We have demonstrated that herpes simplex 1 (HSV1) thymidine kinase (TK) shows no stereospecificity for D- and L-beta-nucleosides. In vitro, L enantiomers are not recognized by human TK, but function as specific substrates for the viral enzyme in the order: L-thymidine (L-T) >> 2'-deoxy-L-guanosine (L-dG) > 2'-deoxy-L-uridine (L-dU) > 2'-deoxy-L-cytidine (L-dC) > 2'-deoxy- L-adenosine (L-dA). HSV1 TK phosphorylates both thymidine enantiomers to their corresponding monophosphates with identical efficiency and the Ki of L-T (2 microM) is almost identical to the Km for the natural substrate D-T (2.8 microM). The L enantiomer reduces the incorporation of exogenous [3H]T into cellular DNA in HeLa TK-/HSV1 TK+ but not in wild-type HeLa cells, without affecting RNA, protein synthesis, cell growth, and viability. L-T markedly reduces HSV1 multiplication in HeLa cells. Our observations could lead to the development of a novel class of antiviral drugs characterized by low toxicity.
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.
Point mutations in the cAMP-responsive element (CRE) of the rat somatostatin gene promoter/enhancer sequence (TGACGTCA) were used as a model for assessing the effect of uracil, deriving either from misincorporation during DNA synthesis (T----U) or cytosine deamination (C----U), on the binding of sequence/specific regulatory proteins. The results show that the T----U conversion in both strands of the CRE palindromic sequence reduces its affinity for the CRE binding factor(s), suggesting the crucial role of the methyl group contributed by T for the correct recognition of the sequence. On the other hand, deamination of C in the CpG central dinucleotide (CpG----UpG) causes an increase of binding affinity which is further enhanced by the contemporary deamination in both strands. Then, both uracil misincorporation and cytosine deamination alter the binding to CRE sequence in vitro, suggesting that uracil, if not removed by uracil DNA-glycosylase, could be dangerous for cellular functions.
A series of N2-substituted guanine derivatives was screened against mammalian thymidine kinase and the thymidine kinase encoded by type I herpes simplex virus to examine their capacity to selectivity inhibit the viral enzyme. Several bases, nucleosides, and nucleotides displayed selective activity. The mechanism of action of the most potent derivative, N2-phenyl-2'-deoxyguanosine (PhdG) was studied in detail. PhdG (a) inhibited the viral enzyme competitively with respect to the substrates thymidine and deoxycytidine, (b) was completely resistant to phosphorylation, (c) displayed limited toxicity for the HeLa cell lines employed as hosts for viral infection, and (d) selectively inhibited viral thymidine kinase function in intact cultured cells. The results indicate that the PhdG drug prototype has potential as a selective anti-herpes agent and as a novel molecular probe of the structure and function of herpes simplex thymidine kinase.
We have recently demonstrated that mammalian uracil-DNA glycosylase activity is undetectable in adult neurons. On the basis of this finding we hypothesized that uracil, derived either from oxidative deamination of cytosine or misincorporation of dUMP in place of dTMP during DNA repair by the unique nuclear DNA polymerase present in adult neurons, DNA polymerase beta, might accumulate in neuronal DNA. Uracil residues could also arise in the herpes simplex 1 (HSV1) genome during latency in nerve cells. We therefore suggest a role for the virus encoded uracil-DNA glycosylase in HSV1 reactivation and in the first steps of DNA replication. We show here 1) that the viral DNA polymerase incorporates dUTP in place of dTTP with a comparable efficiency in vitro; 2) that virus specific DNA/protein interactions between the virus encoded origin binding protein and its target DNA sequence is altered by the presence of uracil residues in its central region TCGCA. Thus uracil, present in viral OriS or other key sequences could hamper the process leading to viral reactivation. Hence, HSV1 uracil-DNA glycosylase, dispensable in viral proliferation in tissue culture, could be essential in neurons for the "cleansing" of the viral genome of uracil residues before the start of replication.
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