Publication: Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target
Issued Date
2014
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eng
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Mahidol University
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BioMed Central
Bibliographic Citation
Malaria Journal. Vol.13, (2014), 149
Suggested Citation
Thidarat Suksangpleng, Ubolsree Leartsakulpanich, Saengduen Moonsom, Saranya Siribal, Usa Boonyuen, Wright, George E, Porntip Chavalitshewinkoon-Petmitr Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target. Malaria Journal. Vol.13, (2014), 149. Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/3069
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Title
Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target
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Abstract
Background: Based on resistance of currently used anti-malarials, a new anti-malarial drug target against Plasmodium
falciparum is urgently needed. Damaged DNA cannot be transcribed without prior DNA repair; therefore, uracil-DNA
glycosylase, playing an important role in base excision repair, may act as a candidate for a new anti-malarial drug
target.
Methods: Initially, the native PfUDG from parasite crude extract was partially purified using two columns, and the
glycosylase activity was monitored. The existence of malarial UDG activity prompted the recombinant expression
of PfUDG for further characterization. The PfUDG from chloroquine and pyrimethamine resistant P. falciparum strain
K1 was amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant PfUDG was
analysed by SDS-PAGE and identified by LC-MS/MS. The three dimensional structure was modelled. Biochemical
properties were characterized. Inhibitory effects of 12 uracil-derivatives on PfUDG activity were investigated. Inhibition
of parasite growth was determined in vitro using SYBR Green I and compared with results from human cytotoxicity
tests.
Results: The native PfUDG was partially purified with a specific activity of 1,811.7 units/mg (113.2 fold purification). After
cloning of 966-bp PCR product, the 40-kDa hexa-histidine tagged PfUDG was expressed and identified. The amino acid
sequence of PfUDG showed only 24.8% similarity compared with the human enzyme. The biochemical characteristics
of PfUDGs were quite similar. They were inhibited by uracil glycosylase inhibitor protein as found in other organisms.
Interestingly, recombinant PfUDG was inhibited by two uracil-derived compounds; 1-methoxyethyl-6-(p-n-octylanilino)
uracil (IC50 of 16.75 μM) and 6-(phenylhydrazino)uracil (IC50 of 77.5 μM). Both compounds also inhibited parasite growth
with IC50s of 15.6 and 12.8 μM, respectively. Moreover, 1-methoxyethyl-6-(p-n-octylanilino)uracil was not toxic to HepG2
cells, with IC50 of > 160 μM while 6-(phenylhydrazino)uracil exhibited cytoxicity, with IC50 of 27.5 μM.
Conclusions: The recombinant PfUDG was expressed, characterized and compared to partially purified native PfUDG.
Their characteristics were not significantly different. PfUDG differs from human enzyme in its size and predicted
amino acid sequence. Two uracil derivatives inhibited PfUDG and parasite growth; however, only one non-cytotoxic
compound was found. Therefore, this selective compound can act as a lead compound for anti-malarial development
in the future.