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

Abstract

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...

High-throughput Screening for Potent and Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase

“…This is encouraging, because DHODH also has a mitochondrial location. In addition, diphenyl ureas that inhibit plasmepsins from P. falciparum and Plasmodium vivax show selective killing of Plasmodium versus mammalian cell lines (33). On this basis, a second generation library of the most potent inhibitors is likely to lead to compounds that are effective in whole cells.…”

QSAR Studies on Ant Malarial Activity of Diverse Set of Compounds for D6 Strain of P. falciparum

“…One of them is artemisine [2][3][4]. Also few potentially antimalarial drugs are used as chemotherapeutics which are (i) quinoline derivatives, e.g., primaquine, chloroquine, and mefloquine [5], and/ or (ii) simple sulfonamides, e.g., sulfadoxine [6,7], pyrimidine derivatives; pyrimethamine [8] .…”

Analysis of Binding Interactions of Pepsin Inhibitor-3 To Mammalian and Malarial Aspartic Proteases

“…been shown to impede growth of the parasite in culture (3,(6)(7)(8)(9). Therefore, the PfPM4 orthologs would be excellent targets for a single drug therapy directed at all four plasmodium species (4).…”

“…The four plasmepsins found in the food vacuole of P. falciparum are PfPM1, PfPM2, PfPM4, and the histoaspartic protease (HAP) (3,4). It is now believed that the enzymes from P. vivax (PvPM4), P. ovale (PoPM4), and P. malariae (PmPM4) are orthologs of PfPM4 (5 (3,(6)(7)(8)(9). Therefore, the PfPM4 orthologs would be excellent targets for a single drug therapy directed at all four plasmodium species (4).…”