Adenosine Triphosphate-Binding Cassette Transporters Are Not Involved In the Detoxification of Azadirachta indica Extracts In Anopheles stephensi Larvae.

Detoxifying pathways of mosquitoes against the neem (Azadirachta indica) extracts are still unclear. The aim of the present study was to investigate the role of adenosine triphosphate-binding cassette (ABC) transporters in this process in Anopheles stephensi, one of the main malaria vectors in southern Asia. Third-stage larvae of An. stephensi were fed with fish food alone or in combination with neem extract at 0.5%, 1%, 5%, and 10%. Six ABC-transporter genes from 3 different subfamilies (B, C, and G) were analyzed to assess their relative expression compared with controls. A bioassay was also performed to assess larval mortality rate at different concentrations and in combination with verapamil, an ABC-transporter inhibitor. No significant variation in the expression levels of any transporter belonging to the B, C, and G subfamilies was detected. Furthermore, the use of verapamil did not induce an increase in mortality at any of the tested neem extract concentrations, indicating that ABC transporters are not involved in the detoxification of neem extracts in An. stephensi larvae.

Open Source Drug Discovery with the Malaria Box Compound Collection for Neglected Diseases and Beyond.

A major cause of the paucity of new starting points for drug discovery is the lack of interaction between academia and industry. Much of the global resource in biology is present in universities, whereas the focus of medicinal chemistry is still largely within industry. Open source drug discovery, with sharing of information, is clearly a first step towards overcoming this gap. But the interface could especially be bridged through a scale-up of open sharing of physical compounds, which would accelerate the finding of new starting points for drug discovery. The Medicines for Malaria Venture Malaria Box is a collection of over 400 compounds representing families of structures identified in phenotypic screens of pharmaceutical and academic libraries against the Plasmodium falciparum malaria parasite. The set has now been distributed to almost 200 research groups globally in the last two years, with the only stipulation that information from the screens is deposited in the public domain. This paper reports for the first time on 236 screens that have been carried out against the Malaria Box and compares these results with 55 assays that were previously published, in a format that allows a meta-analysis of the combined dataset. The combined biochemical and cellular assays presented here suggest mechanisms of action for 135 (34%) of the compounds active in killing multiple life-cycle stages of the malaria parasite, including asexual blood, liver, gametocyte, gametes and insect ookinete stages. In addition, many compounds demonstrated activity against other pathogens, showing hits in assays with 16 protozoa, 7 helminths, 9 bacterial and mycobacterial species, the dengue fever mosquito vector, and the NCI60 human cancer cell line panel of 60 human tumor cell lines. Toxicological, pharmacokinetic and metabolic properties were collected on all the compounds, assisting in the selection of the most promising candidates for murine proof-of-concept experiments and medicinal chemistry programs. The data for all of these assays are presented and analyzed to show how outstanding leads for many indications can be selected. These results reveal the immense potential for translating the dispersed expertise in biological assays involving human pathogens into drug discovery starting points, by providing open access to new families of molecules, and emphasize how a small additional investment made to help acquire and distribute compounds, and sharing the data, can catalyze drug discovery for dozens of different indications. Another lesson is that when multiple screens from different groups are run on the same library, results can be integrated quickly to select the most valuable starting points for subsequent medicinal chemistry efforts.

A chemical susceptibility profile of the Plasmodium falciparum transmission stages by complementary cell-based gametocyte assays.

OBJECTIVES: As most available antimalarial drugs are ineffective against the Plasmodium falciparum transmission stages, new drugs against the parasite's gametocytes are urgently needed to combat malaria globally. The unique biology of gametocytes requires assays that need to be specific, to faithfully monitor anti-gametocyte activity, and to be easy to perform, cheap and scalable to high-throughput screening (HTS). METHODS: We developed an HTS cell-based assay with P. falciparum gametocytes specifically expressing a potent luciferase. To confirm HTS hit activity for several parasite genotypes, the luciferase assay and the gametocyte lactate dehydrogenase (LDH) assay, usable on any parasite isolate, were compared by screening antimalarial drugs and determining IC50 values of anti-gametocyte hits from the 'Malaria Box' against early- and late-stage gametocytes. RESULTS: Comparison of the two assays, conducted on the early and on late gametocyte stages, revealed an excellent correlation (R(2) > 0.9) for the IC50 values obtained by the respective readouts. Differences in susceptibility to drugs and compounds between the two parasite developmental stages were consistently measured in both assays. CONCLUSIONS: This work indicates that the luciferase and gametocyte LDH assays are interchangeable and that their specific advantages can be exploited to design an HTS pipeline leading to new transmission-blocking compounds. Results from these assays consistently defined a gametocyte chemical susceptibility profile, relevant to the planning of future drug discovery strategies.

A Plasmodium falciparum screening assay for anti-gametocyte drugs based on parasite lactate dehydrogenase detection.

OBJECTIVES: Plasmodium gametocytes, responsible for malaria parasite transmission from humans to mosquitoes, represent a crucial target for new antimalarial drugs to achieve malaria elimination/eradication. We developed a novel colorimetric screening method for anti-gametocyte compounds based on the parasite lactate dehydrogenase (pLDH) assay, already standardized for asexual stages, to measure gametocyte viability and drug susceptibility. METHODS: Gametocytogenesis of 3D7 and NF54 Plasmodium falciparum strains was induced in vitro and asexual parasites were depleted with N-acetylglucosamine. Gametocytes were treated with dihydroartemisinin, epoxomicin, methylene blue, primaquine, puromycin or chloroquine in 96-well plates and the pLDH activity was evaluated using a modified Makler protocol. Mosquito infectivity was measured by the standard membrane feeding assay (SMFA). RESULTS: A linear correlation was found between gametocytaemia determined by Giemsa staining and pLDH activity. A concentration-dependent reduction in pLDH activity was observed after 72 h of drug treatment, whereas an additional 72 h of incubation without drugs was required to obtain complete inhibition of gametocyte viability. SMFA on treated and control gametocytes confirmed that a reduction in pLDH activity translates into reduced oocyst development in the mosquito vector. CONCLUSIONS: The gametocyte pLDH assay is fast, easy to perform, cheap and reproducible and is suitable for screening novel transmission-blocking compounds, which does not require parasite transgenic lines.

Antiplasmodial and anti-inflammatory activities of Canthium henriquesianum (K. Schum), a plant used in traditional medicine in Burkina Faso.

ETHNOPHARMACOLOGICAL RELEVANCE: Canthium henriquesianum (K. Schum) is traditionally used in Burkina Faso for the treatment of malaria, but has not been properly investigated, yet. The aim of this study was to characterize in vitro the antiplasmodial and the anti-inflammatory activity of extracts from Canthium henriquesianum (K. Schum). In parallel, extracts of Gardenia sokotensis (Hutch) and Vernonia colorata (Willd), also traditionally used together in Burkina Faso and already reported with antimalarial activity, were compared. MATERIALS AND METHODS: Plant extracts were tested in vitro for antimalarial activity against chloroquine susceptible (D10) and resistant (W2) strains of Plasmodium falciparum using the lactate dehydrogenase assay. Cell cytotoxicity was assessed on human dermal fibroblast (HDF) by the MTT assay. The selectivity index (SI) was used as the ratio of the activity against the parasites compared to the toxicity of the plant extract against HDF. In vitro cytokine production was assessed by ELISA technique. RESULTS: Canthium henriquesianum aqueous extract had a moderate antimalarial activity (IC50<50 µg/ml) with a good selectivity index (SI=HDF/D10>7). Canthium henriquesianum diisopropyl ether extract was the most potent inhibitor of parasite growth with an IC50 9.5 µg/ml on W2 and 8.8 µg/ml on D10 and limited toxicity (SI>2). Gardenia sokotensis and Vernonia colorata aqueous extracts were shown to be significantly less active (IC50≥50 µg/ml) with substantial toxicity. In addition, when the production of IL-1ß and TNFα by lipopolysaccharide (LPS) or hemozoin (malaria pigment) stimulated human THP-1 monocytes was assayed, it was found that the extract of Canthium henriquesianum induced a dose-dependent inhibition of IL-1ß, but not of TNFα production, thus confirming its traditional use as antipyretic. By NMR analysis, the chromone was identified as the mostly represented compound in the diisopropyl ether extract of Canthium henriquesianum. Chromone however, was less active as antimalarial than the crude extract and it did not inhibit cytokine production at not toxic doses, indicating that other molecules in the total extracts contribute to the antiplasmodial and anti-inflammatory activity. CONCLUSION: Canthium henriquesianum seems to possess antimalarial activity in vitro and the ability to inhibit the production of the pyrogenic cytokine IL-1ß.

Antiplasmodial activity of Punica granatum L. fruit rind.

AIM OF THE STUDY: Sun-dried rind of the immature fruit of Punica granatum L. (Punicaceae) (Pg) is presently used as a herbal formulation (OMARIA) in Orissa, India, for the therapy and prophylaxis of malaria. The aims of this study were (i) to assess in vitro the antiplasmodial activity of the methanolic extract, of a tannin enriched fraction and of compounds/metabolites of the antimalarial plant, (ii) to estimate the curative efficacy of the Pg extracts and (iii) to explore the mechanism of action of the antiplasmodial compounds. Urolithins, the ellagitannin metabolites, were also investigated for antiplasmodial activity. MATERIALS AND METHODS: Chloroquine-susceptible (D10) and -resistant (W2) strains of Pf were used for in vitro studies and the rodent malaria model Plasmodium berghei-BALB/c mice was used for in vivo assessments. Recombinant plasmepsins 2 and 4 were used to investigate the interference of Pg compounds with the metabolism of haemoglobin by malaria parasites. RESULTS: The Pg methanolic extract (Pg-MeOH) inhibited parasite growth in vitro with a IC(50) of 4.5 and 2.8 microg/ml, for D10 and W2 strain, respectively. The activity was found to be associated to the fraction enriched with tannins (Pg-FET, IC(50) 2.9 and 1.5 microg/ml) in which punicalagins (29.1%), punicalins, ellagic acid (13.4%) and its glycoside could be identified. Plasmepsin 2 was inhibited by Pg-MeOH extract and by Pg-FET (IC(50) 7.3 and 3.0 microg/ml), which could partly explain the antiparasitic effect. On the contrary, urolithins were inactive. Both Pg-MeOH extract and Pg-FET did not show any in vivo efficacy in the murine model. CONCLUSIONS: The in vitro studies support the use of Pg as antimalarial remedy. Possible explanations for the negative in vivo results are discussed.

A novel DNA-based microfluorimetric method to evaluate antimalarial drug activity.

This paper describes the development of a novel microfluorimetric assay to measure the inhibition of Plasmodium falciparum based on the detection of parasitic DNA by intercalation with PicoGreen. The method was used to determine parasite inhibition profiles and 50% inhibitory concentration values of known or potential antimalarial drugs. Values for parasite inhibition with known anti-malarial drugs using the PicoGreen assay were comparable with those determined by the standard method based upon the uptake of 3H-hypoxanthine and the Giemsa stain microscopic technique. The PicoGreen assay is rapid, sensitive, reproducible, easily interpreted, and ideally suited for screening of large numbers of samples for anti-malarial drug development.