Polyamine uptake in the malaria parasite, Plasmodium falciparum, is dependent on the parasite's membrane potential

Polyamines are present at high levels in proliferating cells, including cancerous cells and protozoan parasites and the inhibition of their synthesis has been exploited in antiproliferative strategies. Inhibition of the malaria parasite’s polyamine biosynthetic pathway causes cytostatic arrest in the trophozoite stage but does not cure in vivo infections in the murine model of malaria. This is possibly due to exogenous polyamine salvage from the host, which replenishes the intracellular polyamine pool. This implies that disruption of polyamine metabolism as an antimalarial chemotherapy strategy may require targeting both polyamine biosynthesis and transport simultaneously

Discovering new transmission-blocking antimalarial compounds : challenges and opportunities

The ability to target human-mosquito parasite transmission challenges global malaria elimination. However, it is not obvious what a transmission-blocking drug will look like; should it 1) target only parasite transmission stages; 2) be combined with a partner drug killing the pathogenic asexual stages or 3) kill both the sexual and asexual blood stages, preferably displaying polypharmacology. The development of transmission-blocking anti-malarials requires objective analyses of the current strategies. Here, pertinent issues and unanswered questions regarding the target candidate profile of a transmission-blocking compound, and its role in malaria elimination strategies are highlighted and novel perspectives proposed. The essential role of a test cascade that integrates screening and validation strategies to identify next generation transmission-blocking anti-malarials is emphasised.Koen Dechering (TROPIQ, The Netherlands), Francesco Silvestrini (Istituto Superiore di Sanità, Rome, Italy), Sarah D’Alessandro and Donatella Taramelli (University of Milan, Italy), Robert Sauerwein (University of Nijmegen, The Netherlands) and Omar Vandal (The Bill & Melinda Gates Foundation) are acknowledged for their contributions towards the screening cascade, which they developed in the course of the Bill and Melinda Gates Foundation Project OPP1040394 ‘Gametocyte Assays for Malaria (GAM) for novel transmission blocking drugs’, coordinated by PA. The South African Transmission-blocking Consortium is funded by the Medical Research Council of South Africa as a Strategic Health Innovation Partnership (MRC SHIP) project.http://www.journals.elsevier.com/trends-in-parasitology2017-09-30hb2016Biochemistr

The ecdysone receptor regulates several key physiological factors in Anopheles funestus

BACKGROUND : Malaria is a devastating disease, transmitted by female Anopheles mosquitoes infected with Plasmodium parasites. Current insecticide-based strategies exist to control the spread of malaria by targeting vectors. However, the increase in insecticide resistance in vector populations hinder the efficacy of these methods. It is, therefore, essential to develop novel vector control methods that efficiently target transmission reducing factors such as vector density and competence. A possible vector control candidate gene, the ecdysone receptor, regulates longevity, reproduction, immunity and other physiological processes in several insects, including malaria vectors. Anopheles funestus is a prominent vector in sub-Saharan Africa, however, the function of the ecdysone receptor in this mosquito has not previously been studied. This study aimed to determine if the ecdysone receptor depletion impacts An. funestus longevity, reproduction and susceptibility to Plasmodium falciparum infection. METHODS : RNA interference was used to reduce ecdysone receptor expression levels in An. funestus females and investigate how the above-mentioned phenotypes are influenced. Additionally, the expression levels of the ecdysone receptor, and reproduction genes lipophorin and vitellogenin receptor as well as the immune gene, leucine rich immune molecule 9 were determined in ecdysone receptor-depleted mosquitoes using quantitative polymerase chain reaction. RESULTS : Ecdysone receptor-depleted mosquitoes had a shorter lifespan, impaired oogenesis, were less fertile, and had reduced P. falciparum infection intensity. CONCLUSIONS : Overall, this study provides the first experimental evidence that supports ecdysone receptor as a potential target in the development of vector control measures targeting An. funestus.Additional file 1: Figure S1. Relative EcR expression levels in dsEcR injected An. funestus females compared to dsGFP injected An. funestus females. The EcR gene was knocked down in dsEcR injected An. funestus females as EcR expression levels were drastically reduced compared to the GFP control. Statistically significant knockdown was evident in dsEcR injected An. funestus females 24, 48 and 72 h after injection as EcR expression in dsEcR injected An. funestus females was 0.11 ± 0.006 (p < 0.05), 0.01 ± 0.001 (p < 0.01) and 0.2 ± 0.06 (p < 0.05) respectively when compared to the GFP injected control of 1. This data confirmed EcR knockdown in An. funestus females injected with dsEcR. Data is representative of 2 biological replicates and normalised using an average of RPS7 and RPL19 reference genes. Expression levels calculated using relative quantification method (∆∆Ct). At each time point statistical significance was assessed with the unpaired student’s t-test. *p < 0.05, **p < 0.01. Error bars represent standard deviation.Additional file 2: Figure S2. The highest mating success rate was achieved when An. funestus males and females are combined for 12 days after which no further increases are observed. The percentage mating success rate increased progressively until it reached its highest value of 62.2% after 12 days of mating. After this point, the mating success rate reached a plateau until day 20. Statistical significance was calculated using one-way ANOVA with Tukey’s post hoc analysis to correct for multiple comparison. Data represents the means of 3 biological replicates. Error bars represent standard deviation of means. ns = not statistically significant p > 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001. (n) = number of females per time point across 3 biological replicates.Additional file 3: Figure S3. Blood feeding rates did not differ between treatment groups. Insignificant differences amongst treatment groups confirmed that the blood feeding rates did not influence any changes observed in the phenotypes of dsEcR treated An. funestus females (p > 0.05). Statistical significance calculated using an unpaired student’s t-test. Error bars represent standard deviation. ns = not statistically significant p > 0.05.Additional file 4: Table S1. Statistical significance between dsGFP and uninjected controls from the various biological assays conducted.The Department of Science and Innovation (DSI); the National Research Foundation (NRF) South African Research Chairs Initiative; the NRF Communities of Practice and the South African Medical Research Council Strategic Health Innovation Partnerships (SHIP) with funds from DSI.https://malariajournal.biomedcentral.comhj2022BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC

Heterologous expression of plasmodial proteins for structural studies and functional annotation

Malaria remains the world's most devastating tropical infectious disease with as many as 40% of the world population living in risk areas. The widespread resistance of Plasmodium parasites to the cost-effective chloroquine and antifolates has forced the introduction of more costly drug combinations, such as Coartem®. In the absence of a vaccine in the foreseeable future, one strategy to address the growing malaria problem is to identify and characterize new and durable antimalarial drug targets, the majority of which are parasite proteins. Biochemical and structure-activity analysis of these proteins is ultimately essential in the characterization of such targets but requires large amounts of functional protein. Even though heterologous protein production has now become a relatively routine endeavour for most proteins of diverse origins, the functional expression of soluble plasmodial proteins is highly problematic and slows the progress of antimalarial drug target discovery. Here the status quo of heterologous production of plasmodial proteins is presented, constraints are highlighted and alternative strategies and hosts for functional expression and annotation of plasmodial proteins are reviewed

Identification of 2,4-Disubstituted Imidazopyridines as Hemozoin Formation Inhibitors with Fast-Killing Kinetics and In Vivo Efficacy in the Plasmodium falciparum NSG Mouse Model

A series of 2,4-disubstituted imidazopyridines, originating from a SoftFocus Kinase library, was identified from a high throughput phenotypic screen against the human malaria parasite Plasmodium falciparum. Hit compounds showed moderate asexual blood stage activity. During lead optimization, several issues were flagged such as cross-resistance against the multidrug-resistant K1 strain, in vitro cytotoxicity, and cardiotoxicity and were addressed through structure–activity and structure–property relationship studies. Pharmacokinetic properties were assessed in mice for compounds showing desirable in vitro activity, a selectivity window over cytotoxicity, and microsomal metabolic stability. Frontrunner compound 37 showed good exposure in mice combined with good in vitro activity against the malaria parasite, which translated into in vivo efficacy in the P. falciparum NOD-scid IL-2Rγnull (NSG) mouse model. Preliminary mechanistic studies suggest inhibition of hemozoin formation as a contributing mode of action

Antimalarial and antitumour activities of the steroidal quinone-methide celastrol and its combinations with artemiside, artemisone and methylene blue

Artemisinin, isolated from the traditional Chinese medicinal plant qīng hāo 青蒿 (Artemisia annua) and its derivatives are used for treatment of malaria. With treatment failures now being recorded for the derivatives and companion drugs used in artemisinin combination therapies new drug combinations are urgently required. The amino-artemisinins artemiside and artemisone display optimal efficacies in vitro against asexual and sexual blood stages of the malaria parasite Plasmodium falciparum and are active against tumour cell lines. In continuing the evolution of combinations of the amino-artemisinins with new drugs, we examine the triterpenoid quinone methide celastrol isolated from the traditional Chinese medicinal plant léi gōng téng 雷公藤 (Tripterygium wilfordii). This compound is redox active, and has attracted considerable attention because of potent biological activities against manifold targets. We report that celastrol displays good IC50 activities ranging from 0.50–0.82 µM against drug-sensitive and resistant asexual blood stage Pf, and 1.16 and 0.28 µM respectively against immature and late stage Pf NF54 gametocytes. The combinations of celastrol with each of artemisone and methylene blue against asexual blood stage Pf are additive. Given that celastrol displays promising antitumour properties, we examined its activities alone and in combinations with amino-artemisinins against human liver HepG2 and other cell lines. IC50 values of the aminoartemisinins and celastrol against HepG2 cancer cells ranged from 0.55–0.94 µM. Whereas the amino-artemisinins displayed notable selectivities (SI > 171) with respect to normal human hepatocytes, in contrast, celastrol displayed no selectivity (SI < 1). The combinations of celastrol with artemiside or artemisone against HepG2 cells are synergistic. Given the promise of celastrol, judiciously designed formulations or structural modifications are recommended for mitigating its toxicity.https://www.frontiersin.org/journals/pharmacologyBiochemistr

Discovery: an interactive resource for the rational selection and comparison of putative drug target proteins in malaria

<p>Abstract</p> <p>Background</p> <p>Up to half a billion human clinical cases of malaria are reported each year, resulting in about 2.7 million deaths, most of which occur in sub-Saharan Africa. Due to the over-and misuse of anti-malarials, widespread resistance to all the known drugs is increasing at an alarming rate. Rational methods to select new drug target proteins and lead compounds are urgently needed. The Discovery system provides data mining functionality on extensive annotations of five malaria species together with the human and mosquito hosts, enabling the selection of new targets based on multiple protein and ligand properties.</p> <p>Methods</p> <p>A web-based system was developed where researchers are able to mine information on malaria proteins and predicted ligands, as well as perform comparisons to the human and mosquito host characteristics. Protein features used include: domains, motifs, EC numbers, GO terms, orthologs, protein-protein interactions, protein-ligand interactions and host-pathogen interactions among others. Searching by chemical structure is also available.</p> <p>Results</p> <p>An <it>in silico</it> system for the selection of putative drug targets and lead compounds is presented, together with an example study on the bifunctional DHFR-TS from <it>Plasmodium falciparum</it>.</p> <p>Conclusion</p> <p>The Discovery system allows for the identification of putative drug targets and lead compounds in Plasmodium species based on the filtering of protein and chemical properties.</p

Nowhere to hide : interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination

BACKGROUND : The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions. METHODS : Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable. RESULTS : A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4 %. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident. CONCLUSIONS : With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broaddiversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.South African Medical Research Council Strategic Health Initiatives Partnerships with the Medicines for Malaria Venture as well as the Council for Scientific and Industrial Research, and the 3R Foundation (project 118–10).http://www.malariajournal.comhb201