Thymidine kinase and uridine-cytidine kinase from Entamoeba histolytica: cloning, characterization, and search for specific inhibitors.

Entamoeba histolytica is an intestinal parasite and the causative agent of amoebiasis, which is a significant source of morbidity and mortality in developing countries. Although anti-amoebic drugs such as metronidazole, emetine, chloroquine and nitazoxanide are generally effective, there is always potential for development of drug resistance. In order to find novel targets to control E. histolytica proliferation we cloned, expressed and purified thymidine kinase (Eh-TK) and uridine-cytidine kinase (Eh-UCK) from E. histolytica. Eh-TK phosphorylates thymidine with a Km of 0.27 microm, whereas Eh-UCK phosphorylates uridine and cytidine with Km of 0.74 and 0.22 mM, respectively. For both enzymes, ATP acts as specific phosphate donor. In order to find alternative treatments of E. histolytica infection we tested numerous nucleoside analogues and related compounds as inhibitors and/or substrates of Eh-TK and Eh-UCK, and active compounds against E. histolytica in cell culture. Our results indicate that inhibitors or alternative substrates of the enzymes, although partially reducing protozoan proliferation, are reversible and not likely to become drugs against E. histolytica infections.

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

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 beta-thymidine. Here we show that, after phosphorylation by HSV-TK, tritiated L-beta-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.

Impaired 2',3'-dideoxy-3'-thiacytidine accumulation in T-lymphoblastoid cells as a mechanism of acquired resistance independent of multidrug resistant protein 4 with a possible role for ATP-binding cassette C11.

Cellular factors may contribute to the decreased efficacy of chemotherapy in HIV infection. Indeed, prolonged treatment with nucleoside analogues, such as azidothymidine (AZT), 2',3'-deoxycytidine or 9-(2-phosphonylmethoxyethyl)adenine, induces cellular resistance. We have developed a human T lymphoblastoid cell line (CEM 3TC) that is selectively resistant to the antiproliferative effect of 2',3'-dideoxy-3'-thiacytidine (3TC) because the CEM 3TC cells were equally sensitive to AZT, as well as the antimitotic agent, vinblastine. The anti-retroviral activity of 3TC against HIV-1 was also severely impaired in the CEM 3TC cells. Despite similar deoxycytidine kinase activity and unchanged uptake of nucleosides such as AZT and 2'-deoxycytidine, CEM 3TC had profoundly impaired 3TC accumulation. Further studies indicated that CEM 3TC retained much less 3TC. However, despite a small overexpression of multidrug resistance protein (MRP) 4, additional studies with cells specifically engineered to overexpress MRP4 demonstrated there was no impact on either 3TC accumulation or efflux. Finally, an increased expression of the MRP5 homologue, ATP-binding cassette C11 (ABCC11) was observed in the CEM 3TC cells. We speculate that the decreased 3TC accumulation in the CEM 3TC might be due to the upregulation of ABCC11.

Synthesis and in vitro activity of D- and L-enantiomers of 5-(trifluoromethyl)uracil nucleoside derivatives.

Recently, beta-L-nucleoside analogues have emerged as a new class of sugar modified nucleosides with potential antiviral and/or antitumoral activity. As a part of our ongoing research on this topic, we decided to synthesize 5-CF3-beta-L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic drug approved by FDA but only used in topical applications due to concomitant cytotoxicity. 5-CF3-beta-L-dUrd (7) as well as some other related L-nucleoside derivatives were stereospecifically prepared and tested in vitro against viral (HSV-1 and HSV-2) and human thymidine kinases (TK).

5-(Trifluoromethyl)-beta-l-2'-deoxyuridine, the L-enantiomer of trifluorothymidine: stereospecific synthesis and antiherpetic evaluations.

As a part of our ongoing work on beta-L-nucleoside analogues as potential antiviral drugs, we have synthesized 5-(trifluoromethyl)-beta-L-2'-deoxyuridine (L-TFT), the hitherto unknown L-enantiomer of trifluorothymidine (CF(3)dUrd, TFT). We have also studied the effect of L-TFT on human and herpes simplex virus (HSV) type 1 and 2 thymidine kinases, and human thymidine phosphorylase, as well as its anti-HSV-1 and anti-HSV-2 activities in cell cultures. L-TFT has been found: (i) to inhibit HSV-1 TK with activity comparable to TFT, with no effect on human TK, (ii) to be phosphorylated by the viral enzyme with similar efficiency to TFT, (iii) to be resistant, in contrast to TFT, to hydrolysis by human thymidine phosphorylase. Unfortunately, when evaluated in cell cultures, L-TFT did not show any anti-HSV-1 and anti-HSV-2 activities.

Thymidine phosphorylase: a two-face Janus in anticancer chemotherapy.

Several cytokines and growth factors modulate angiogenesis through a fine tuned paracrine or autocrine mode of action. Among them is plateled-derived endothelial cell growth factor (PD-ECGF), which is highly is expressed in tumors, and is angiogenic by stimulation of endothelial cell migration. Studies have shown that PD-ECGF is identical to the well known enzyme thymidine phosphorylase (TP), which is involved in thymidine metabolism and homeostasis. Interestingly, PD-ECGF plays an angiogenic role as a result of its TP enzyme activity. In light of these findings, PD-ECGF/TP should not be considered a true growth factor, and its PD-ECGF name is now actually a misnomer. Recently, TP activity was thought of as an interesting potential two-face target for controling tumor-dependent angiogenesis. In fact, on one hand, its high levels of expression in tumors compared to non-neoplastic regions, and its broad substrate specificity suggested that TP could be used as an enzymatic tool to locally activate anticancer nucleoside bases or base analogs. On the other hand, its enzyme-dependent angiogenic activity engendered the search for specific inhibitors to reduce TP-dependent angiogenesis. This review will describe TP, its activity, its possible mechanisms of action and its role in angiogenesis. Particular attention will be focused on the design and biological characterization of novel TP inhibitors which recently showed promising anticancer activity.

Anti-(herpes simplex virus) activity of 4'-thio-2'-deoxyuridines: a biochemical investigation for viral and cellular target enzymes.

The antiviral activity of several nucleoside analogues is often limited by their rapid degradation by pyrimidine nucleoside phosphorylases. In an attempt to avoid this degradation, several modified nucleosides have been synthesized. A series of 4'-thio-2'-deoxyuridines exhibits an anti-[herpes simplex virus (HSV)] activity significantly higher (20-600 times) than that shown by the corresponding 4'-oxy counterpart. We investigated the mode of action of these compounds and we found that: (i) several 4'-thio-2'-deoxyuridines are phosphorylated to the mono- and di-phosphates by HSV-1 thymidine kinase (TK) more efficiently than their corresponding 4'-oxy counterpart; (ii) both are inhibitors of cellular thymidylate synthase; (iii) 4'-thio-2'-deoxyuridines are resistant to phosphorolysis by human thymidine phosphorylase; (iv) both 4'-oxy- and 4'-thio-2'-deoxyuridines are phosphorylated to deoxyribonucleotide triphosphate in HSV-1-infected cells and are incorporated into viral DNA; (v) 4'-thio-2'-deoxyuridines are better inhibitors than their 4'-oxy counterparts of [(3)H]thymidine incorporation in HSV-1-infected cells; (vi) 4'-thio-2'-deoxyuridines are not recognized by HSV-1 and human uracil-DNA glycosylases. Our data suggest that 4'-thio-2'-deoxyuridines, resistant to pyrimidine phosphorylase, can be preferentially or selectively phosphorylated by viral TK in HSV-infected cells, where they are further converted into triphosphate by cellular nucleotide kinases. Once incorporated into viral DNA, they are better inhibitors of viral DNA synthesis than their corresponding 4'-oxy counterpart, either because they are not recognized, and thus not removed, by viral uracil-DNA glycosylase, or because they preferentially interfere with viral DNA polymerase.

Novel nonsubstrate inhibitors of human thymidine phosphorylase, a potential target for tumor-dependent angiogenesis.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) is an enzyme involved in thymidine metabolism and homeostasis, and its catalytic activity appears to play an important role in angiogenesis. Here we describe the cloning and expression of a His-tagged human TP/PD-ECGF and its assay with uracil and thymine analogues. We present the design, synthesis, and biological evaluation of novel 6-(phenylalkylamino)uracil derivatives which, at micromolar concentrations, inhibit both catabolic and anabolic reactions of human TP in vitro. These base analogues are not converted by the enzyme into the nucleoside form, thus representing pure nonsubstrate inhibitors of the enzyme.