In vitro inhibition of Plasmodium falciparum by pyrazofurin, an inhibitor of pyrimidine biosynthesis de novo

Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (δ-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.

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Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda

Nature Medicine ◽

10.1038/s41591-020-1005-2 ◽

2020 ◽

Vol 26 (10) ◽

pp. 1602-1608 ◽

Cited By ~ 28

Author(s):

Aline Uwimana ◽

Eric Legrand ◽

Barbara H. Stokes ◽

Jean-Louis Mangala Ndikumana ◽

Marian Warsame ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Plasmodium Falciparum ◽

Gene Editing ◽

De Novo ◽

Artemisinin Resistance ◽

Clonal Expansion ◽

Efficacy Trials ◽

Cure Rates ◽

Antimalarial Chemotherapy

Abstract Artemisinin resistance (delayed P. falciparum clearance following artemisinin-based combination therapy), is widespread across Southeast Asia but to date has not been reported in Africa1–4. Here we genotyped the P. falciparum K13 (Pfkelch13) propeller domain, mutations in which can mediate artemisinin resistance5,6, in pretreatment samples collected from recent dihydroarteminisin-piperaquine and artemether-lumefantrine efficacy trials in Rwanda7. While cure rates were >95% in both treatment arms, the Pfkelch13 R561H mutation was identified in 19 of 257 (7.4%) patients at Masaka. Phylogenetic analysis revealed the expansion of an indigenous R561H lineage. Gene editing confirmed that this mutation can drive artemisinin resistance in vitro. This study provides evidence for the de novo emergence of Pfkelch13-mediated artemisinin resistance in Rwanda, potentially compromising the continued success of antimalarial chemotherapy in Africa.

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MCT4 regulates de novo pyrimidine biosynthesis in GBM in a lactate-independent manner

Neuro-Oncology Advances ◽

10.1093/noajnl/vdz062 ◽

2020 ◽

Vol 2 (1) ◽

Author(s):

Raffaella Spina ◽

Dillon M Voss ◽

Xiaohua Yang ◽

Jason W Sohn ◽

Robert Vinkler ◽

...

Keyword(s):

De Novo ◽

Apoptotic Cell ◽

Pyrimidine Biosynthesis ◽

Monocarboxylate Transporter ◽

Independent Manner ◽

Pyrimidine Nucleosides ◽

Significant Prolongation ◽

Orthotopic Xenografts

Abstract Background Necrotic foci with surrounding hypoxic cellular pseudopalisades and microvascular hyperplasia are histological features found in glioblastoma (GBM). We have previously shown that monocarboxylate transporter 4 (MCT4) is highly expressed in necrotic/hypoxic regions in GBM and that increased levels of MCT4 are associated with worse clinical outcomes. Methods A combined transcriptomics and metabolomics analysis was performed to study the effects of MCT4 depletion in hypoxic GBM neurospheres. Stable and inducible MCT4-depletion systems were used to evaluate the effects of and underlining mechanisms associated with MCT4 depletion in vitro and in vivo, alone and in combination with radiation. Results This study establishes that conditional depletion of MCT4 profoundly impairs self-renewal and reduces the frequency and tumorigenicity of aggressive, therapy-resistant, glioblastoma stem cells. Mechanistically, we observed that MCT4 depletion induces anaplerotic glutaminolysis and abrogates de novo pyrimidine biosynthesis. The latter results in a dramatic increase in DNA damage and apoptotic cell death, phenotypes that were readily rescued by pyrimidine nucleosides supplementation. Consequently, we found that MCT4 depletion promoted a significant prolongation of survival of animals bearing established orthotopic xenografts, an effect that was extended by adjuvant treatment with focused radiation. Conclusions Our findings establish a novel role for MCT4 as a critical regulator of cellular deoxyribonucleotide levels and provide a new therapeutic direction related to MCT4 depletion in GBM.

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Cytometric measurement of in vitro inhibition of Plasmodium falciparum field isolates by drugs: a new approach for re-invasion inhibition study

Malaria Journal ◽

10.1186/1475-2875-13-110 ◽

2014 ◽

Vol 13 (1) ◽

pp. 110 ◽

Cited By ~ 2

Author(s):

Marie Varela ◽

Romy Razakandrainibe ◽

Delphine Aldebert ◽

Jean Barale ◽

Ronan Jambou

Keyword(s):

Plasmodium Falciparum ◽

Inhibition Study ◽

New Approach ◽

Field Isolates ◽

In Vitro Inhibition ◽

Invasion Inhibition

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Biosynthesis of GDP-fucose and Other Sugar Nucleotides in the Blood Stages of Plasmodium falciparum

Journal of Biological Chemistry ◽

10.1074/jbc.m112.439828 ◽

2013 ◽

Vol 288 (23) ◽

pp. 16506-16517 ◽

Cited By ~ 27

Author(s):

Sílvia Sanz ◽

Giulia Bandini ◽

Diego Ospina ◽

Maria Bernabeu ◽

Karina Mariño ◽

...

Keyword(s):

Plasmodium Falciparum ◽

De Novo ◽

Cell Communication ◽

Developmental Cycle ◽

Metabolic Labeling ◽

Sugar Nucleotides ◽

Liquid Chromatography Tandem Mass ◽

Intraerythrocytic Developmental Cycle

Carbohydrate structures play important roles in many biological processes, including cell adhesion, cell-cell communication, and host-pathogen interactions. Sugar nucleotides are activated forms of sugars used by the cell as donors for most glycosylation reactions. Using a liquid chromatography-tandem mass spectrometry-based method, we identified and quantified the pools of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, GDP-mannose, and GDP-fucose in Plasmodium falciparum intraerythrocytic life stages. We assembled these data with the in silico functional reconstruction of the parasite metabolic pathways obtained from the P. falciparum annotated genome, exposing new active biosynthetic routes crucial for further glycosylation reactions. Fucose is a sugar present in glycoconjugates often associated with recognition and adhesion events. Thus, the GDP-fucose precursor is essential in a wide variety of organisms. P. falciparum presents homologues of GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase enzymes that are active in vitro, indicating that most GDP-fucose is formed by a de novo pathway that involves the bioconversion of GDP-mannose. Homologues for enzymes involved in a fucose salvage pathway are apparently absent in the P. falciparum genome. This is in agreement with in vivo metabolic labeling experiments showing that fucose is not significantly incorporated by the parasite. Fluorescence microscopy of epitope-tagged versions of P. falciparum GDP-mannose 4,6-dehydratase and GDP-l-fucose synthase expressed in transgenic 3D7 parasites shows that these enzymes localize in the cytoplasm of P. falciparum during the intraerythrocytic developmental cycle. Although the function of fucose in the parasite is not known, the presence of GDP-fucose suggests that the metabolite may be used for further fucosylation reactions.

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Effect of deoxycytidine on the in vitro response of human leukemia cells to inhibitors of de novo pyrimidine biosynthesis

Cancer Chemotherapy and Pharmacology ◽

10.1007/bf00252977 ◽

1987 ◽

Vol 19 (3) ◽

Cited By ~ 2

Author(s):

Kapil Bhalla ◽

Steven Grant

Keyword(s):

De Novo ◽

Pyrimidine Biosynthesis ◽

Leukemia Cells ◽

Human Leukemia ◽

Human Leukemia Cells ◽

In Vitro Response

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Plants of the Araceae family for malaria and related diseases: a review

Revista Brasileira de Plantas Medicinais ◽

10.1590/1983-084x/14_024 ◽

2015 ◽

Vol 17 (4) ◽

pp. 657-666 ◽

Cited By ~ 3

Author(s):

G. FRAUSIN ◽

R. B. S. LIMA ◽

A. F. HIDALGO ◽

L. C. MING ◽

A.M. POHLIT

Keyword(s):

Plasmodium Falciparum ◽

Malaria Parasite ◽

Preparation Method ◽

Antimalarial Activity ◽

Composition Studies ◽

In Vitro Growth ◽

Human Malaria Parasite ◽

Google Books ◽

In Vitro Inhibition

ABSTRACTIn the current work we performed a review of the Araceae family species traditionally used to treat malaria and its symptoms. The aim is to reveal the large number of antimalarial Araceae species used worldwide and their great unexplored potential as sources of antimalarial natural products. The SciFinder Scholar, Scielo, PubMed, ScienceDirect and Google books search engines were consulted. Forty-three records of 36 species and 23 genera of Araceae used for malaria and symptoms treatment were found. The neotropical genera Philodendron Schott and Anthurium Schott were the best represented for the use in the treatment of malaria, fevers, liver problems and headaches. Leaves and tubers were the most used parts and decoction was the most common preparation method. The extracts of Araceae species inhibit the in vitro growth of the human malaria parasite, the Plasmodium falciparum Welch, and significant median inhibitory concentrations (IC50) for extracts of guaimbê-sulcado (Rhaphidophora decursiva (Roxb.) Schott), aninga (Montrichardia linifera (Arruda) Schott), Culcasia lancifolia N.E. Br. and forest anchomanes (Anchomanes difformis (Blume) Engl.) have been reported demonstrating the antimalarial and cytotoxicity potential of the extracts and sub-fractions. In the only report about the antimalarial components of this family, the neolignan polysyphorin and the benzoperoxide rhaphidecurperoxin presented strong in vitro inhibition of the D6 and W2 strains of Plasmodiumfalciparum (IC50 = 368-540 ng/mL). No live study about antimalarial activity in animal models has been conducted on a species of Araceae. More bioguided chemical composition studies about the in vitro and also thein vivo antimalarial activity of the Araceae are needed in order to enhance the knowledge about the antimalarial potential of this family.

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