Studies on Plasmepsins I and II from the Malarial Parasite Plasmodium falciparum and their Exploitation as Drug Targets

Moon, Richard P.; Bur, Daniel; Loetscher, Hansruedi; D’Arcy, Allan; Tyas, Lorraine; Oefner, Christian; Grueninger-Leitch, Fiona; Mona, Daniel; Rupp, Katherina; Dorn, Arnulf; Matile, Hugues; Certa, Ulrich; Berry, Colin; Kay, John; Ridley, Robert G.

doi:10.1007/978-1-4615-5373-1_56

Cited by 19 publications

(11 citation statements)

References 19 publications

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“…Hemoglobin-degrading enzymes of hematophagous parasites are being targeted for the rational development of novel antiparasitic compounds (26,27). Given that Hb is the natural substrate of these enzymes, comparison of their cleavage sites in the molecule with the cleavage site profile of homologous mammalian-host enzymes, such as human cathepsin D, is likely to provide specific leads that could be exploited in inhibitor design.…”

Section: Discussionmentioning

confidence: 99%

Hemoglobin-degrading, Aspartic Proteases of Blood-feeding Parasites

Brinkworth¹,

Prociv²,

Loukas³

et al. 2001

Journal of Biological Chemistry

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Blood-feeding parasites, including schistosomes, hookworms, and malaria parasites, employ aspartic proteases to make initial or early cleavages in ingested host hemoglobin. To better understand the substrate affinity of these aspartic proteases, sequences were aligned with and/or three-dimensional, molecular models were constructed of the cathepsin D-like aspartic proteases of schistosomes and hookworms and of plasmepsins of Plasmodium falciparum and Plasmodium vivax, using the structure of human cathepsin D bound to the inhibitor pepstatin as the template. The catalytic subsites S5 through S4 were determined for the modeled parasite proteases. Subsequently, the crystal structure of mouse renin complexed with the nonapeptidyl inhibitor t-butyl-CO-His-Pro-Phe-His-Leu [CHOHCH 2 ]Leu-Tyr-Tyr-Ser-NH 2 (CH-66) was used to build homology models of the hemoglobin-degrading peptidases docked with a series of octapeptide substrates. The modeled octapeptides included representative sites in hemoglobin known to be cleaved by both Schistosoma japonicum cathepsin D and human cathepsin D, as well as sites cleaved by one but not the other of these enzymes. The peptidase-octapeptide substrate models revealed that differences in cleavage sites were generally attributable to the influence of a single amino acid change among the P5 to P4 residues that would either enhance or diminish the enzymatic affinity. The difference in cleavage sites appeared to be more profound than might be expected from sequence differences in the enzymes and hemoglobins. The findings support the notion that selective inhibitors of the hemoglobin-degrading peptidases of blood-feeding parasites at large could be developed as novel anti-parasitic agents.Blood flukes, hookworms, and the malaria parasites are among the most important pathogens of humans in terms of both numbers of people infected and the consequent morbidity and mortality (1). Although phylogenetically unrelated, these parasites all share the same food source; they are obligate blood feeders, or hematophages. Hb from ingested or parasitized erythrocytes is their major source of exogenous amino acids for growth, development, and reproduction; the Hb, a ϳ64-kDa tetrameric polypeptide, is comprehensively catabolized by parasite enzymes to free amino acids or small peptides. Intriguingly, all these parasites appear to employ cathepsin D-like aspartic proteases to make initial or early cleavages in the Hb substrate (2-4).The vertebrate endopeptidase, cathepsin D (EC 3.4.23.5), is a member of the aspartic protease category of hydrolases, which also includes renin, pepsin, chymosin, cathepsin E, HIV 1 protease, and several other enzymes (5, 6). Cathepsin D is expressed in a diverse range of mammalian cells and tissues and is located predominantly in lysosomes (6). The molecule comprises two rather similar lobes, each incorporating a homologous Asp-Thr-Gly catalytic site motif, with the substrate binding groove located between these lobes. In aspartic proteases generally, the nucleophile that attacks...

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Section: Discussionmentioning

confidence: 99%

Hemoglobin-degrading, Aspartic Proteases of Blood-feeding Parasites

Brinkworth¹,

Prociv²,

Loukas³

et al. 2001

Journal of Biological Chemistry

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“…While residing in erythrocytes, the parasites rely on human hemoglobin as a food source, digesting it with a series of proteases. The aspartic proteases, called plasmepsins, are critical for hemoglobin degradation and are thus logical targets for antimalarial drug development (14,19,25). At least four plasmepsins have been identified and cloned from P. falciparum (26; R. Banerjee and D. E. Goldberg, Mol.…”

mentioning

confidence: 99%

New Class of Small Nonpeptidyl Compounds Blocks Plasmodium falciparum Development In Vitro by Inhibiting Plasmepsins

Jiang

Prigge

Wei

et al. 2001

Antimicrob Agents Chemother

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Malarial parasites rely on aspartic proteases called plasmepsins to digest hemoglobin during the intraerythrocytic stage. Plasmepsins from Plasmodium falciparum and Plasmodium vivax have been cloned and expressed for a variety of structural and enzymatic studies. Recombinant plasmepsins possess kinetic similarity to the native enzymes, indicating their suitability for target-based antimalarial drug development. We developed an automated assay of P. falciparum plasmepsin II and P. vivax plasmepsin to quickly screen compounds in the Walter Reed chemical database. A low-molecular-mass (346 Da) diphenylurea derivative (WR268961) was found to inhibit plasmepsins with a K i of 1 to 6 M. This compound appears to be selective for plasmepsin, since it is a poor inhibitor of the human aspartic protease cathepsin D (K i greater than 280 M). WR268961 inhibited the growth of P. falciparum strains W2 and D6, with 50% inhibitory concentrations ranging from 0.03 to 0.16 g/ml, but was much less toxic to mammalian cells. The Walter Reed chemical database contains over 1,500 compounds with a diphenylurea core structure, 9 of which inhibit the plasmepsins, with K i values ranging from 0.05 to 0.68 M. These nine compounds show specificity for the plasmepsins over human cathepsin D, but they are poor inhibitors of P. falciparum growth in vitro. Computational docking experiments indicate how diphenylurea compounds bind to the plasmepsin active site and inhibit the enzyme.Malaria, the most severe parasitic disease, infects nearly 300 million people and kills more than a million each year (28). Plasmodium falciparum and Plasmodium vivax are the two malaria species responsible for the most infections and deaths. Although several very effective antimalarial drugs have been used to control this disease, P. falciparum has developed resistance to nearly all available antimalarial drugs (27). Recently, P. vivax from Southeast Asia has developed resistance to the most widely used antimalarial drug, chloroquine. The search for novel antimalarial drugs against specific parasitic targets is thus an urgent task to pursue. In the last decade, many potential targets for new antimalarial drugs have been discovered, such as dihydropteroate synthase, hemoglobin degradation enzymes, and shikimate pathway enzymes (17). Our work focuses on the discovery of new inhibitors of hemoglobin degradation enzymes called plasmepsins.Malarial parasites invade human erythrocytes in the asexual stage of infection. While residing in erythrocytes, the parasites rely on human hemoglobin as a food source, digesting it with a series of proteases. The aspartic proteases, called plasmepsins, are critical for hemoglobin degradation and are thus logical targets for antimalarial drug development (14,19,25). At least four plasmepsins have been identified and cloned from P. falciparum (26; R. Banerjee and D. E. Goldberg, Mol. Parasite Meet., MBL, Woods Hole, Mass., 1999). Active recombinant plasmepsin II has been successfully obtained in large enough quantities (3, 10) to facilit...

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“…4B). In addition, costaining was performed with antibodies to MSP-1 19 and the micronemal protein EBA-175. Merging of the two staining patterns revealed that compartments containing falcipain-1 are distinct from the MSP-1-and EBA-175-containing compartments (Fig.…”

Section: Large-scale Expression and Refolding Of F14mentioning

confidence: 99%

“…Depending upon their mode of action, these proteases have been classified into five different groups (aspartic, cysteine, serine, metallo-, and threonine proteases). Among these different proteases, the roles of cysteine proteases (falcipains) and aspartic proteases (plasmepsins) have been best characterized through the use of their specific inhibitors (2,8,19,20,36).…”

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confidence: 99%

Falcipain-1, aPlasmodium falciparumCysteine Protease with Vaccine Potential

Kumar

Korde

et al. 2007

Infect Immun

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Cysteine proteases (falcipains) of Plasmodium falciparum are potential targets for antimalarial chemotherapy, since they have been shown to be involved in important cellular functions such as hemoglobin degradation and invasion/rupture of red blood cells during parasite life cycle. The role of falcipain-1 at the asexual blood stages of the parasite still remains uncertain. This is mainly due to a lack of methods to prepare this protein in an active form. In order to obtain biologically active falcipain-1, a number of falcipain-1 constructs were designed and a systematic assessment of the refolding conditions was done. We describe here the expression, purification, and characterization of a falcipain-1 construct encoding mature falcipain-1 and 35 amino acids from the C-terminal of the pro domain. Recombinant falcipain-1 was overexpressed in the form of inclusion bodies, solubilized, and purified by Ni 2؉ -nitrilotriacetic acid affinity chromatography under denaturing conditions. A systemic approach was then followed to optimize refolding parameters. An optimum refolding condition was obtained, and the yield of the purified refolded falcipain-1 was ϳ1 mg/liter. Activity of the protein was analyzed by fluorometric and gelatin degradation assays. Immunolocalization studies using anti-falcipain-1 sera revealed a distinct staining at the apical end of the P. falciparum merozoites. Previous studies using falcipain-1-specific inhibitors have suggested a role of falcipain-1 in merozoite invasion. Based on its localization and its role in invasion, we analyzed the immunogenicity of falcipain-1 in mice, followed by heterologous challenge with Plasmodium yoelii sporozoites. Our results suggest a possible role of falcipain-1 in merozoite invasion.

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Studies on Plasmepsins I and II from the Malarial Parasite Plasmodium falciparum and their Exploitation as Drug Targets

Cited by 19 publications

References 19 publications

Hemoglobin-degrading, Aspartic Proteases of Blood-feeding Parasites

Hemoglobin-degrading, Aspartic Proteases of Blood-feeding Parasites

New Class of Small Nonpeptidyl Compounds Blocks Plasmodium falciparum Development In Vitro by Inhibiting Plasmepsins

Falcipain-1, aPlasmodium falciparumCysteine Protease with Vaccine Potential

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