Abstract
BackgroundMalaria 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.MethodsTo 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.ResultsDifferent 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.ConclusionThese results strongly confirm the pivotal and essential role of dUTPase in pyrimidine biosynthesis of P. falciparum intraerythrocytic stages.
Highlights
Malaria remains as a major global problem, being one of the infectious diseases that engender highest mortality across the world
New anti-malarial strategies have included evaluation of the enzyme Deoxyuridine 5′-triphosphate nucleotido-hydrolase (dUTPase) as a potential drug target. This enzyme is essential in both eukaryotes [5] and prokaryotes [6] and several inhibitors of the enzyme have been described that exhibit anti-malarial activity [7,8,9,10] for certain derivatives the correlation between dUTPase inhibition and anti-malarial activity was poor. dUTPase performs a dual role by catalyzing the hydrolysis of dUTP to dUMP and PPi
(See figure on page.) Fig. 1 Efforts for disruption of the Plasmodium falciparum dUTPase coding sequence (Pfdut) gene by single crossover recombination. a After a single crossover event of the plasmid with the dut locus, the KO construct would lead to the generation of two truncated inactive copies of the gene. b The control knockout (KOkon) plasmid should generate a functional copy of the dut gene and a non-functional pseudogene upon single crossover recombination. c Southern blot analysis of the pHH1-dutKO transfected line after drug cycles
Summary
Malaria remains as a major global problem, being one of the infectious diseases that engender highest mortality across the world. The biosynthesis of nucleotides has been highlighted as a promising pathway in the search for new anti-malarial targets, due to the high dependence of nucleotides in the intraerythrocytic stages [2] Certain enzymes, such as dihydroorotate dehydrogenase or purine nucleoside phosphorylase, have been extensively studied as drug targets yet the potential of other steps of the pathway remains unclear [3, 4]. New anti-malarial strategies have included evaluation of the enzyme dUTPase (deoxyuridine 5′-triphosphate nucleotidohydrolase, E.C. 3.6.1.23) as a potential drug target This enzyme is essential in both eukaryotes [5] and prokaryotes [6] and several inhibitors of the enzyme have been described that exhibit anti-malarial activity [7,8,9,10] for certain derivatives the correlation between dUTPase inhibition and anti-malarial activity was poor. The crystal structures of different dUTPases including the P. falciparum and human enzymes have been published to date [13, 14] and the molecular and structural basis for the specific inhibition of a series of triphenyl uridine derivatives has been established [13]
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