Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH 2]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite. drug design ͉ malaria ͉ structural biology ͉ protease T here are 300-500 million cases of clinical malaria annually, and 1.4-2.6 million deaths. Malaria is caused by apicomplexan parasites of the genus Plasmodium, with Plasmodium falciparum the most lethal of the 4 species that infect humans. Clinical manifestations begin when parasites enter erythrocytes, and most antimalaria drugs, such as chloroquine, exert their action by preventing the parasite development within these cells (1). As a result of the rapid spread of drug-resistant parasites, there is a constant need to identify and validate new antimalarial targets.Intraerythrocytic parasites have limited capacity for de novo amino acid synthesis and rely on degradation of host hemoglobin (Hb) to maintain protein metabolism and synthesis, and an osmotically stable environment within the erythrocyte (1-4). Within the erythrocytes, malaria parasites consume as much as 75% of the cellular Hb (1). Hb is initially degraded by the concerted action of cysteine-, aspartyl-, and metalloendoproteases, and a dipeptidase (cathepsin C) within a digestive vacuole (DV) to di-and tripeptide fragments (5, 6). These fragments are suggested to be exported to the parasite cytoplasm, where further hydrolysis to release free amino acids takes place [supporting information (SI) Fig. S1; see refs. 7 and 8].The release of amino acids involves 2 metallo-exopeptidases: an alanyl aminopeptidase, PfA-M1 (9, 10), and a leucine aminopeptidase, PfA-M17 (7,11,12). We have demonstrated that the aminopeptidase inhibitor bestatin, an antibiotic and natural analogue of the dipeptide Phe-Leu derived from the fungus Streptomyces olivoretticul, prevents P. falciparum malaria growth in culture (13,14). More recently, it was shown not only that synthetic phosphinate dipeptide analogues that inhibit metallo-aminopeptidases prevented the growth of wildty...
The Kluyveromyces lactis toxin causes an arrest of sensitive yeast cells in the G1 phase of the cell division cycle. Two complementary genetic approaches have been undertaken in the yeast Saccharomyces cerevisiae to understand the mode of action of this toxin. First, two sequences conferring toxin resistance specifically in high copy number have been isolated and shown to encode a tRNA(Glu3) and a novel polypeptide. Disruption of the latter sequence in the yeast genome conferred toxin resistance and revealed that it was nonessential, while the effect of the tRNA(Glu)3 was highly specific and mediated resistance by affecting the toxin's target. An alpha-specific, copy number-independent suppressor of toxin sensitivity was also isolated and identified as MATa, consistent with the observation that diploid cells are partially resistant to the toxin. Second, in a comprehensive screen for toxin-resistant mutants, representatives of 13 complementation groups have been obtained and characterized to determine whether they are altered in the toxin's intracellular target. Of 10 genes found to affect the target process, one (KTI12) was found to encode the novel polypeptide previously identified as a multicopy resistance determinant. Thus, both loss of KTI12 function and elevated KTI12 copy number can cause resistance to the K. lactis toxin.
Blood stages of Plasmodium falciparum export proteins into their erythrocyte host, thereby inducing extensive host cell modifications that become apparent after the first half of the asexual development cycle (ring stage). This is responsible for a major part of parasite virulence. Export of many parasite proteins depends on a sequence motif termed Plasmodium export element (PEXEL) or vacuolar transport signal (VTS). This motif has allowed the prediction of the Plasmodium exportome. Using published genome sequence, we redetermined the boundaries of a previously studied region linked to P. falciparum virulence, reducing the number of candidate genes in this region to 13. Among these, we identified a cluster of four ring stage-specific genes, one of which is known to encode an exported protein. We demonstrate that all four genes code for proteins exported into the host cell, although only two genes contain an obvious PEXEL/VTS motif. We propose that the systematic analysis of ring stage-specific genes will reveal a cohort of exported proteins not present in the currently predicted exportome. Moreover, this provides further evidence that host cell remodeling is a major task of this developmental stage. Biochemical and photobleaching studies using these proteins reveal new properties of the parasiteinduced membrane compartments in the host cell. This has important implications for the biogenesis and connectivity of these structures. INTRODUCTIONPlasmodium falciparum is the causative agent of the most severe form of human malaria, killing 1-2 million people each year (Snow et al., 2005). The symptoms of this disease are associated with the continuous asexual multiplication of P. falciparum parasites within red blood cells (RBCs) (for review, see Miller et al., 2002). In this part of the life cycle, the parasite develops, within 48 h from the ring stage, to the trophozoite stage and finally to the schizont stage after which the infected RBCs (IRBCs) rupture, releasing up to 32 merozoite stage parasites that infect new RBCs.The ring stage lasts for almost half of this cycle (ϳ0-20 h after invasion) and shows low metabolic activity with little change in size and morphology (Zolg et al., 1984;de Rojas and Wasserman, 1985). P. falciparum ring forms are the only stages apparent in the blood circulation of infected humans, because more mature parasites adhere to the endothelium of various organs to avoid clearance by the spleen (Langreth and Peterson, 1985). The subsequent accumulation of infected RBCs in the microvasculature is largely responsible for malaria-associated morbidity and mortality. This sequestration of IRBCs is mediated by the parasite protein P. falciparum erythrocyte membrane protein 1 (PfEMP-1), which is exported to the surface of the IRBCs and is encoded by a family of variant antigens (Baruch et al., 1995;Smith et al., 1995;Su et al., 1995). Because human RBCs are devoid of a functional protein trafficking system, the parasite has to establish a protein export system in the host cell before PfEMP-1 can b...
Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, Pf A-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the x-ray crystal structure of the monomeric Pf A-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here, we present the x-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with antimalarial activity. The six active sites of the Pf A-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to Pf A-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centers in these enzymes (two in Pf A-M17 and one in Pf A-M1) in both substrate and drug binding. Our detailed understanding of the Pf A-M1 and Pf A-M17 active sites now permits a rational approach in the development of a unique class of two-target and/or combination antimalarial therapy.drug design | malaria | protease | structural biology | neutral aminopeptidases
Parasite resistance to antimalarial drugs is a serious threat to human health, and novel agents that act on enzymes essential for parasite metabolism, such as proteases, are attractive targets for drug development. Recent studies have shown that clinically utilized human immunodeficiency virus (HIV) protease inhibitors can inhibit the in vitro growth of Plasmodium falciparum at or below concentrations found in human plasma after oral drug administration. The most potent in vitro antimalarial effects have been obtained for parasites treated with saquinavir, ritonavir, or lopinavir, findings confirmed in this study for a genetically distinct P. falciparum line (3D7). To investigate the potential in vivo activity of antiretroviral protease inhibitors (ARPIs) against malaria, we examined the effect of ARPI combinations in a murine model of malaria. In mice infected with Plasmodium chabaudi AS and treated orally with ritonavir-saquinavir or ritonavir-lopinavir, a delay in patency and a significant attenuation of parasitemia were observed. Using modeling and ligand docking studies we examined putative ligand binding sites of ARPIs in aspartyl proteases of P. falciparum (plasmepsins II and IV) and P. chabaudi (plasmepsin) and found that these in silico analyses support the antimalarial activity hypothesized to be mediated through inhibition of these enzymes. In addition, in vitro enzyme assays demonstrated that P. falciparum plasmepsins II and IV are both inhibited by the ARPIs saquinavir, ritonavir, and lopinavir. The combined results suggest that ARPIs have useful antimalarial activity that may be especially relevant in geographical regions where HIV and P. falciparum infections are both endemic.
The malaria parasite Plasmodium falciparum has at least five putative histone deacetylase (HDAC) enzymes, which have been proposed as new antimalarial drug targets and may play roles in regulating gene transcription, like the better-known and more intensively studied human HDACs (hHDACs). Fourteen new compounds derived from L-cysteine or 2-aminosuberic acid were designed to inhibit P. falciparum HDAC-1 (PfHDAC-1) based on homology modeling with human class I and class II HDAC enzymes. The compounds displayed highly potent antiproliferative activity against drug-resistant (Dd2) or drug sensitive (3D7) strains of P. falciparum in vitro (50% inhibitory concentration of 13 to 334 nM). Unlike known hHDAC inhibitors, some of these new compounds were significantly more toxic to P. falciparum parasites than to mammalian cells. The compounds inhibited P. falciparum growth in erythrocytes at both the early and late stages of the parasite's life cycle and caused altered histone acetylation patterns (hyperacetylation), which is a marker of HDAC inhibition in mammalian cells. These results support PfHDAC enzymes as being promising targets for new antimalarial drugs.
Recent studies have indicated that antiretroviral protease inhibitors may affect outcome in malarial disease. We have investigated the antimalarial activities of 6 commonly used antiretroviral agents. Our data indicate that, in addition to the previously published effects on cytoadherence and phagocytosis, the human immunodeficiency virus (HIV)-1 protease inhibitors saquinavir, ritonavir, and indinavir directly inhibit the growth of Plasmodium falciparum in vitro at clinically relevant concentrations. These findings are particularly important in light of both the high rate of malaria and HIV-1 coinfection in sub-Saharan Africa and the effort to employ highly active antiretroviral therapy in these regions.
Previous studies have pinpointed the M17 leucyl aminopeptidase of Plasmodium falciparum (PfLAP) as a target for the development of new antimalarials. This metallo-exopeptidase functions in the terminal stages of hemoglobin digestion and is inhibited by bestatin, a natural analog of Phe-Leu. By screening novel phosphinate dipeptide analogues for inhibitory activity against recombinant PfLAP, we have discovered two compounds, 4 (hPheP[CH2]Phe) and 5 (hPheP[CH2]Tyr), with inhibitory constants better than bestatin. These compounds are fast, tight-binding inhibitors that make improved contacts within the active site of PfLAP. Both compounds inhibit the growth of P. falciparum in vitro, exhibiting IC50 values against the chloroquine-resistant clone Dd2 of 20-40 and 12-23 muM, respectively. While bestatin exhibited some in vivo activity against Plasmodium chabaudi chabaudi, compound 4 reduced parasite burden by 92%. These studies establish the PfLAP as a prime target for the development of antimalarial drugs and provide important new lead compounds.
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