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Publication Open Access Molecular characterization of Plasmodium falciparum uracil-DNA glycosylase and its potential as a new anti-malarial drug target(2014) Thidarat Suksangpleng; Ubolsree Leartsakulpanich; Saengduen Moonsom; Saranya Siribal; Usa Boonyuen; Wright, George E; Porntip Chavalitshewinkoon-Petmitr; Mahidol University. Faculty of Tropical Medicine. Department of Protozoology,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.Publication Open Access Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I(2014) Krittikorn Kümpornsin; Namfon Kotanan; Pornpimol Chobson; Theerarat Kochakarn; Piyaporn Jirawatcharadech; Peera Jaru-ampornpan; Yongyuth Yuthavong; Thanat Chookajorn; Mahidol University. Faculty of Science. Department of BiochemistryBackground: Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. Methods: Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ΔfolE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. Results: Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. Conclusions: Functional assays for P. falciparum GCH1 based on enzymatic activity and genetic complementation were successfully developed. The assays in combination with a homology model characterized the enzymatic activity of P. falciparum GCH1 and the importance of its key amino acid residues. The potential to use the assay for inhibitor screening was validated by 8-oxo-GTP, a known GTP analogue inhibitor.Publication Open Access Laboratory prediction of the requirement for renal replacement in acute falciparum malaria.(2011-08-03) Hanson, Josh; Hasan, Md Mahtab Uddin; Royakkers, Annick A; Alam, Shamsul; Prakaykaew Charunwatthana; ประกายแก้ว จรูญวรรธนะ; Maude, Richard J; Douthwaite, Sam T; Yunus, Emran Bin; Mantha, Murty L; Schultz, Marcus J; Faiz, M Abul; White, Nicholas J; Day, Nicholas P; Dondorp, Arjen M; Hanson, Josh; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit. Shoklo Malaria Research Unit.BACKGROUND: Acute renal failure is a common complication of severe malaria in adults, and without renal replacement therapy (RRT), it carries a poor prognosis. Even when RRT is available, delaying its initiation may increase mortality. Earlier identification of patients who will need RRT may improve outcomes. METHOD: Prospectively collected data from two intervention studies in adults with severe malaria were analysed focusing on laboratory features on presentation and their association with a later requirement for RRT. In particular, laboratory indices of acute tubular necrosis (ATN) and acute kidney injury (AKI) that are used in other settings were examined. RESULTS: Data from 163 patients were available for analysis. Whether or not the patients should have received RRT (a retrospective assessment determined by three independent reviewers) was used as the reference. Forty-three (26.4%) patients met criteria for dialysis, but only 19 (44.2%) were able to receive this intervention due to the limited availability of RRT. Patients with impaired renal function on admission (creatinine clearance < 60 ml/min) (n = 84) had their laboratory indices of ATN/AKI analysed. The plasma creatinine level had the greatest area under the ROC curve (AUC): 0.83 (95% confidence interval 0.74-0.92), significantly better than the AUCs for, urinary sodium level, the urea to creatinine ratio (UCR), the fractional excretion of urea (FeUN) and the urinary neutrophil gelatinase-associated lipocalcin (NGAL) level. The AUC for plasma creatinine was also greater than the AUC for blood urea nitrogen level, the fractional excretion of sodium (FeNa), the renal failure index (RFI), the urinary osmolality, the urine to plasma creatinine ratio (UPCR) and the creatinine clearance, although the difference for these variables did not reach statistical significance. CONCLUSIONS: In adult patients with severe malaria and impaired renal function on admission, none of the evaluated laboratory indices was superior to the plasma creatinine level when used to predict a later requirement for renal replacement therapy.