Phosphorylation of Pyrimidine Deoxynucleoside Analog Diphosphates

Krishnan, Preethi; Fu, Qin; Lam, Wing; Liou, Jieh-Yuan; Dutschman, Ginger E.; Cheng, Yung‐Chi

doi:10.1074/jbc.m109025200

Cited by 86 publications

(35 citation statements)

References 40 publications

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“…The last step in the phosphorylation of L-deoxynucleoside analog diphosphates to the respective triphosphates, which are the pharmacologically active metabolites, remained largely unexplored. Through comparison of phosphorylation of several clinically relevant D-deoxynucleoside, D-dideoxynucleoside, and L-deoxynucleoside analog diphosphates by nucleoside-metabolizing enzymes purified from the human hepatic carcinoma cell line HepG2, we recently reported that L-deoxynucleoside analog diphosphates could be phosphorylated by 3-phosphoglycerate kinase (PGK); D-deoxynucleoside analog diphosphates were likely to be phosphorylated by nucleoside diphosphate kinase; and D-dideoxynucleoside analog diphosphates were excellent substrates for creatine kinase (16). Moreover, L-deoxynucleoside analog diphosphates were better substrates for PGK than the corresponding D-deoxynucleoside and D-dideoxynucleoside analog diphosphates (at 200 M).…”

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Phosphorylation of Pyrimidine l-Deoxynucleoside Analog Diphosphates

Krishnan

Liou

Cheng

2002

Journal of Biological Chemistry

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Human PGK (ϳ46 kDa) is a glycolytic enzyme that catalyzes the conversion of 1,3-biphosphoglycerate to 3-phosphoglycerate and during the process generates one molecule of ATP (17). The reaction is reversible. In addition to its participation in the glycolytic cycle, cytoplasmic PGK is known to stimulate viral mRNA synthesis (18). It is also expressed in the nuclei where it modulates DNA synthesis and repair (19 -21). Since PGK in nuclei retains its ability to bind to its natural substrates, it has been proposed that its activity in nuclei may be regulated by the energy state of the cell (21). Extracellular PGK was recently shown to have a thiol-reductase activity (22). Reduction of plasmin by PGK results in proteolysis of plasmin to angiostatin fragments, which are inhibitors of angiogenesis (23-25). ATP and 3-phosphoglycerate could inhibit reduction of plasmin, presumably through inducing a conformational change that was not favorable to the reduction of plasmin (25). This suggested that the level of nucleoside diphosphates or triphosphates (natural or otherwise) might have a regulatory impact on the multiple cellular functions of PGK.The sequences of mammalian PGKs are conserved over 96%, and there is also a high level of tertiary structure homology (26). The crystal structures of horse muscle PGK in

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confidence: 99%

Phosphorylation of Pyrimidine l-Deoxynucleoside Analog Diphosphates

Krishnan

Liou

Cheng

2002

Journal of Biological Chemistry

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“…Similarly, L-nucleoside diphosphates are phosphorylated to triphosphates by pathways different from those responsible for D-nucleoside diphosphate phosphorylation. 20,21 The limited extent of the background phosphorylation of LT in cells not transduced by HSV-TK-containing vectors 15 does not interfere with the histoautoradiographic imaging of the cells expressing HSV-TK.…”

Section: Discussionmentioning

confidence: 99%

Lack of enantioselectivity of herpes virus thymidine kinase allows safer imaging of gene delivery

et al. 2003

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Herpes simplex virus thymidine kinase (HSV-TK) is widely used in gene therapy. The enzymatic activity of HSV-TK may be traced in vivo by specific radiopharmaceuticals in order to image transgene expression. However, most of these radiopharmaceuticals are toxic per se or after activation by HSV-TK, and therefore do not represent ideal molecules for clinical applications and repeated imaging. Unlike human cytosolic TK, HSV-TK is not enantioselective and can efficiently phosphorylate both D and L enantiomers of b-thymidine. Here we show that, after phosphorylation by HSV-TK, tritiated L-b-thymidine (LT) is selectively retained inside the cells in vitro and in vivo. We used the in vivo accumulation of radioactive phosphorylated LT to image the HSV-TK-positive cells inside a transplantable murine brain tumour after inoculation of cells producing retroviruses carrying HSV-TK. Owing to their unnatural enantiomeric conformation, phosphorylated LT metabolites are very poorly processed by mammalian enzymes, thus leading to increased cellular retention and minimal toxicity. The ability to image cells expressing the HSV-TK gene by using radiolabelled LT, without damaging the cells accumulating the phosphorylated L-nucleoside, will be important to monitor the levels and spatial distribution of therapeutic vectors carrying HSV-TK.

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“…This study also emphasizes that contrary to the assumptions that NDPKs phosphorylate all nucleoside analog diphosphates, the structure of the analog is just another variable in determining the substrate specificity of the enzyme. Our earlier studies had in fact suggested that pyrimidine D-dideoxynucleoside analog diphosphates are phosphorylated by creatine kinase (32). Given the diversity of nucleoside analogs currently used or under clinical trials for the treatment of cancer and viral infections, it is important to understand the structure-based distinction in the metabolism of these analogs.…”

Section: Discussionmentioning

confidence: 99%

“…The cells were harvested in cold phosphate-buffered saline containing 20 M dipyridamole (Sigma) and extracted with 15% trichloroacetic acid for 10 min on ice. The supernatant containing the nucleoside and its phosphorylated forms was extracted with a 45/55 ratio of trioctylamine and 1,1,2-trichlorotrifluroethane (32,33). The trichloroacetic acid insoluble pellet representing the nucleotide incorporated into the DNA was washed twice and resolubilized in Me 2 SO prior to evaluation on a Beckman LS5000TD scintillation counter.…”

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confidence: 99%

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Novel Role of 3-Phosphoglycerate Kinase, a Glycolytic Enzyme, in the Activation of L-Nucleoside Analogs, a New Class of Anticancer and Antiviral Agents

Krishnan

Gullen

Lam

et al. 2003

Journal of Biological Chemistry

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L-Nucleoside analogs are a new class of clinically active antiviral and anticancer agents. The phosphorylation of these analogs from diphosphate to triphosphate metabolites is crucial for their biological action. We studied the role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the metabolism of L-nucleoside analogs, using small interfering RNAs to down-regulate the amount of this enzyme in HelaS3 and 2.2.15 cells, chosen as models for studying the impact of the enzyme on the anticancer and antihepatitis B virus activities of these analogs. Decrease in the expression of 3-phosphoglycerate kinase led to a corresponding decrease in the formation of the triphosphate metabolites of L-nucleoside analogs (but not D-nucleoside analogs), resulting in detrimental effects on their activity. The enzyme is important for generating as well as maintaining the steady state levels of L-nucleotides in the cells, thereby playing a key role in the activity of L-nucleoside analogs against human immunodeficiency virus, hepatitis B virus, and cancer. This study also indicates a structure-based distinction in the metabolism of L-and D-nucleoside analogs, disputing the classic notion that nucleoside diphosphate kinases are responsible for the phosphorylation of all classes of nucleoside analog diphosphates.L-Nucleoside analogs are an emerging class of anticancer and antiviral agents that are being used clinically or are under advanced clinical trials (1, 2). Structurally, L-nucleoside analogs are mirror images of the natural D-nucleosides. Despite the unnatural L-configuration, these analogs are able to utilize the salvage pathway of deoxynucleoside metabolism to be phosphorylated to the active triphosphate form (3, 4). Similar to the D-nucleoside analog counterparts, L-nucleoside analog triphosphates exert their pharmacological activity through incorporation into cellular or viral DNA, leading to chain termination or inhibition of viral reverse transcriptase or DNA polymerase (5-10). In the past decade, the realization that L-nucleosides are capable of being activated in cells has led to the discovery of several antiviral pyrimidine L-deoxynucleoside analogs. The first two steps in the metabolism of L-nucleoside analogs seem to be similar to those of its D-nucleoside counterparts. The cytidine analogs, 3TC, L-OddC, L-Fd4C, and gemcitabine, are phosphorylated by cytoplasmic deoxycytidine kinase to the monophosphate metabolite, whereas L-FMAU can be phosphorylated by both deoxycytidine kinase and thymidine kinase (4, 10, 23-25). The cytidine analogs are then phosphorylated by cytidine/uridine monophosphate kinase to the respective diphosphates, and thymidine analogs are phosphorylated by thymidylate kinase (4, 26 -28). The last step of pyrimidine nucleoside analog metabolism was assumed to be carried out by nucleoside diphosphate kinase (NDPK); however, its role in the phosphorylation of L-nucleoside analog diphosphates remained doubtful (29 -31). We examined this in further detail by studying the phosphorylation of pyrimidine D...

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Phosphorylation of Pyrimidine Deoxynucleoside Analog Diphosphates

Cited by 86 publications

References 40 publications

Phosphorylation of Pyrimidine l-Deoxynucleoside Analog Diphosphates

Phosphorylation of Pyrimidine l-Deoxynucleoside Analog Diphosphates

Lack of enantioselectivity of herpes virus thymidine kinase allows safer imaging of gene delivery

Novel Role of 3-Phosphoglycerate Kinase, a Glycolytic Enzyme, in the Activation of L-Nucleoside Analogs, a New Class of Anticancer and Antiviral Agents

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