Functional genetic evaluation of DNA house-cleaning enzymes in the malaria parasite: dUTPase and Ap4AH are essential in <i>Plasmodium berghei</i> but ITPase and NDH are dispensable

Kumar, Hirdesh; Kehrer, Jessica; Singer, Mirko; Reinig, Miriam; Santos, Jorge M.; Mair, Gunnar R.; Frischknecht, Friedrich

doi:10.1080/14728222.2019.1575810

Cited by 10 publications

(7 citation statements)

References 68 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…If dUTP levels are high in the cellular milieu, repair synthesis will reintroduce uracils, creating a hyperactive futile cycle leading to chromosome fragmentation and cell death (thymine‐less cell death). Correct and efficient action of dUTPase is therefore of crucial importance to prevent thymine‐less cell death .…”

mentioning

confidence: 99%

Evaluation of critical design parameters for RT‐qPCR‐based analysis of multiple dUTPase isoform genes in mice

et al. 2019

View full text Add to dashboard Cite

The coupling of nucleotide biosynthesis and genome integrity plays an important role in ensuring faithful maintenance and transmission of genetic information. The enzyme dUTPase is a prime example of such coupling, as it generates dUMP for thymidylate biosynthesis and removes dUTP for synthesis of uracil‐free DNA. Despite its significant role, the expression patterns of dUTPase isoforms in animals have not yet been described. Here, we developed a detailed optimization procedure for RT‐qPCR‐based isoform‐specific analysis of dUTPase expression levels in various organs of adult mice. Primer design, optimal annealing temperature, and primer concentrations were specified for both nuclear and mitochondrial dUTPase isoforms, as well as two commonly used reference genes, GAPDH and PPIA. The linear range of the RNA concentration for the reverse transcription reaction was determined. The PCR efficiencies were calculated using serial dilutions of cDNA. Our data indicate that organs involved in lymphocyte production, as well as reproductive organs, are characterized by high levels of expression of the nuclear dUTPase isoform. On the other hand, we observed that expression of the mitochondrial dUTPase isoform is considerably increased in heart, kidney, and ovary. Despite the differences in expression levels among the various organs, we also found that the mitochondrial dUTPase isoform shows a much more uniform expression pattern as compared to the reference genes GAPDH and PPIA.

show abstract

mentioning

confidence: 99%

Evaluation of critical design parameters for RT‐qPCR‐based analysis of multiple dUTPase isoform genes in mice

et al. 2019

View full text Add to dashboard Cite

show abstract

“…Separately, we recently reported the discovery of intervenolin and its derivatives as potent and specific H. pylori DHODH inhibitors; intervenolin shows excellent antibacterial activity in both in vitro and in vivo models [23]. Other enzyme from nucleotide biosynthesis such as dUTPase (deoxyuridine 5 -triphosphate nucleotidohydrolase, E.C.36.1.23), a house-keeping enzyme that catalyzes the hydrolysis of dUTP to PPi and dUMP, which in turn, is used as precursor dTMP biosynthesis, have been reported to be indispensable for parasite survival within erythrocytes and a druggable target [52][53][54].…”

Section: Discussionmentioning

confidence: 99%

Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target

Sato

Hartuti

Inaoka

et al. 2020

Genes

View full text Add to dashboard Cite

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial monotopic membrane protein that plays an essential role in the pyrimidine de novo biosynthesis and electron transport chain pathways. In Eimeria tenella, an intracellular apicomplexan parasite that causes the most severe form of chicken coccidiosis, the activity of pyrimidine salvage pathway at the intracellular stage is negligible and it relies on the pyrimidine de novo biosynthesis pathway. Therefore, the enzymes of the de novo pathway are considered potential drug target candidates for the design of compounds with activity against this parasite. Although, DHODHs from E. tenella (EtDHODH), Plasmodium falciparum (PfDHODH), and human (HsDHODH) show distinct sensitivities to classical DHODH inhibitors, in this paper, we identify ferulenol as a potent inhibitor of both EtDHODH and HsDHODH. Additionally, we report the crystal structures of EtDHODH and HsDHODH in the absence and presence of ferulenol. Comparison of these enzymes showed that despite similar overall structures, the EtDHODH has a long insertion in the N-terminal helix region that assumes a disordered configuration. In addition, the crystal structures revealed that the ferulenol binding pocket of EtDHODH is larger than that of HsDHODH. These differences can be explored to accelerate structure-based design of inhibitors specifically targeting EtDHODH.

show abstract

“…Since the absence of insertions in the CDS was considered as an indicator that disruptions are lethal, the data is also indicative of Pfdut being essential [30]. In addition, the enzyme appears to be essential in P. berghei since deletion of dUTPase failed after several attempts suggesting a crucial role during intraerythrocytic development [15].…”

Section: Discussionmentioning

confidence: 99%

“…While dUTPase has been extensively studied with the aim of inhibitor discovery, little information exists regarding its biological role and essentiality for cell survival in P. falciparum . A recent study performed in Plasmodium berghei has suggested that dUTPase null mutants are not viable [15]. It is reasonable to assume that dUTPase is crucial to the parasite given the high AT/CG ratio (approximately 80%) in its genome and the fact that Plasmodium lacks dCMP/dCTP deaminase activities which would confer a central role to dUTPase in dUMP formation.…”

Section: Introductionmentioning

confidence: 99%

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

Pérez-Moreno

Sánchez-Carrasco

Ruíz-Pérez

et al. 2019

Malar J

View full text Add to dashboard Cite

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.

show abstract

Functional genetic evaluation of DNA house-cleaning enzymes in the malaria parasite: dUTPase and Ap4AH are essential in Plasmodium berghei but ITPase and NDH are dispensable

Abstract: Supplementary Figure 2. Alignment of human (Q9BY32) and Plasmodium berghei (PBANKA_0618300) ITPases. The catalytic glutamate residue is indicated by an 'x' above the alignment. Accession numbers are from uniprot.org and plasmodb.org.

Cited by 10 publications

References 68 publications

Evaluation of critical design parameters for RT‐qPCR‐based analysis of multiple dUTPase isoform genes in mice

Evaluation of critical design parameters for RT‐qPCR‐based analysis of multiple dUTPase isoform genes in mice

Structural and Biochemical Features of Eimeria tenella Dihydroorotate Dehydrogenase, a Potential Drug Target

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

Contact Info

Product

Resources

About