Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Penciclovir is a potent antiherpesvirus agent which is highly selective due to its phosphorylation only in virus infected cells. Phosphorylation of one of the hydroxymethyl groups of penciclovir (PCV) creates a chiral centre leading to the possible formation of (R)- and (S)-enantiomers. The absolute configuration and stereospecificity of the PCV-phosphates produced in cells infected with herpes simplex viruses types 1 and 2 (HSV-1 and HSV-2), as well as by HSV-1-encoded thymidine kinase, were determined using isotopically chiral [4'-13C]PCV precursors and 13C NMR spectroscopy of the isolated metabolites. The absolute configuration of penciclovir-triphosphate (PCV-TP) produced in HSV-1 infected cells was shown to be S with an enantiomeric purity of greater than 95%. However, in contrast fo HSV-1-infected cells in which none of the (R) enantiomer was detected, about 10% of (R)-PCV-TP was produced in HSV-2-infected cells. Phosphorylation of PCV by HSV-1-encoded thymidine kinase was found to give 75% (S)- and 25% (R)-PCV-monophosphate. The proportion of the (S)-isomer appears to be amplified in the subsequent phosphorylations leading to the triphosphate.
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PMID:Use of isotopically chiral [4'-13C]penciclovir and 13C NMR to determine the specificity and absolute configuration of penciclovir phosphate esters formed in HSV-1 and HSV-2 infected cells and by HSV-1-encoded thymidine kinase. 830 85

The three anti-herpes nucleoside analogues ganciclovir, penciclovir and aciclovir were investigated as to their recombinogenic [sister chromatid exchange (SCE) inducing] and clastogenic activity in CHO cells expressing the thymidine kinase gene of HSV-1, which is a precondition of therapeutic activity of these drugs. The compounds were applied for the duration of one cell cycle and cytogenetic end-points were measured between 0 and 42 h after exposure. Although the nucleoside analogues are quite similar with respect to chemical structure, they differ basically in their genotoxic potency, aberration types induced as well as the time course of chromosomal damage. Aciclovir induced SCEs and chromosomal aberrations immediately after exposure but only in a concentration range much higher than that reached in blood plasma during anti-herpes therapy. The direct genotoxic activity is explained by the obligate chain terminating property of aciclovir upon incorporation into genomic DNA. On the other hand, genotoxic damage caused by ganciclovir and penciclovir is of the delayed type requiring at least one post-exposure cell cycle for its expression. Unlike aciclovir, ganciclovir is an extremely potent inducer of SCEs and chromosome breaks and translocations at concentrations far below those impairing the proliferative activity and triggering apoptosis of the target cells (as shown by our previous investigation). Penciclovir is essentially devoid of genotoxic activity. It induces SCEs only at cytotoxic/apoptotic concentrations, is only weakly clastogenic and induces premature chromosome condensation which appears to result from uncoupling of karyokinesis and cytokinesis. The genotoxic activity of ganciclovir is explained as due to repair processes triggered in the second post-exposure replication cycle at the sites of nucleoside analogue incorporation into genomic DNA. The findings have considerable implications with respect to the use of ganciclovir or other antiviral drugs in suicide gene therapy of malignant diseases.
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PMID:Cytogenetic genotoxicity of anti-herpes purine nucleoside analogues in CHO cells expressing the thymidine kinase gene of herpes simplex virus type 1: comparison of ganciclovir, penciclovir and aciclovir. 1071 44

Penciclovir (PCV), an antiherpesvirus agent in the same class as acyclovir (ACV), is phosphorylated in herpes simplex virus (HSV)-infected cells by the viral thymidine kinase (TK). Resistance to ACV has been mapped to mutations within either the TK or the DNA polymerase gene. An identical activation pathway, the similarity in mode of action, and the invariant cross-resistance of TK-negative mutants argue that the mechanisms of resistance to PCV and ACV are likely to be analogous. A total of 48 HSV type 1 (HSV-1) and HSV-2 isolates were selected after passage in the presence of increasing concentrations of PCV or ACV in MRC-5 cells. Phenotypic analysis suggested these isolates were deficient in TK activity. Moreover, sequencing of the TK genes from ACV-selected mutants identified two homopolymeric G-C nucleotide stretches as putative hot spots, thereby confirming previous reports examining Acv(r) clinical isolates. Surprisingly, mutations identified in PCV-selected mutants were generally not in these regions but distributed throughout the TK gene and at similar frequencies of occurrence within A-T or G-C nucleotides, regardless of virus type. Furthermore, HSV-1 isolates selected in the presence of ACV commonly included frameshift mutations, while PCV-selected HSV-1 mutants contained mostly nonconservative amino acid changes. Data from this panel of laboratory isolates show that Pcv(r) mutants share cross-resistance and only limited sequence similarity with HSV mutants identified following ACV selection. Subtle differences between PCV and ACV in the interaction with viral TK or polymerase may account for the different spectra of genotypes observed for the two sets of mutants.
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PMID:Characterization of herpes simplex viruses selected in culture for resistance to penciclovir or acyclovir. 1116 Jun 74

Homodimeric protein interactions are potent regulators of cellular functions, but are particularly challenging to study in vivo. We used a split synthetic renilla luciferase (hRLUC) complementation-based bioluminescence assay to study homodimerization of herpes simplex virus type 1 thymidine kinase (TK) in mammalian cells and in living mice. We quantified and imaged homodimerization of TK chimeras containing N-terminal (N-hRLUC) or C-terminal (C-hRLUC) fragments of hRLUC in the upstream and downstream positions, respectively (tail-to-head homodimer). This was monitored using luminometry (68-fold increase, and was significantly [P<0.01] above background light emission) and by CCD camera imaging of living mice implanted with ex vivo transfected 293T cells (2.7-fold increase, and is significantly [P<0.01] above background light emission). We also made a mutant-TK to generate N-hRLUC mutant TK and mutant TK-C-hRLUC by changing a single amino acid at position 318 from arginine to cysteine, a key site that has previously been reported to be essential for TK homo-dimerization, to support the specificity of the hRLUC complementation signal from TK homodimerization. Ex vivo substrate (8-3H Penciclovir) accumulation assays in 293T cells expressing the TK protein chimeras showed active TK enzyme. We also devised an experimental strategy by constructing variant TK chimeras (possessing extra N-hRLUC or C-hRLUC 'spacers') to monitor incremental lack of association of the tail-to-head TK homodimer. Application of this potentially generalizable assay to screen for molecules that promote or disrupt ubiquitous homodimeric protein-protein interactions could serve not only as an invaluable tool to understand biological networks but could also be applied to drug discovery and validation in living subjects.
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PMID:Molecular imaging of homodimeric protein-protein interactions in living subjects. 1513 89

Famciclovir is converted rapidly and efficiently after oral administration to the selective antiviral compound, penciclovir. In cell culture, penciclovir is a potent inhibitor of herpes simplex virus (HSV) types 1 and 2, varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and hepatitis B virus (HBV). Phosphorylation of penciclovir and aciclovir in uninfected cells is limited, and penciclovir, like aciclovir, has minimal effect on replicating cells in culture as expected for a selective antiviral agent. Mode of action studies with VZV and HSV have shown that the phosphorylation of penciclovir in infected cells is far more efficient than for aciclovir. This compensates for differences observed between penciclovir triphosphate and aciclovir triphosphate in the inhibition of HSV and VZV DNA polymerases. Because HBV is not known to encode a thymidine kinase, a different rationale for the selective inhibition of this virus by penciclovir is required. Recent data indicate that the DNA polymerase of HBV is far more sensitive to inhibition by penciclovir triphosphate than cellular DNA polymerases, suggesting that for this virus, selectivity operates at the DNA polymerase. Penciclovir triphosphate is more stable within infected cells than aciclovir triphosphate, and consequently penciclovir has more prolonged antiviral activity than aciclovir. Similarly, famciclovir is more effective than aciclovir or valaciclovir in suppressing HSV replication when given at a lower dosing frequency in certain animal models. These preclinical properties have helped to provide the foundation for the famciclovir clinical programme.
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PMID:Famciclovir, from the bench to the patient--a comprehensive review of preclinical data. 1861 46