Journal Article

Validation of Plasmodium falciparum dUTPase as the target of 5′-tritylated deoxyuridine analogues with anti-malarial activity

Sánchez-Carrasco, Paula, Baragaña, Beatriz, Johansson, Nils Gunnar, Kumar, Vijeesh, Gilbert, Ian Hugh, Sarkar, Sandipan, Müller, Sylke, Ruiz-Pérez, Luis Miguel, Hampton, Shahienaz Emma, Pandurangan, Thiyagamurthy, Kaiser, Marcel, Pérez-Moreno, Guiomar, González-Pacanowska, Dolores

2019

Overview

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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: 5′-tritylated deoxyuridine analogues / Antimalarials - pharmacology / Antiprotozoal agents / Biomedical and Life Sciences / Biomedicine / Biosynthesis / Cell survival / Cloning / Communicable diseases / Cytosol / Deoxyribonucleic acid / Deoxyuridine - analogs & derivatives / Deoxyuridine - pharmacology / Deoxyuridine 5′-triphosphate nucleotido-hydrolase / Disease control / DNA / DNA-directed DNA polymerase / dUTP pyrophosphatase / Entomology / Enzymes / Erythrocytes / Fluorescent antibody technique / Fusion protein / Genetic research / Health aspects / Human diseases / Hydrolase / Imaging techniques / Immunofluorescence / Incubation period / Infectious Diseases / Inhibitors / Localization / Malaria / Metabolism / Microbiology / Microscopy / Mode of action / Mortality / Novels / Parasites / Parasitology / Physiological aspects / Plasmids / Plasmodium falciparum / Plasmodium falciparum - drug effects / Plasmodium falciparum - enzymology / Plasmodium falciparum - genetics / Protozoan Proteins - genetics / Protozoan Proteins - metabolism / Public Health / Pyrimidines / Pyrophosphatases - genetics / Pyrophosphatases - metabolism / Survival / Tropical Medicine / Vaccines / Vector-borne diseases