Future antimalarials from Artemisia? A rationale for natural product mining against drug-refractory Plasmodium stages.

Covering: up to 2023Infusions of the plants Artemisia annua and A. afra are gaining broad popularity to prevent or treat malaria. There is an urgent need to address this controversial public health question by providing solid scientific evidence in relation to these uses. Infusions of either species were shown to inhibit the asexual blood stages, the liver stages including the hypnozoites, but also the sexual stages, the gametocytes, of Plasmodium parasites. Elimination of hypnozoites and sterilization of mature gametocytes remain pivotal elements of the radical cure of P. vivax, and the blockage of P. vivax and P. falciparum transmission, respectively. Drugs active against these stages are restricted to the 8-aminoquinolines primaquine and tafenoquine, a paucity worsened by their double dependence on the host genetic to elicit clinical activity without severe toxicity. Besides artemisinin, these Artemisia spp. contain many natural products effective against Plasmodium asexual blood stages, but their activity against hypnozoites and gametocytes was never investigated. In the context of important therapeutic issues, we provide a review addressing (i) the role of artemisinin in the bioactivity of these Artemisia infusions against specific parasite stages, i.e., alone or in association with other phytochemicals; (ii) the mechanisms of action and biological targets in Plasmodium of ca. 60 infusion-specific Artemisia phytochemicals, with an emphasis on drug-refractory parasite stages (i.e., hypnozoites and gametocytes). Our objective is to guide the strategic prospecting of antiplasmodial natural products from these Artemisia spp., paving the way toward novel antimalarial "hit" compounds either naturally occurring or Artemisia-inspired.

Artemisinin-independent inhibitory activity of Artemisia sp. infusions against different Plasmodium stages including relapse-causing hypnozoites.

Artemisinin-based combination therapies (ACT) are the frontline treatments against malaria worldwide. Recently the use of traditional infusions from Artemisia annua (from which artemisinin is obtained) or Artemisia afra (lacking artemisinin) has been controversially advocated. Such unregulated plant-based remedies are strongly discouraged as they might constitute sub-optimal therapies and promote drug resistance. Here, we conducted the first comparative study of the anti-malarial effects of both plant infusions in vitro against the asexual erythrocytic stages of Plasmodium falciparum and the pre-erythrocytic (i.e., liver) stages of various Plasmodium species. Low concentrations of either infusion accounted for significant inhibitory activities across every parasite species and stage studied. We show that these antiplasmodial effects were essentially artemisinin-independent and were additionally monitored by observations of the parasite apicoplast and mitochondrion. In particular, the infusions significantly incapacitated sporozoites, and for Plasmodium vivax and P. cynomolgi, disrupted the hypnozoites. This provides the first indication that compounds other than 8-aminoquinolines could be effective antimalarials against relapsing parasites. These observations advocate for further screening to uncover urgently needed novel antimalarial lead compounds.

Imaging of Plasmodium liver stages to drive next-generation antimalarial drug discovery.

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Isolation and antimalarial activity of new morphinan alkaloids on Plasmodium yoelii liver stage.

Decoction of Strychnopsis thouarsii is used in the Malagasy traditional medicine to combat malaria. We have shown that this traditional remedy prevents malaria infection by targeting Plasmodium at its early liver stage. Bioassay-guided fractionation of S. thouarsii stem barks extracts, using a rodent Plasmodium yoelii liver stage parasites inhibition assay, led to isolate the new morphinan alkaloid tazopsine (1) together with sinococuline (2) and two other new related morphinan analogs, 10-epi-tazopsine (3) and 10-epi-tazoside (4). Structures were characterized by 2D NMR, MS, and CD spectral analysis. Compounds 1-3 were found to fully inhibit the rodent P. yoelii liver stage parasites in vitro.

A plant-derived morphinan as a novel lead compound active against malaria liver stages.

BACKGROUND: The global spread of multidrug-resistant malaria parasites has led to an urgent need for new chemotherapeutic agents. Drug discovery is primarily directed to the asexual blood stages, and few drugs that are effective against the obligatory liver stages, from which the pathogenic blood infection is initiated, have become available since primaquine was deployed in the 1950s. METHODS AND FINDINGS: Using bioassay-guided fractionation based on the parasite's hepatic stage, we have isolated a novel morphinan alkaloid, tazopsine, from a plant traditionally used against malaria in Madagascar. This compound and readily obtained semisynthetic derivatives were tested for inhibitory activity against liver stage development in vitro (P. falciparum and P. yoelii) and in vivo (P. yoelii). Tazopsine fully inhibited the development of P. yoelii (50% inhibitory concentration [IC50] 3.1 muM, therapeutic index [TI] 14) and P. falciparum (IC50 4.2 muM, TI 7) hepatic parasites in cultured primary hepatocytes, with inhibition being most pronounced during the early developmental stages. One derivative, N-cyclopentyl-tazopsine (NCP-tazopsine), with similar inhibitory activity was selected for its lower toxicity (IC50 3.3 muM, TI 46, and IC50 42.4 muM, TI 60, on P. yoelii and P. falciparum hepatic stages in vitro, respectively). Oral administration of NCP-tazopsine completely protected mice from a sporozoite challenge. Unlike the parent molecule, the derivative was uniquely active against Plasmodium hepatic stages. CONCLUSIONS: A readily obtained semisynthetic derivative of a plant-derived compound, tazopsine, has been shown to be specifically active against the liver stage, but inactive against the blood forms of the malaria parasite. This unique specificity in an antimalarial drug severely restricts the pressure for the selection of drug resistance to a parasite stage limited both in numbers and duration, thus allowing researchers to envisage the incorporation of a true causal prophylactic in malaria control programs.

Submicroscopic Plasmodium falciparum infections before and after sulfadoxine-pyrimethamine and artesunate association treatment in Dienga, Southeastern Gabon.

BACKGROUND: It has been shown that Plasmodium falciparum submicroscopic infections (SMI) can contribute to malaria-associated anemia as well as to cerebral malaria. Polymerase chain reaction (PCR) assays are usually used as an alternative to microscopy in detecting subpatently infected individuals. OBJECTIVES: The main objective of this study was to investigate the occurrence of SMI before and after a suppressive antimalarial treatment in the population of the village of Dienga in Gabon. METHODS: Nested PCR was used to detect SMI and to determine genotypes. RESULTS: The prevalence rates of SMI were 13.67% (38/278) at day 0 and 8.99% (25/278) at day 14 after sulfadoxine-pyrimethamine-artesunate treatment. Genotype analysis of two polymorphic regions of the merozoite surface protein (MSP)-1 block 2, MSP-2 and a dimorphic region of the erythrocyte binding antigen (EBA-175) revealed that as many as 88% (22/25) of SMI detected after treatment were completely new alleles, indicating either previously sequestered parasites or newly acquired infections. CONCLUSION: These results demonstrate the usefulness of sulfadoxine-pyrimethamine-artesunate association treatment in the population of Dienga and confirmed early parasite genotype change after a suppressive antimalarial treatment in endemic areas.

Transient supplementation of superoxide dismutase protects endothelial cells against Plasmodium falciparum-induced oxidative stress.

The pathogenesis of cerebral malaria, a major complication of Plasmodium falciparum infection, relies on mechanisms such as cytokine production and cytoadherence of parasitized red blood cells (PRBCs) on microvascular endothelial cells. In this way parasites avoid spleen clearance by sequestration in post-capillary venules of various organs including the brain. Infected erythrocytes adhesion has also been shown to have molecular signaling consequences providing insight on how tissue homeostasis could be comprised by endothelium perturbation. Our previous work demonstrated that PRBCs adhesion to human lung endothelial cells (HLEC) induces caspases activation, oxidative stress and apoptosis. Cytoplasmic Cu/Zn superoxide dismutase (SOD1), which provides the first line of defense against oxidative stress within a cell, is now used as a treatment of numerous diseases including traumatic brain injury and ischemic stroke. In this report, we demonstrated that transient supplementation of SOD1 protects endothelial cells against P. falciparum induced oxidative stress and apoptosis. We also showed a significant decrease in PRBCs cytoadherence through a downregulation of ICAM-1 and an induction of iNOS. Protection of endothelium via antioxidant delivery may constitute a relevant strategy in cerebral malaria treatment.

Identification of new antimalarial drugs by linear discriminant analysis and topological virtual screening.

OBJECTIVES: A quantitative structure-activity relationship study using a database of 395 compounds previously tested against chloroquine-susceptible strains of the blood stages of Plasmodium falciparum to predict new in vitro antimalarial drugs has been developed. METHODS: Topological indices were used as structural descriptors and were related to antimalarial activity by using linear discriminant analysis (LDA) and multilinear regression (MLR). Two discriminant equations were obtained (FD1 and FD2), which allowed us to carry out successful classification of 90% and 80% of compounds, respectively. The IC50 values of the compounds were introduced to get an MLR equation model suitable to predict their in vitro activities. RESULTS: Using this model, a set of 27 drugs against a chloroquine-susceptible clone (3D7) of P. falciparum have been selected and evaluated in vitro. Among these drugs are monensin, nigericin, vincristine, vindesine, ethylhydrocupreine and salinomycin with in vitro IC50s at nanomolar concentrations (0.3, 0.4, 2, 6, 26 and 188 nM, respectively). Other compounds such as hycanthone, amsacrine, aphidicolin, bepridil, amiodarone, ranolazine and triclocarban showed in vitro IC50 values below 5 microM in the mathematical model. CONCLUSIONS: These results demonstrate the usefulness of the approach for the selection and design of new lead drugs active against P. falciparum.

Antioxidant and anti-inflammatory properties of a Cucumis melo LC. extract rich in superoxide dismutase activity.

The present study was conducted to evaluate in vitro and in vivo the antioxidant and anti-inflammatory properties of a cantaloupe melon (Cucumis melo LC., Cucurbitaceae) extract (CME) selected for its high superoxide dismutase activity. Peritoneal macrophages were pre-activated in vitro with 300 IU of interferon-gamma (IFN-gamma) and were then challenged in culture with IgGl/anti-IgG1 immune complexes (IgG1IC) in presence of various CME extracts. The subsequent production of free radicals (superoxide anion, nitric oxide, and peroxynitrite) and of pro-(TNF-alpha) and anti-(IL-10) inflammatory cytokines was evaluated. The CME inhibited in a dose-dependent manner the production of superoxide anion with a maximal effect at 100 microg/ml. This inhibitory effect of CME appeared to be closely linked to the SOD activity because it was dramatically decreased after heat inactivation of the SOD activity (HI-CME). In addition, the CME inhibited the production of peroxynitrite strengthening the antioxidant properties of this CME rich in SOD activity. The production of the pro- and anti-inflammatory cytokines, namely TNF-alpha and IL-10, being conditioned by the redox status of macrophages we also evaluated the effect of CME and HI-CME on the IgG1IC-induced cytokine production. When the SOD activity was present in the CME it promoted the IgG1IC-induced production of IL-10 instead of TNF-alpha. These data demonstrated that, in addition to its antioxidant properties, the anti-inflammatory properties of the CME extract were principally related to its capacity to induce the production of IL-10 by peritoneal macrophages. The particular properties of wheat gliadin (Triticum vulgare, Poaceae) for the oral delivery of functional proteins led us to test it in a new nutraceutical formula based on its combination with the CME thus monitoring the SOD activity release during the gastro-intestinal digestive process. In these experiments C57BL/6 mice were supplemented orally everyday during 28 days with: (1) the placebo, (2) the CME extract alone, (3) the gliadin, (4) the CME/gliadin combination, or (5) the HI-CME/gliadin combination (SOD inactivated). At the end of the supplementation period all the animals were injected intra-peritoneal (i.p.) with the pro-inflammatory cytokine IFN-gamma (300 IU) and peritoneal macrophages were harvested 24 h after to test their capacities to produce free radicals, TNF-alpha and IL-10 after triggering with IgG1IC. We demonstrated that animals supplemented during 28 days with the CME/gliadin combination were protected against the pro-inflammatory properties of IFN-gamma while the other products were inefficient. These data did not only indicate that the SOD activity is important for the antioxidant and anti-inflammatory properties of the CME extract, but also demonstrated that when the SOD activity is preserved during the digestive process by its combination with wheat gliadin it is possible to elicit in vivo the pharmacological effects of this antioxidant enzyme.

Supplementation with gliadin-combined plant superoxide dismutase extract promotes antioxidant defences and protects against oxidative stress.

The potential benefits to health of antioxidant enzymes supplied either through dietary intake or supplementation is still a matter of controversy. The development of dietary delivery systems using wheat gliadin biopolymers as a natural carrier represents a new alternative. Combination of antioxidant enzymes with this natural carrier not only delayed their degradation (i.e. the superoxide dismutase, SOD) during the gastrointestinal digestive process, but also promoted, in vivo, the cellular defences by strengthening the antioxidant status. The effects of supplementation for 28 days with a standardized melon SOD extract either combined (Glisodin) or not with gliadin, were evaluated on various oxidative-stress biomarkers. As already described there was no change either in superoxide dismutase, catalase or glutathione peroxidase activities in blood circulation or in the liver following non-protected SOD supplementation. However, animals supplemented with Glisodin showed a significant elevation in circulated antioxidant enzymes activities, correlated with an increased resistance of red blood cells to oxidative stress-induced hemolysis. In the presence of Sin-1, a chemical donor of peroxynitrites, mitochondria from hepatocytes regularly underwent membrane depolarization as the primary biological event of the apoptosis cascade. Hepatocytes isolated from animals supplemented with Glisodin presented a delayed depolarization response and an enhanced resistance to oxidative stress-induced apoptosis. It is concluded that supplementation with gliadin-combined standardized melon SOD extract (Glisodin) promoted the cellular antioxidant status and protected against oxidative stress-induced cell death.