Anti-plasmodial polyvalent interactions in Artemisia annua L. aqueous extract – possible synergistic and resistance mechanisms

Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L-1), dihydroartemisinic acid (70.0±0.3 mg L-1), arteannuin B (1.3±0.0 mg L-1), isovitexin (105.0±7.2 mg L-1) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B’s potentiation of artemisinin

Persistence of Plasmodium falciparum parasitemia after artemisinin combination therapy: evidence from a randomized trial in Uganda

Artemisinin resistance is rapidly spreading in Southeast Asia. The efficacy of artemisinin-combination therapy (ACT) continues to be excellent across Africa. We performed parasite transcriptional profiling and genotyping on samples from an antimalarial treatment trial in Uganda. We used qRT-PCR and genotyping to characterize residual circulating parasite populations after treatment with either ACT or ACT-primaquine. Transcripts suggestive of circulating ring stage parasites were present after treatment at a prevalence of >25% until at least 14 days post initiation of treatment. Greater than 98% of all ring stage parasites were cleared within the first 3 days, but subsequently persisted at low concentrations until day 14 after treatment. Genotyping demonstrated a significant decrease in multiplicity of infection within the first 2 days in both ACT and ACT-primaquine arms. However, multiple clone infections persisted until day 14 post treatment. Our data suggest the presence of genetically diverse persisting parasite populations after ACT treatment. Although we did not demonstrate clinical treatment failures after ACT and the viability and transmissibility of persisting ring stage parasites remain to be shown, these findings are of relevance for the interpretation of parasite clearance transmission dynamics and for monitoring drug effects in Plasmodium falciparum parasites

Ca2+ monitoring in Plasmodium falciparum using the yellow cameleon-Nano biosensor

Calcium (Ca2+)-mediated signaling is a conserved mechanism in eukaryotes, including the human malaria parasite, Plasmodium falciparum. Due to its small size (300?nM). We determined that the mammalian SERCA inhibitor thapsigargin and antimalarial dihydroartemisinin did not perturb SERCA activity. The change of the cytosolic Ca2+ level in P. falciparum was additionally detectable by flow cytometry. Thus, we propose that the developed YC-Nano-based system is useful to study Ca2+ signaling in P. falciparum and is applicable for drug screening.We are grateful to Japanese Red Cross Blood Society for providing human RBC and plasma. We also thank Tanaka R, Ogoshi (Sakura) M and Matsumoto N for technical assistance and Templeton TJ for critical reading. This study was conducted at the Joint Usage / Research Center on Tropical Disease, Institute of Tropical Medicine, Nagasaki University, Japan. KP was a Tokyo Biochemical Research Foundation (TBRF, http://www.tokyobrf.or.jp) post-doctoral fellow and PEF was a Japanese Society of Promotion Sciences (JSPS) post-doctoral fellow. This work was supported in part by the TBRF (K.P.), JSPS (P.E.F.), Takeda Science Foundation (K.Y.), Grants-in-Aids for Scientific Research 24590509 (K.Y.), 22390079 (O.K.), and for Scientific Research on Innovative Areas 23117008 (O.K.), MEXT, Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Evidence for the Contribution of the Hemozoin Synthesis Pathway of the Murine Plasmodium yoelii to the Resistance to Artemisinin-Related Drugs

Plasmodium falciparum malaria is a major global health problem, causing approximately 780,000 deaths each year. In response to the spreading of P. falciparum drug resistance, WHO recommended in 2001 to use artemisinin derivatives in combination with a partner drug (called ACT) as first-line treatment for uncomplicated falciparum malaria, and most malaria-endemic countries have since changed their treatment policies accordingly. Currently, ACT are often the last treatments that can effectively and rapidly cure P. falciparum infections permitting to significantly decrease the mortality and the morbidity due to malaria. However, alarming signs of emerging resistance to artemisinin derivatives along the Thai-Cambodian border are of major concern. Through long-term in vivo pressures, we have been able to select a murine malaria model resistant to artemisinins. We demonstrated that the resistance of Plasmodium to artemisinin-based compounds depends on alterations of heme metabolism and on a loss of hemozoin formation linked to the down-expression of the recently identified Heme Detoxification Protein (HDP). These artemisinins resistant strains could be able to detoxify the free heme by an alternative catabolism pathway involving glutathione (GSH)-mediation. Finally, we confirmed that artemisinins act also like quinolines against Plasmodium via hemozoin production inhibition. The work proposed here described the mechanism of action of this class of molecules and the resistance to artemisinins of this model. These results should help both to reinforce the artemisinins activity and avoid emergence and spread of endoperoxides resistance by focusing in adequate drug partners design. Such considerations appear crucial in the current context of early artemisinin resistance in Asia

Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies

Antimalarial chemotherapy, globally reliant on artemisinin-based combination therapies (ACTs), is threatened by the spread of drug resistance in Plasmodium falciparum parasites. Here we use zinc-finger nucleases to genetically modify the multidrug resistance-1 transporter PfMDR1 at amino acids 86 and 184, and demonstrate that the widely prevalent N86Y mutation augments resistance to the ACT partner drug amodiaquine and the former first-line agent chloroquine. In contrast, N86Y increases parasite susceptibility to the partner drugs lumefantrine and mefloquine, and the active artemisinin metabolite dihydroartemisinin. The PfMDR1 N86 plus Y184F isoform moderately reduces piperaquine potency in strains expressing an Asian/African variant of the chloroquine resistance transporter PfCRT. Mutations in both digestive vacuole-resident transporters are thought to differentially regulate ACT drug interactions with host haem, a product of parasite-mediated haemoglobin degradation. Global mapping of these mutations illustrates where the different ACTs could be selectively deployed to optimize treatment based on regional differences in PfMDR1 haplotypes.This work was funded in part by the National Institutes of Health (R01 AI50234, AI124678 and AI109023) and a Burroughs Wellcome Fund Investigator in Pathogenesis of Infectious Diseases award to D.A.F. This research also received funding from the Portuguese Fundacao para a Ciencia e Tecnologia (FCT), cofunded by Programa Operacional Regional do Norte (ON.2-O Novo Norte); from the Quadro de Referencia Estrategico Nacional (QREN) through the Fundo Europeu de Desenvolvimento Regional (FEDER) and from the Projeto Estrategico - LA 26 - 2013-2014 (PEst-C/SAU/LA0026/2013). M.I.V. is the recipient of a postdoctoral fellowship from FCT/Ministerio da Ciencia e Ensino Superior, Portugal-MCES (SFRH/BPD/76614/2011). A.M.L. was supported by an Australian National Health and Medical Research Council (NHMRC) Overseas Biomedical Fellowship (585519). R.E.M. was supported by an NHMRC RD Wright Biomedical Fellowship (1053082). A.C.U. was supported by an Irving scholarship from Columbia University. We thank Dr Andrea Ecker for her help with plasmid design and Pedro Ferreira for his expert help with Fig. 6.info:eu-repo/semantics/publishedVersio

Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4

Plasmodium vivax affects hundreds of millions each year and results in severe morbidity and mortality. Plasmodial cysteine proteases (CPs) play crucial roles during the progression of malaria since inhibition of these molecules impairs parasite growth. These CPs might be targeted for new antimalarial drugs. We characterized a novel P. vivax CP, vivapain-4 (VX-4), which appeared to evolve differentially among primate Plasmodium species. VX-4 showed highly unique substrate preference depending on surrounding micro-environmental pH. It effectively hydrolyzed benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA) and Z-Phe-Arg-MCA at acidic pH and Z-Arg-Arg-MCA at neutral pH. Three amino acids (Ala90, Gly157 and Glu180) that delineate the S2 pocket were found to be substituted in VX-4. Alteration of Glu180 abolished hydrolytic activity against Z-Arg-Arg-MCA at neutral pH, indicating Glu180 is intimately involved in the pH-dependent substrate preference. VX-4 hydrolyzed actin at neutral pH and hemoglobin at acidic pH, and participated in plasmepsin 4 activation at neutral/acidic pH. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. The differential substrate preferences depending on pH suggested a highly efficient mechanism to enlarge biological implications of VX-4, including hemoglobin degradation, maturation of plasmepsin, and remodeling of the parasite architecture during growth and development of P. vivax

The Activities of Current Antimalarial Drugs on the Life Cycle Stages of Plasmodium: A Comparative Study with Human and Rodent Parasites

Michael Delves and colleagues compare the activity of 50 current and experimental antimalarials against liver, sexual blood, and mosquito stages of selected human and nonhuman parasite species, including Plasmodium falciparum, Plasmodium berghei, and Plasmodium yoelii