alternative pathway. Functional validation of common putative export domains of malaria parasites in P. berghei provides an alternative and simpler system to investigate export mechanisms.