A Role for the Protease Falcipain 1 in Host Cell Invasion by the Human Malaria Parasite

Greenbaum, Doron C.; Baruch, Amos; Grainger, Munira; Bozdech, Zbynek; Medzihradszky, Katlin F.; Engel, Juan C.; DeRisi, Joseph L.; Holder, Anthony A.; Bogyo, Matthew

doi:10.1126/science.1077426

Cited by 263 publications

(188 citation statements)

References 23 publications

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“…The findings reported here, coupled with the findings of previous work demonstrating an important role of cysteine proteases in trophozoite growth, merozoite release, oocyst rupture, and sporozoite invasiveness, demonstrate the therapeutic potential of cysteine protease inhibitors to significantly reduce both the morbidity from malaria and the transmission of the malaria parasite (1,4,10,23,25,28,30,34,37). Identifying the proteases expressed in both the asexual and the sexual stages and their in vivo accessibilities to inhibitors is an important component of drug development.…”

Section: Discussionmentioning

confidence: 60%

“…To identify a more specific P. falciparum inhibitor, a number of other compounds are being screened for their abilities to block asexual growth and/or falcipain activity (5,16,22). Several compounds that effectively inhibit recombinant falcipains 2A, 2B, and 3 have been developed and have been tested for their effects on asexual growth, but they have never been screened against sexual stage parasites (10,17,29). Two compounds, Mu-Leu-hPhe-VSPh and Mu-Leu-hPhe-FMK (generously provided by P. Rosenthal), that effectively inhibit falcipain 2B and 3, as well as YA29, which was reported to preferentially inhibit falcipain 1, were used to treat mature gametocyte cultures (10,17,29).…”

Section: Resultsmentioning

confidence: 99%

“…Several compounds that effectively inhibit recombinant falcipains 2A, 2B, and 3 have been developed and have been tested for their effects on asexual growth, but they have never been screened against sexual stage parasites (10,17,29). Two compounds, Mu-Leu-hPhe-VSPh and Mu-Leu-hPhe-FMK (generously provided by P. Rosenthal), that effectively inhibit falcipain 2B and 3, as well as YA29, which was reported to preferentially inhibit falcipain 1, were used to treat mature gametocyte cultures (10,17,29). However, none of these significantly inhibited oocyst production following a 1-to 2-h incubation (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

Ekşi

Czesny

Gemert³

et al. 2007

Antimicrob Agents Chemother

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During asexual intraerythrocytic growth, Plasmodium falciparum utilizes hemoglobin obtained from the host red blood cell (RBC) as a nutrient source. Papain-like cysteine proteases, falcipains 2 and 3, have been reported to be involved in hemoglobin digestion and are targets of current antimalarial drug development efforts. However, their expression during gametocytogenesis, which is required for malaria parasite transmission, has not been studied. Many of the available antimalarials do not inhibit development of sexual stage parasites, and therefore, the persistence of gametocytes after drug treatment allows continued transmission of the disease. In the work reported here, incubation of stage V gametocytes with membrane-permeant cysteine protease inhibitor E64d significantly inhibited oocyst production (80 to 100%). The same conditions inhibited processing of gametocyte-surface antigen Pfs230 during gametogenesis but did not alter the morphology of the food vacuole in gametocytes, inhibit emergence, or block male exflagellation. E64d reduced the level of oocyst production more effectively than that reported previously for falcipain 1-knockout parasites, suggesting that falcipains 2 and 3 may also be involved in malaria parasite transmission. However, in this study only falcipain 3 and not falcipain 2 was found to be expressed in stage V gametocytes. Interestingly, during gametocytogenesis falcipain 3 was transported into the red blood cell and by stage V was localized in vesicles along the RBC surface, consistent with a role during gamete emergence. The ability of a membrane-permeant cysteine protease inhibitor to significantly reduce malaria parasite transmission suggests that future drug design should include evaluation of gametogenesis and sporogonic development.The clinical symptoms of malaria are caused by asexually replicating parasites, but malaria parasite transmission requires that a subpopulation of parasites undergo sexual differentiation. In the species of parasite responsible for the most virulent form of malaria, Plasmodium falciparum, complete maturation into mature stage V gametocytes takes 10 to 12 days in the human host. Once stage V gametocytes are taken up by a mosquito, gametogenesis is stimulated and, after fertilization, the zygote differentiates into an ookinete. The ookinete then migrates to the basal surface of the midgut and forms an oocyst, where tens of thousands of infectious sporozoites are produced.Both asexual and sexual stage parasites are present concurrently in an infected individual and therefore would both be exposed to antimalarial drugs. However, gametocytes have been found to be resistant to many of the commonly used antimalarials, such as the 4-aminoquinolines; and sulfadoxinepyrimethamine has been reported to increase the production of gametocytes, which could enhance transmission (2, 12, 21, 36). Consequently, even after successful treatment for clinical symptoms, an individual can still transmit the malaria parasite for at least a week. Therefore, it is important to d...

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Section: Discussionmentioning

confidence: 60%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

Ekşi

Czesny

Gemert³

et al. 2007

Antimicrob Agents Chemother

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“…This probe covalently modifies papain family cysteine proteases and allows their direct isolation by virtue of a biotin tag on the probe. The detailed protocol for the purification is outlined elsewhere (13,14). Isolated proteins were separated on SDS-PAGE gels followed by excision and in-gel digestion prior to analysis by tandem mass spectrometry.…”

Section: Methodsmentioning

confidence: 99%

O-Sulfonation of Serine and Threonine

Medzihradszky

Darula

Perlson

et al. 2004

Molecular & Cellular Proteomics

130

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Protein sulfonation on serine and threonine residues is described for the first time. This post-translational modification is shown to occur in proteins isolated from organisms representing a broad span of eukaryote evolution, including the invertebrate mollusk Lymnaea stagnalis, the unicellular malaria parasite Plasmodium falciparum, and humans. Detection and structural characterization of this novel post-translational modification was carried out using liquid chromatography coupled to electrospray tandem mass spectrometry on proteins including a neuronal intermediate filament and a myosin light chain from the snail, a cathepsin-C-like enzyme from the parasite, and the cytoplasmic domain of the human orphan receptor tyrosine kinase Ror-2. These findings suggest that sulfonation of serine and threonine may be involved in multiple functions including protein assembly and signal transduction. Molecular & Cellular Proteomics 3:429 -443, 2004.Sulfonation occurs as a common enzymatic modification of endogenous substances including proteins, carbohydrates, catecholamines, and estrogenic steroids as well as xenobiotic chemicals (1). Sulfonation refers to the transfer of the sulfonate group (SO 3 Ϫ1 ) from 3Ј-phosphoadenosine-5Ј-phosphosulfate (PAPS), 1 the only known sulfonate donor (2), and can occur through several types of linkages, such as esters and anhydrides (O-sulfonation), amides (N-sulfonation), and thioesters (S-sulfonation), of which O-sulfonation is the most prominent (3). The transfer of SO 3 Ϫ1 to a hydroxyl or phenolic acceptor (O-sulfonation) generates a sulfono-derivative, and this reaction has commonly been referred to as sulfation rather than the more accurate O-sulfonation. The majority of cellular sulfonation is of the O type and occurs primarily on polysaccharides, steroids, catecholamines, and thyroid hormones (1). These reactions are catalyzed by the soluble cytosolic sulfotransferases and appear to alter their bioactivity. For example, estrogen, testosterone, and thyroid hormones (T 3 and T 4 ) can interact with their respective receptors to regulate transcription, whereas their sulfate-containing moieties cannot. Furthermore, the half-life of these compounds in blood is significantly shorter than that of their conjugated counterparts, suggesting that sulfonation maintains these compounds in an inactive state ready for rapid deployment by the removal of the sulfonyl group.While the cytosolic sulfotransferases conjugate cell-permeable or intracellular compounds, the membrane-bound Golgiassociated sulfotransferases are primarily responsible for sulfonation of extracellular proteins via a co-or post-translational mechanism. The membrane-bound sulfotransferases are responsible for the sulfonation of various glycosaminoglycans, such as heparin and heparan sulfate. Additionally, these enzymes catalyze the direct sulfonation of proteins on the O 4 position of tyrosine residues (4). It is one of the last modifications to occur during protein transiting the trans-Golgi and thus has been found almost ex...

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“…Chemical biology approaches are increasingly being used to glean new insights into P. falciparum biology. Activity based probes have been used to identify and investigate the role of falcipain-1 in parasite invasion of RBCs (16) and that of the subtilisin-family serine protease, PfSUB1, and cysteine protease dipeptidyl peptidase in parasite release from infected RBCs (17). Similarly, selected enzyme targets are also being screened against large chemical libraries to identify specific inhibitors that can be used to modulate the activity of this target in situ and potentially serve as lead compounds in drug discovery efforts.…”

mentioning

confidence: 99%

Inhibiting Plasmodium falciparum growth and heme detoxification pathway using heme-binding DNA aptamers

Niles¹,

DeRisi

Marletta

2009

Proc. Natl. Acad. Sci. U.S.A.

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The human parasite Plasmodium falciparum enzymatically digests hemoglobin during its intra-erythrocytic developmental stages in acidic food vacuole compartments. The released heme is rapidly detoxified by polymerization into the chemically inert pigment, hemozoin. Several heme-binding anti-malarial compounds, such as chloroquine, efficiently inhibit this process, and this is believed to be the predominant mechanism by which these drugs induce parasite toxicity. In an effort to expand the biochemical tools available for exploration of this pathogen's basic biology, we chose this heme-detoxification pathway as a model system for exploring the suitability of DNA aptamers for modulating this essential parasite biochemical pathway. In this report, we demonstrate that heme-binding DNA aptamers efficiently inhibit in vitro hemozoin formation catalyzed by either a model lipid system or parasite-derived extracts just as or more potently than chloroquine. Furthermore, when parasites are grown in red cells loaded with heme-binding aptamers, their growth is significantly inhibited relative to parasites exposed to non-heme-binding DNA oligonucleotides. Both the timing of parasite-induced toxicity and the concentration of heme-binding aptamer required for inducing toxicity correlate well with the uptake of red cell cytosolic components by the parasite, and the requirement for compounds with similar in vitro hemozoin inhibitory potency to preconcentrate within the parasite before observing toxicity. Thus, these hemebinding aptamers recapitulate the in vitro hemozoin inhibition activity and induce parasite toxicity in a manner consistent with inhibition of this pathway. Altogether, these data demonstrate that aptamers can be versatile tools with applicability in functionally dissecting important P. falciparum-specific pathways both in vitro and in vivo.

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A Role for the Protease Falcipain 1 in Host Cell Invasion by the Human Malaria Parasite

Cited by 263 publications

References 23 publications

Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

Inhibition of Plasmodium falciparum Oocyst Production by Membrane-Permeant Cysteine Protease Inhibitor E64d

O-Sulfonation of Serine and Threonine

Inhibiting Plasmodium falciparum growth and heme detoxification pathway using heme-binding DNA aptamers

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