Falciparum Dd2 Research Articles

Synthesis of spiro-1,2-dioxolanes and their activity against Plasmodium falciparum

Artemisinin-derived compounds play an integral role in current malaria chemotherapy. Given the virtual certainty of emerging resistance, we have investigated spiro-1,2-dioxolanes as an alternative scaffold. The endoperoxide functionality was generated by the SnCl 4-mediated annulation of a bis-silylperoxide and an alkene. The first set of eight analogs gave EC 50 values of 50–150 nM against Plasmodium falciparum 3D7 and Dd2 strains, except for the carboxylic acid analog. A second series, synthesized by coupling a spiro-1,2-dioxolane carboxylic acid to four separate amines, afforded the most potent compound (EC 50 ∼5 nM).

Comprehensive study of proteasome inhibitors against Plasmodium falciparum laboratory strains and field isolates from Gabon

BackgroundThe emergence and spread of Plasmodium falciparum resistance to almost all available antimalarial drugs necessitates the search for new chemotherapeutic compounds. The ubiquitin/proteasome system plays a major role in overall protein turnover, especially in fast dividing eukaryotic cells including plasmodia. Previous studies show that the 20S proteasome is expressed and catalytically active in plasmodia and treatment with proteasome inhibitors arrests parasite growth. This is the first comprehensive screening of proteasome inhibitors with different chemical modes of action against laboratory strains of P. falciparum. Subsequently, a selection of inhibitors was tested in field isolates from Lambaréné, Gabon.MethodsEpoxomicin, YU101, YU102, MG132, MG115, Z-L3-VS, Ada-Ahx3-L3-VS, lactacystin, bortezomib (Velcade®), gliotoxin, PR11 and PR39 were tested and compared to chloroquine- and artesunate-activities in a standardized in vitro drug susceptibility assay against P. falciparum laboratory strains 3D7, D10 and Dd2. Freshly obtained field isolates from Lambaréné, Gabon, were used to measure the activity of chloroquine, artesunate, epoxomicin, MG132, lactacystin and bortezomib. Parasite growth was detected through histidine-rich protein 2 (HRP2) production. Raw data were fitted by a four-parameter logistic model and individual inhibitory concentrations (50%, 90%, and 99%) were calculated.ResultsAmongst all proteasome inhibitors tested, epoxomicin showed the highest activity in chloroquine-susceptible (IC50: 6.8 nM [3D7], 1.7 nM [D10]) and in chloroquine-resistant laboratory strains (IC50: 10.4 nM [Dd2]) as well as in field isolates (IC50: 8.5 nM). The comparator drug artesunate was even more active (IC50: 1.0 nM), whereas all strains were chloroquine-resistant (IC50: 113 nM).ConclusionThe peptide α',β'-epoxyketone epoxomicin is highly active against P. falciparum regardless the grade of the parasite's chloroquine susceptibility. Therefore, inhibition of the proteasome is a highly promising strategy to develop new antimalarials. Epoxomicin can serve as a standard to compare new inhibitors with species-specific activity.

Design, Synthesis, and Evaluation of 10-N-Substituted Acridones as Novel Chemosensitizers in Plasmodium falciparum

A series of novel 10-N-substituted acridones, bearing alkyl side chains with tertiary amine groups at the terminal position, were designed, synthesized, and evaluated for the ability to enhance the potency of quinoline drugs against multidrug-resistant (MDR) Plasmodium falciparum malaria parasites. A number of acridone derivatives, with side chains bridged three or more carbon atoms apart between the ring nitrogen and terminal nitrogen, demonstrated chloroquine (CQ)-chemosensitizing activity against the MDR strain of P. falciparum (Dd2). Isobologram analysis revealed that selected candidates demonstrated significant synergy with CQ in the CQ-resistant (CQR) parasite Dd2 but only additive (or indifferent) interaction in the CQ-sensitive (CQS) D6. These acridone derivatives also enhanced the sensitivity of other quinoline antimalarials, such as desethylchloroquine (DCQ) and quinine (QN), in Dd2. The patterns of chemosensitizing effects of selected acridones on CQ and QN were similar to those of verapamil against various parasite lines with mutations encoding amino acid 76 of the P. falciparum CQ resistance transporter (PfCRT). Unlike other known chemosensitizers with recognized psychotropic effects (e.g., desipramine, imipramine, and chlorpheniramine), these novel acridone derivatives exhibited no demonstrable effect on the uptake or binding of important biogenic amine neurotransmitters. The combined results indicate that 10-N-substituted acridones present novel pharmacophores for the development of chemosensitizers against P. falciparum.

Roxithromycin potentiates the effects of chloroquine and mefloquine on multidrug-resistant Plasmodium falciparum in vitro

Chloroquine (CQ) and mefloquine (MQ) are no longer potent antimalarial drugs due to the emergence of resistant Plasmodium falciparum. Combination therapy has become the standard for many regimes in overcoming drug resistance. Roxithromycin (ROM), a known p-glycoprotein inhibitor, is reported to have antimalarial activity and it is hoped it will potentiate the effects of both CQ/MQ and reverse CQ/MQ-resistance. We assayed the effects of CQ and MQ individually and in combination with ROM on synchronized P. falciparum (Dd2 strain) cultures. The IC 50 values of CQ and MQ were 60.0 ± 5.0 and 16.0 ± 3.0 ng/ml; these were decreased substantially when combined with ROM. Isobolograms indicate that CQ–ROM combinations were relatively more synergistic (mean FICI 0.70) than MQ–ROM (mean FICI 0.85) with their synergistic effect at par with CQ–verapamil (VRP) (mean FICI 0.64) and MQ–VRP (mean FICI 0.60) combinations. We conclude that ROM potentiates the CQ/MQ response on multidrug-resistant P. falciparum.

The course of infections and pathology in immunomodulated NOD/LtSz-SCID mice inoculated with Plasmodium falciparum laboratory lines and clinical isolates

Human chimeras are potentially invaluable models for hemoprotozoan parasites such as Plasmodium falciparum. The work presented assesses the susceptibility of immunomodulated NOD/LtSz-SCID mice to genetically distinct P. falciparum parasites. To this end, mice grafted with human erythrocytes were inoculated with two P. falciparum laboratory lines, 3D7 and Dd2 and four clinical isolates, ISCIII-230, ISCIII-231, ISCIII-381 and ISCIII-399. The results showed that, without a previous period of parasite adaptation, 100% of the inoculated mice developed an infection, generally self-limited, though some mice died. The parasitemias ranged from 0.05 to 8% and lasted an average of 19 days (15–26 days) depending on the line or isolate studied. Sexual forms of different maturity, stage II–IV and mature gametocytes were observed in the peripheral blood of mice in 22, 50, 25, 72 and 80% of the mice infected with Dd2, ISCIII-399, ISCIII-230, ISCIII-231 and ISCIII-381 isolates, respectively. The study of the clinical symptoms, the haematological parameters and the histopathological changes in the infected mice showed that most of the malaria features were present in the infected mice except that the sequestration of infected erythrocytes was absent or at most a minor phenomenon, as also indicated by the presence of mature forms of the parasites in the peripheral blood. This study shows that the human chimeras allow the complete asexual and sexual erythrocytic cycle of different P. falciparum lines and clinical isolates to be observed in vivo. It opens a new way to investigate any parasite population in terms of infectivity, transmission, and drug resistance.

Isolation and functional characterization of a dynamin-like gene from Plasmodium falciparum

A novel dynamin-like GTPase gene, Pfdyn1, was cloned from an asexual stage cDNA library of Plasmodium falciparum Dd2 strain. Pfdyn1 contains a highly conserved N-terminal tripartite GTPase domain, a coiled-coil region, and a C-terminal 129 aa unknown function domain. Like yeast Vps1p, it lacks pleckstrin homology domain and proline-rich region. Western blot analysis showed that Pfdyn1 is a Triton X-100 insoluble protein expressed only in the mature sub-stage. Morphological studies indicated that Pfdyn1 is partly co-localized with PfGRP, a known ER-resident protein, and localizes diffusely with several membrane structures and a 60–100 nm vesicle both inside and on surface of the parasites and also in the cytoplasm of infected erythrocytes. The dsRNA originated by C-terminus fragment of Pfdyn1 inhibits markedly the growth of P. falciparum parasite at the erythrocyte stage. Those data showed that Pfdyn1 is a conservative, membrane related protein and plays an essential role for the survival of Plasmodium parasite.

Regulation of intracellular glutathione levels in erythrocytes infected with chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum.

Malaria is one of the most devastating tropical diseases despite the availability of numerous drugs acting against the protozoan parasite Plasmodium in its human host. However, the development of drug resistance renders most of the existing drugs useless. In the malaria parasite the tripeptide glutathione is not only involved in maintaining an adequate intracellular redox environment and protecting the cell against oxidative stress, but it has also been shown that it degrades non-polymerized ferriprotoporphyrin IX (FP IX) and is thus implicated in the development of chloroquine resistance. Glutathione levels in Plasmodium -infected red blood cells are regulated by glutathione synthesis, glutathione reduction and glutathione efflux. Therefore the effects of drugs that interfere with these metabolic processes were studied to establish possible differences in the regulation of the glutathione metabolism of a chloroquine-sensitive and a chloroquine-resistant strain of Plasmodium falciparum. Growth inhibition of P. falciparum 3D7 by D,L-buthionine-( S, R )sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase (gamma-GCS), and by Methylene Blue (MB), an inhibitor of gluta thione reductase (GR), was significantly more pronounced than inhibition of P. falciparum Dd2 growth by these drugs. These results correlate with the higher levels of total glutathione in P. falciparum Dd2. Short-term incubations of Percoll-enriched trophozoite-infected red blood cells in the presence of BSO, MB and N, N (1)-bis(2-chloroethyl)- N -nitrosourea and subsequent determinations of gamma-GCS activities, GR activities and glutathione disulphide efflux revealed that maintenance of intracellular glutathione in P. falciparum Dd2 is mainly dependent on glutathione synthesis whereas in P. falciparum 3D7 it is regulated via GR. Generally, P. falciparum Dd2 appears to be able to sustain its intracellular glutathione more efficiently than P. falciparum 3D7. In agreement with these findings is the differential susceptibility to oxidative stress of both parasite strains elicited by the glucose/glucose oxidase system.

Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on­Acacia nilotica

Twenty-two plant organs from eleven plants comprising five families were extracted and screened for antiplasmodial activity in vitro against Plasmodium falciparum 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant and pyrimethamine sensitive). Fifty nine percent of plant extracts from 22 extracts exerted activity on P. falciparum strain 3D7 with an IC50 less than 50 µg/mL, whereas 43% of plant extracts showed an IC50 value within 50 µg/mL on Dd2 strains. Plant extracts from Gardenia lutea, Haplophyllum tuberculatum, Cassia tora, Acacia nilotica and Aristolochia bracteolata possessed IC50 values less than 5 µg/mL on both tested strains. Bioassay guided fractionation of A. nilotica revealed that the ethyl acetate extract possessed the highest activity (IC50 = 1.5 µg/mL). Fraction 2 (Rf = 0.75) prepared by preparative chromatography showed the highest activity on P. falciparum (IC50 = 1.7 µg/mL). Phytochemical analysis indicated that the most active phase contained terpenoids and tannins and was devoid of alkaloids and saponins. The effect of plant extracts on lymphocyte proliferation showed low toxicity to the human cells. This plant has been subjected to long term clinical trials in folk medicine and is a promising plant. Copyright © 1999 John Wiley & Sons, Ltd.

Antiplasmodial activity of selected sudanese medicinal plants with emphasis on Acacia nilotica.

Twenty-two plant organs from eleven plants comprising five families were extracted and screened for antiplasmodial activity in vitro against Plasmodium falciparum 3D7 (chloroquine sensitive) and Dd2 (chloroquine resistant and pyrimethamine sensitive). Fifty nine percent of plant extracts from 22 extracts exerted activity on P. falciparum strain 3D7 with an IC(50) less than 50 microg/mL, whereas 43% of plant extracts showed an IC(50) value within 50 microg/mL on Dd2 strains. Plant extracts from Gardenia lutea, Haplophyllum tuberculatum, Cassia tora, Acacia nilotica and Aristolochia bracteolata possessed IC(50) values less than 5 microg/mL on both tested strains. Bioassay guided fractionation of A. nilotica revealed that the ethyl acetate extract possessed the highest activity (IC(50) = 1.5 microg/mL). Fraction 2 (R(f) = 0.75) prepared by preparative chromatography showed the highest activity on P. falciparum (IC(50) = 1.7 microg/mL). Phytochemical analysis indicated that the most active phase contained terpenoids and tannins and was devoid of alkaloids and saponins. The effect of plant extracts on lymphocyte proliferation showed low toxicity to the human cells. This plant has been subjected to long term clinical trials in folk medicine and is a promising plant.

A developmental defect in Plasmodium falciparum male gametogenesis.

Asexually replicating populations of Plasmodium parasites, including those from cloned lines, generate both male and female gametes to complete the malaria life cycle through the mosquito. The generation of these sexual forms begins with the induction of gametocytes from haploid asexual stage parasites in the blood of the vertebrate host. The molecular processes that govern the differentiation and development of the sexual forms are largely unknown. Here we describe a defect that affects the development of competent male gametocytes from a mutant clone of P. falciparum (Dd2). Comparison of the Dd2 clone to the predecessor clone from which it was derived (W2'82) shows that the defect is a mutation that arose during the long-term cultivation of asexual stages in vitro. Light and electron microscopic images, and indirect immunofluorescence assays with male-specific anti-alpha-tubulin II antibodies, indicate a global disruption of male development at the gametocyte level with at least a 70-90% reduction in the proportion of mature male gametocytes by the Dd2 clone relative to W2'82. A high prevalence of abnormal gametocyte forms, frequently containing multiple and unusually large vacuoles, is associated with the defect. The reduced production of mature male gametocytes may reflect a problem in processes that commit a gametocyte to male development or a progressive attrition of viable male gametocytes during maturation. The defect is genetically linked to an almost complete absence of male gamete production and of infectivity to mosquitoes. This is the first sex-specific developmental mutation identified and characterized in Plasmodium.