Phosphorylation of uridine and cytidine nucleoside analogs by two human uridine-cytidine kinases.
ABSTRACT Uridine-cytidine kinases (UCK) have important roles for the phosphorylation of nucleoside analogs that are being investigated for possible use in chemotherapy of cancer. We have cloned the cDNA of two human UCKs. The approximately 30-kDa proteins, named UCK1 and UCK2, were expressed in Escherichia coli and shown to catalyze the phosphorylation of Urd and Cyd. The enzymes did not phosphorylate deoxyribonucleosides or purine ribonucleosides. UCK1 mRNA was detected as two isoforms of approximately 1.8 and approximately 2.7 kb. The 2.7-kb band was ubiquitously expressed in the investigated tissues. The band of approximately 1.8 kb was present in skeletal muscle, heart, liver, and kidney. The two isoforms of UCK2 mRNA of 1.2 and 2.0 kb were only detected in placenta among the investigated tissues. The genes encoding UCK1 and UCK2 were mapped to chromosome 9q34.2-9q34.3 and 1q22-1q23.2, respectively. We tested 28 cytidine and uridine nucleoside analogs as possible substrates of the enzymes. The enzymes phosphorylated several of the analogs, such as 6-azauridine, 5-fluorouridine, 4-thiouridine, 5-bromouridine, N(4)-acetylcytidine, N(4)-benzoylcytidine, 5-fluorocytidine, 2-thiocytidine, 5-methylcytidine, and N(4)-anisoylcytidine. The cloning and recombinant expression of the two human UCKs will be important for development of novel pyrimidine ribonucleoside analogs and the characterization of their pharmacological activation.
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ABSTRACT: Chronic infection with hepatitis C virus (HCV) is a major global health burden and is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma. Current therapy for HCV infection has limited efficacy, particularly against genotype 1 virus, and is hampered by a range of adverse effects. Therefore, there is a clear unmet medical need for efficacious and safe direct antiviral drugs for use in combination with current treatments to increase cure rates and shorten treatment times. The broad genotypic coverage achievable with nucleosides or nucleotides and the high genetic barrier to resistance of these compounds observed in vitro and in vivo suggest that this class of inhibitors could be a valuable component of future therapeutic regimens. Here, we report the in vitro inhibitory activity and mode of action of 2'-deoxy-2'-spirocyclopropylcytidine (TMC647078), a novel and potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase that causes chain termination of the nascent HCV RNA chain. In vitro combination studies with a protease inhibitor resulted in additive efficacy in the suppression of HCV RNA replication, highlighting the potential for the combination of these two classes in the treatment of chronic HCV infection. No cytotoxic effects were observed in various cell lines. Biochemical studies indicated that TMC647078 is phosphorylated mainly by deoxycytidine kinase (dCK) without inhibiting the phosphorylation of the natural substrate, and high levels of triphosphate were observed in Huh7 cells and in primary hepatocytes in vitro. TMC647078 is a potent novel nucleoside inhibitor of HCV replication with a promising in vitro virology and biology profile.Antimicrobial Agents and Chemotherapy 05/2011; 55(8):3812-20. · 4.57 Impact Factor
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ABSTRACT: TAS-106 [1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine] is a new anticancer ribo-nucleoside with promising antitumor activity. We have previously presented evidence suggesting that the TAS-106 sensitivity of cells is correlated with intracellular accumulation of the triphosphate of TAS-106, which may be affected both by cellular membrane transport mechanisms and uridine-cytidine kinase (UCK) activity. Since the presence of a UCK family consisting of two members, UCK1 and UCK2, has recently been reported in human cells, we investigated the relation between expression of UCK1 and UCK2 at both the mRNA and protein levels and UCK activity (TAS-106 phosphorylation activity) in a panel of 10 human cancer cell lines. Measurement of UCK activity in these cell lines revealed that it was well correlated with the cells' sensitivity to TAS-106. In addition, the mRNA or protein expression level of UCK2 was closely correlated with UCK activity in these cell lines, but neither the level of expression of UCK1 mRNA nor that of protein was correlated with enzyme activity. We therefore compared the protein expression level of UCK2 in several human tumor tissues and the corresponding normal tissues. Expression of UCK2 protein was barely detectable in 4 of the 5 human tumor tissues, but tended to be high in the pancreatic tumor tissue. It could not be detected at all in any of the normal tissues. Thus, expression of UCK2 appeared to be correlated with cellular sensitivity to TAS-106, and it may contribute to the tumor-selective cytotoxicity of TAS-106.Japanese journal of cancer research: Gann 08/2002; 93(7):825-33.
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ABSTRACT: The immuno-modulatory properties of nucleotides such as adenosine or inosine, have been described extensively. Recently, the nucleoside uridine and its analogue 4-thiouridine have gained attention for their protective role in acute lung inflammation. In this study, we investigated the influence of uridine on asthmatic airway inflammation. We used the classical ovalbumin (OVA)-alum model, as well as a model of house dust mite-(HDM)-induced airway inflammation. The degree of inflammation was determined by bronchoalveolar lavage (BAL), histology, and measurement of bronchial hyperresponsiveness. Intratracheal treatment of OVA-sensitized animals with uridine before allergen challenge resulted in a reduction in total BAL cells and BAL eosinophils. This was accompanied by reduced tissue infiltration and diminished production of T helper type 2-cytokines by mediastinal lymph node cells. Additionally, mice treated with uridine developed less bronchial hyperresponsiveness. Uridine was also effective in reducing airway inflammation in HDM-induced asthma. The protective effects of uridine were independent of myeloid dendritic cell (mDC) function, because in vitro pre-treatment of allergen-pulsed DCs with uridine did not alter the degree of inflammation. However, uridine inhibited the release of pro-inflammatory mediators in vivo and by cultured lung epithelial cells, suggesting an effect on lung structural cells. In summary, we were able to show that uridine inhibits the classical features of asthmatic airway inflammation. As uridine supplementation is well tolerated in humans, it might be a new therapeutic approach for the treatment of bronchial asthma.Clinical & Experimental Allergy 04/2010; 40(10):1552-60. · 4.79 Impact Factor