Anti-Plasmodium activity of imidazolium and triazolium salts

We have previously reported that tetrazolium salts were both potent and specific inhibitors of Plasmodium replication, and that they appear to interact with a parasite component that is both essential and conserved. The use of tetrazolium salts in vivo is limited by the potential reduction of the tetrazolium ring to form an inactive, neutral acyclic formazan. To address this issue imidazolium and triazolium salts were synthesized and evaluated as Plasmodium inhibitors. Many of the imidazolium and triazolium salts were highly potent with active concentrations in the nanomolar range in Plasmodium falciparum cultures, and specific to Plasmodium with highly favorable therapeutic ratios. The results corroborate our hypothesis that an electron-deficient core is required so that the compound may thereby interact with a negatively charged moiety on the parasite merozoite; the side groups in the compound then form favorable interactions with adjacent parasite components and thereby determine both the potency and selectivity of the compound.     

Falstatin, a cysteine protease inhibitor of Plasmodium falciparum, facilitates erythrocyte invasion

Erythrocytic malaria parasites utilize proteases for a number of cellular processes, including hydrolysis of hemoglobin, rupture of erythrocytes by mature schizonts, and subsequent invasion of erythrocytes by free merozoites. However, mechanisms used by malaria parasites to control protease activity have not been established. We report here the identification of an endogenous cysteine protease inhibitor of Plasmodium falciparum, falstatin, based on modest homology with the Trypanosoma cruzi cysteine protease inhibitor chagasin. Falstatin, expressed in Escherichia coli, was a potent reversible inhibitor of the P. falciparum cysteine proteases falcipain-2 and falcipain-3, as well as other parasite- and nonparasite-derived cysteine proteases, but it was a relatively weak inhibitor of the P. falciparum cysteine proteases falcipain-1 and dipeptidyl aminopeptidase 1. Falstatin is present in schizonts, merozoites, and rings, but not in trophozoites, the stage at which the cysteine protease activity of P. falciparum is maximal. Falstatin localizes to the periphery of rings and early schizonts, is diffusely expressed in late schizonts and merozoites, and is released upon the rupture of mature schizonts. Treatment of late schizionts with antibodies that blocked the inhibitory activity of falstatin against native and recombinant falcipain-2 and falcipain-3 dose-dependently decreased the subsequent invasion of erythrocytes by merozoites. These results suggest that P. falciparum requires expression of falstatin to limit proteolysis by certain host or parasite cysteine proteases during erythrocyte invasion. This mechanism of regulation of proteolysis suggests new strategies for the development of antimalarial agents that specifically disrupt erythrocyte invasion.     

Stage-dependent toxicity of N-acetyl-glucosamine to Plasmodium falciparum

The effects of N-acetyl-glucosamine on growth of synchronized cultures of Plasmodium falciparum were assessed by morphological observations and by measurement of parasite incorporation of 3H-hypoxanthine. Inhibition of 3H-hypoxanthine incorporation was more marked during the later stages of the erythrocytic cycle. At concentrations of the sugar below 20 mM, however, the deleterious effects were mainly a result of failure of released merozoites to invade erythrocytes, rather than a failure of schizonts to mature or release merozoites. These results are compatible with the hypothesis that a lectin-like substance on the merozoite interacts with a surface glycoprotein on the red cell and that sugar residues on this glycoprotein may be involved in this recognition.     

Developmental stage-specific biosynthesis of glycosylphosphatidylinositol anchors in intraerythrocytic Plasmodium falciparum and its inhibition in a novel manner by mannosamine

Glycosylphosphatidylinositols (GPIs) are the major glycoconjugates in intraerythrocytic stage Plasmodium falciparum. Several functional proteins including merozoite surface protein 1 are anchored to the cell surface by GPI modification, and GPIs are vital to the parasite. Here, we studied the developmental stage-specific biosynthesis of GPIs by intraerythrocytic P. falciparum. The parasite synthesizes GPIs exclusively during the maturation of early trophozoites to late trophozoites but not during the development of rings to early trophozoites or late trophozoites to schizonts and merozoites. Mannosamine, an inhibitor of GPI biosynthesis, inhibits the growth of the parasite specifically at the trophozoite stage, preventing further development to schizonts and causing death. Mannosamine has no effect on the development of either rings to early trophozoites or late trophozoites to schizonts and merozoites. The analysis of GPIs and proteins synthesized by the parasite in the presence of mannosamine demonstrates that the effect is because of the inhibition of GPI biosynthesis. The data also show that mannosamine inhibits GPI biosynthesis by interfering with the addition of mannose to an inositol-acylated GlcN-phosphatidylinositol (PI) intermediate, which is distinctively different from the pattern seen in other organisms. In other systems, mannosamine inhibits GPI biosynthesis by interfering with either the transfer of a mannose residue to the Manalpha1-6Manalpha1-4GlcN-PI intermediate or the formation of ManN-Man-GlcN-PI, an aberrant GPI intermediate, which cannot be a substrate for further addition of mannose. Thus, the parasite GPI biosynthetic pathway could be a specific target for antimalarial drug development.     

Type II fatty acid synthesis is essential only for malaria parasite late liver stage development

Intracellular malaria parasites require lipids for growth and replication. They possess a prokaryotic type II fatty acid synthesis (FAS II) pathway that localizes to the apicoplast plastid organelle and is assumed to be necessary for pathogenic blood stage replication. However, the importance of FAS II throughout the complex parasite life cycle remains unknown. We show in a rodent malaria model that FAS II enzymes localize to the sporozoite and liver stage apicoplast. Targeted deletion of FabB/F , a critical enzyme in fatty acid synthesis, did not affect parasite blood stage replication, mosquito stage development and initial infection in the liver. This was confirmed by knockout of FabZ , another critical FAS II enzyme. However, FAS II-deficient Plasmodium yoelii liver stages failed to form exo-erythrocytic merozoites, the invasive stage that first initiates blood stage infection. Furthermore, deletion of FabI in the human malaria parasite Plasmodium falciparum did not show a reduction in asexual blood stage replication in vitro . Malaria parasites therefore depend on the intrinsic FAS II pathway only at one specific life cycle transition point, from liver to blood.     

Plasmodium falciparum: increased and multiple invasion during short periods of time

We describe how to obtain an increased merozoite invasion of Plasmodium falciparum into human erythrocytes during short periods of time. Using this procedure, infected erythrocytes show multiple invasions (2-4 merozoites per erythrocyte), amplifying, several times, the effects of parasite entry into host cells. The procedure yields synchronous cultures (2-h age range) with parasitemia as high as 15%. It is possible to reach parasitemia of 50% or higher allowing for a 6-h invasion period.     

Fractionation and identification of proteins by 2-DE and MS: towards a proteomic analysis of Plasmodium falciparum

Since completion of genome sequencing of the malarial parasite Plasmodium falciparum, proteomic tools for the identification of parasite proteins have become particularly attractive as they allow a more thorough interpretation of these data. Recent advances in 2-D PAGE, MS, and bioinformatics have created great opportunities for mapping and characterization of protein populations. We employed these improvements in a proteomic approach for the analysis of proteins detected in two blood stages of P. falciparum, (i) in the schizont stage and (ii) in the merozoite stage. For the isolation of merozoites, we introduced a new protocol based on the preparation of clustered structures of merozoites upon treatment of cultures with the common cysteine proteinase inhibitor E64. Peptide mass fingerprints of excised and trypsinated protein spots, acquired by MALDI-TOF MS were generated to identify a variety of proteins. Moreover, prefractionation procedures were used to enrich and map low-abundance proteins in protein samples. The data demonstrate that classic proteomic analyses using 2-D PAGE are now feasible for P. falciparum and represent the first step in the direction of creating 2-D reference maps for this medically most relevant protozoon.     

Plasmodium falciparum: stage specific effects of a selective inhibitor of lactate dehydrogenase

Plasmodium falciparum lactate dehydrogenase (PfLDH) is essential for ATP generation. Based on structural differences within the active site between P. falciparum and human LDH, we have identified a series of heterocyclic azole-based inhibitors that selectively bind within the PfLDH but not the human LDH (hLDH) active site and showed anti-malarial activity in vitro and in vivo. Here we expand on an azole, OXD1, from this series and found that the anti-P. falciparum activity was retained against a panel of strains independently of their anti-malarial drug sensitivity profile. Trophozoites had relatively higher PfLDH enzyme activity and PfLDH-RNA expression levels than rings and were the most susceptible stages to OXD1 exposure. This is probably linked to their increased energy requirements and consistent with glycolysis being an essential metabolic pathway for parasite survival within the erythrocyte. Further structural elaboration of these azoles could lead to the identification of compounds that target P. falciparum through such a novel mechanism and with more potent anti-malarial activity.     

Structure-activity relationships of antileishmanial and antimalarial chalcones

A series of oxygenated chalcones which have been evaluated earlier for antimalarial activity (Plasmodium falciparum K1) were tested for antileishmanial activity against Leishmania donovani amastigotes. A comparison of structure-activity relationships reveal that different physicochemical and structural requirements exist for these two activities. Antileishmanial activity is associated with less lipophilic chalcones, in particular those with 4'-hydroxyl-substituted B rings and hetero/polyaromatic A rings. In contrast, chalcones with good antimalarial activity have alkoxylated B rings and electron-deficient A rings. Visualization of the steric and electrostatic fields generated from comparative molecular field analysis (CoMFA) indicate that the ring A of chalcones make a more significant contribution to antileishmanial activity while both rings A and B are important for antimalarial activity. Despite different requirements, two alkoxylated chalcones (8, 19) were identified which combined good antimalarial and antileishmanial activities.     

Anti-Plasmodium activity of imidazole-dioxolane compounds

A series of imidazole-dioxolane compounds, which we hypothesize should bind to heme and thus interfere with heme catabolism in the parasite, were assayed for inhibitory activity in Plasmodium falciparum cultures and the results were compared to those obtained with Chinese hamster ovary (CHO) cells. The majority of the compounds displayed a similar ratio of inhibitory activity in the two culture systems; however, a number of the compounds tested showed promising anti-Plasmodium activity. The mechanism of action of these compounds remains unclear, however their inability to act synergistically with chloroquine suggests that, if they are inhibiting heme detoxification, they do so in a manner that does not complement the action of chloroquine.     

Plasmodium falciparum DNA helicase 60. dsRNA- and antibody-mediated inhibition of malaria parasite growth and downregulation of its enzyme activities by DNA-interacting compounds

Helicases are ubiquitous enzymes that play important roles in all types of DNA transaction in the cells. Recently we have reported the characterization of the first DEAD-box helicase [Plasmodium falciparum DNA helicase 60 (PfDH60)] from Plasmodium falciparum and have shown that it is a unique, dual bipolar helicase expressed in a stage-specific manner. In this study, we show the further characterization of PfDH60. For analyzing the significance of this enzyme in parasite growth, we studied the effect of dsRNA and specific antibodies on growth of the parasite. The studies indicate that the parasite cultures treated with PfDH60 dsRNA exhibited approximately 50% growth inhibition when compared with either untreated cultures or cultures treated with unrelated dsRNA. It was interesting to note that purified immunoglobulins against PfDH60 induced approximately 62% inhibition of in vitro growth of P. falciparum and that this inhibitory effect was associated with morphologic damage to the parasite. DNA-interacting compounds inhibit DNA helicase and ssDNA-dependent ATPase activities of PfDH60. Of various compounds tested, only actinomycin, daunorubicin, ethidium bromide, netropsin and nogalamycin were able to inhibit the enzyme activities of PfDH60, with apparent IC50 values for helicase inhibition of 0.8, 0.3, 2.0, 1.2 and 1.5 microm, respectively. It may be proposed that these compounds form a complex with DNA and specifically inhibit helicases due to obstruction in the translocation of the enzyme. These compounds also inhibited parasite growth in culture. This is the first study to show inhibition of growth of the parasite by the dsRNA of a helicase, and most probably this is due to interference with cognate mRNA expression.     

Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes

The most virulent form of malaria is caused by waves of replication of blood stages of the protozoan pathogen Plasmodium falciparum. The parasite divides within an intraerythrocytic parasitophorous vacuole until rupture of the vacuole and host-cell membranes releases merozoites that invade fresh erythrocytes to repeat the cycle. Despite the importance of merozoite egress for disease progression, none of the molecular factors involved are known. We report that, just prior to egress, an essential serine protease called PfSUB1 is discharged from previously unrecognized parasite organelles (termed exonemes) into the parasitophorous vacuole space. There, PfSUB1 mediates the proteolytic maturation of at least two essential members of another enzyme family called SERA. Pharmacological blockade of PfSUB1 inhibits egress and ablates the invasive capacity of released merozoites. Our findings reveal the presence in the malarial parasitophorous vacuole of a regulated, PfSUB1-mediated proteolytic processing event required for release of viable parasites from the host erythrocyte.     

The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis

The plastid in Plasmodium falciparum asexual stages is a tubular structure measuring about 0.5 micron x 0.15 micron in the merozoite, and 1.6 x 0.35 microns in trophozoites. Each parasite contains a single plastid until this organelle replicates in late schizonts. The plastid always adheres to the (single) mitochondrion, along its whole length in merozoites and early rings, but only at one end in later stages. Regions of the plastid are also closely related to the pigment vacuole, nuclear membrane and endoplasmic reticulum. In merozoites the plastid is anchored to a band of 2-3 subpellicular microtubules. Reconstructions show the plastid wall is characteristically three membranes thick, with regions of additional, complex membranes. These include inner and outer membrane complexes. The inner complex in the interior lumen is probably a rolled invagination of the plastid's inner membrane. The outer complex lies between the outer and middle wall membranes. The interior matrix contains ribosome-like granules and a network of fine branched filaments. Merozoites of P. berghei and P. knowlesi possess plastids similar in structure to those of P. falciparum. A model is proposed for the transfer of membrane lipid from the plastid to other organelles in the parasite.     

Isolation and functional characterization of Plasmodium falciparum merozoite antigens

Merozoite surface proteins are thought to play an important role during the invasion of red blood cells by merozoites. In this article the strategies for the chromatographic isolation and for the functional and molecular characterisation of isolated antigens from freshly harvested Plasmodium falciparum merozoites from cultures are described.     

Plasmodium falciparum: characterization of a 33-kDa soluble antigen

A 33-kDa soluble antigen identified in the culture supernatant by patient serum and monoclonal antibodies was present in rings, trophozoites, schizonts, and merozoites of Plasmodium falciparum. The antigen which is released into the culture supernatant by growing parasites was also observed in the host cells of trophozoites and schizonts and could be localized on the host cell surface. Its specificity for the surface of trophozoites and schizonts was observed to decrease with increased duration without subculture. The antigen could then be detected on the surface of noninfected erythrocytes. The antigenicity of the 33-kDa antigen was destroyed by heating at 65 degrees C. Monoclonal and polyclonal specific antibodies weakly inhibited parasite growth in vitro. The antigen was present in both knob positive and knob negative parasites in all the P. falciparum isolates tested.     

Recombination associated with replication of malarial mitochondrial DNA.

Mitochondrial DNA of the malarial parasite Plasmodium falciparum comprises approximately 20 copies per cell of a 6 kb genome, arranged mainly as polydisperse linear concatemers. In synchronous blood cultures, initiation of mtDNA replication coincides with the start of the 4-5 doublings in nuclear DNA that mark the reproductive phase of the erythrocytic cycle. We show that mtDNA replication coincides with a recombination process reminiscent of the replication mechanism used by certain bacteriophages and plasmids. The few circular forms of mtDNA which are also present do not replicate by a theta mechanism, but are themselves the product of recombination, and we propose they undergo rolling circle activity to generate the linear concatemers.     

Stimulation of the chemiluminescence of human polymorphonuclear leukocytes in various stages of the intraerythrocyte development of Plasmodium falciparum

The synchronized cultures of Plasmodium falciparum were used to stimulate in vitro the chemiluminescence of human polymorphonuclear leukocytes in the presence of immune serum. The schizonts were concentrated by Percoll gradient centrifugation method (density 1.085 and osmolarity 285 mOsmol), and placed in culture, treated 6 hours later by sorbitol. Under incubation at constant temperature and pressure, the rate of synchronization reached 85% for schizonts during 5 replicative cycles. Every asexual stages of Plasmodium falciparum were used separately to stimulate polymorphonuclear leukocytes: merozoites were the most effective, followed by schizonts, trophozoites, and lastly supernatants of cultures containing degradation products of parasites.     

Stage-dependent inhibition of chloroquine on Plasmodium falciparum in vitro

Morphological observation of the life cycle of the malaria parasite, Plasmodium falciparum, in highly synchronous cultures after an exposure to therapeutic concentrations of chloroquine in ring, trophozoite and schizont stages, respectively, were carried out in order to determine the influence of chloroquine on the growth of the different stages of the malarial parasites. It was found that chloroquine could not affect merozoite invasion of the erythrocytes; the ring stage was more sensitive to chloroquine than the trophozoite and schizont stages; and chloroquine in therapeutic concentrations prevented only the transformation of rings to trophozoites and could not affect the transformations of trophozoites to schizonts and schizonts to new rings. The determination of the IC50 of chloroquine showed that the IC50 of trophozoites was about 6 times as high as that of rings.     

Synthesis and in vitro evaluation of new chloroquine-chalcone hybrids against chloroquine-resistant strain of Plasmodium falciparum

The control of malaria has been complicated with increasing resistance of malarial parasite against existing antimalarials. Herein, we report the synthesis of a new series of chloroquine-chalcone based hybrids (8-22) and their antimalarial efficacy against both chloroquine-susceptible (3D7) and chloroquine-resistant (K1) strains of Plasmodium falciparum. Most of the compounds showed enhanced antimalarial activity as compared to chloroquine in chloroquine-resistant (K1) strain of Plasmodium falciparum. Furthermore, to unfold the mechanism of action of these synthesized hybrid molecules, we carried out hemin dependent studies, in which three compounds were found to be active.     

Aryl aryl methyl thio arenes prevent multidrug-resistant malaria in mouse by promoting oxidative stress in parasites

We have synthesized a new series of aryl aryl methyl thio arenes (AAMTAs) and evaluated antimalarial activity in vitro and in vivo against drug-resistant malaria. These compounds interact with free heme, inhibit hemozoin formation, and prevent Plasmodium falciparum growth in vitro in a concentration-dependent manner. These compounds concentration dependently promote oxidative stress in Plasmodium falciparum as evident from the generation of intraparasitic oxidants, protein carbonyls, and lipid peroxidation products. Furthermore, AAMTAs deplete intraparasite GSH levels, which is essential for antioxidant defense and survival during intraerythrocytic stages. These compounds displayed potent antimalarial activity not only in vitro but also in vivo against multidrug-resistant Plasmodium yoelii dose dependently in a mouse model. The mixtures of enantiomers of AAMTAs containing 3-pyridyl rings were found to be more efficient in providing antimalarial activity. Efforts have been made to synthesize achiral AAMTAs 17-23 and among them, compound 18 showed significant antimalarial activity in vivo.