43 research outputs found
Validation of Plasmodium falciparum dUTPase as the target of 5'-tritylated deoxyuridine analogues with anti-malarial activity
BACKGROUND: Malaria remains as a major global problem, being one of the infectious diseases that engender highest mortality across the world. Due to the appearance of resistance and the lack of an effective vaccine, the search of novel anti-malarials is required. Deoxyuridine 5'-triphosphate nucleotido-hydrolase (dUTPase) is responsible for the hydrolysis of dUTP to dUMP within the parasite and has been proposed as an essential step in pyrimidine metabolism by providing dUMP for thymidylate biosynthesis. In this work, efforts to validate dUTPase as a drug target in Plasmodium falciparum are reported. METHODS: To investigate the role of PfdUTPase in cell survival different strategies to generate knockout mutants were used. For validation of PfdUTPase as the intracellular target of four inhibitors of the enzyme, mutants overexpressing PfdUTPase and HsdUTPase were created and the IC50 for each cell line with each compound was determined. The effect of these compounds on dUTP and dTTP levels from P. falciparum was measured using a DNA polymerase assay. Detailed localization studies by indirect immunofluorescence microscopy and live cell imaging were also performed using a cell line overexpressing a Pfdut-GFP fusion protein. RESULTS:Different attempts of disruption of the dut gene of P. falciparum were unsuccessful while a 3' replacement construct could recombine correctly in the locus suggesting that the enzyme is essential. The four 5'-tritylated deoxyuridine analogues described are potent inhibitors of the P. falciparum dUTPase and exhibit antiplasmodial activity. Overexpression of the Plasmodium and human enzymes conferred resistance against selective compounds, providing chemical validation of the target and confirming that indeed dUTPase inhibition is involved in anti-malarial activity. In addition, incubation with these inhibitors was associated with a depletion of the dTTP pool corroborating the central role of dUTPase in dTTP synthesis. PfdUTPase is mainly localized in the cytosol. CONCLUSION: These results strongly confirm the pivotal and essential role of dUTPase in pyrimidine biosynthesis of P. falciparum intraerythrocytic stages
Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum
Fosmidomycin Uptake into Plasmodium and Babesia-Infected Erythrocytes Is Facilitated by Parasite-Induced New Permeability Pathways
., a mouse malaria parasite. and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery
Antimalarial drug targets in Plasmodium falciparum predicted by stage-specific metabolic network analysis
Genome content analysis yields new insights into the relationship between the human malaria parasite Plasmodium falciparum and its anopheline vectors
Effect of fosmidomycin on metabolic and transcript profiles of the methylerythritol phosphate pathway in Plasmodium falciparum
In Plasmodium falciparum , the formation of isopentenyl diphosphate
and dimethylallyl diphosphate, central intermediates in the
biosynthesis of isoprenoids, occurs via the methylerythritol phosphate
(MEP) pathway. Fosmidomycin is a specific inhibitor of the second
enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate
reductoisomerase. We analyzed the effect of fosmidomycin on the levels
of each intermediate and its metabolic requirement for the isoprenoid
biosynthesis, such as dolichols and ubiquinones, throughout the
intraerythrocytic cycle of P. falciparum. The steady-state RNA levels
of the MEP pathway-associated genes were quantified by real-time
polymerase chain reaction and correlated with the related metabolite
levels. Our results indicate that MEP pathway metabolite peak precede
maximum transcript abundance during the intraerythrocytic cycle.
Fosmidomycin-treatment resulted in a decrease of the intermediate
levels in the MEP pathway as well as in ubiquinone and dolichol
biosynthesis. The MEP pathway associated transcripts were modestly
altered by the drug, indicating that the parasite is not strongly
responsive at the transcriptional level. This is the first study that
compares the effect of fosmidomycin on the metabolic and transcript
profiles in P. falciparum, which has only the MEP pathway for
isoprenoid biosynthesis