A Spectrophotometric Assay for Quantitative Determination of kcat of Herpes Simplex Virus Type 1 Thymidine Kinase Substrates

Schelling, Pierre; Folkers, Gerd; Scapozza, Léonardo

doi:10.1006/abio.2001.5191

Cited by 39 publications

(40 citation statements)

References 27 publications

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“…Compounds 1, 2, and 4 were phosphorylated with the highest efficiency. These results contrast with those obtained with the cellular enzyme, where only compounds 1 and 2 were substrates, and these had K m values that exceeded 100 M and were not 50 , effective concentration that reduced plaque formation by 50%. Virus strains used were described previously (47).…”

Section: Antiviral Activity and Tk Dependence In Orthopoxvirusescontrasting

confidence: 91%

“…2D; Tables 3 and 4). K m values were confirmed in a standard spectrophotometric assay (50) and an isothermal calorimetry assay that directly measures the heat of binding of the substrate to the enzyme and generated values of 27 and 24 M, respectively (P. F. Torrence and R. F. Smith, unpublished data). These values agree well with the reported value of 15 M for the human TK1 homolog purified from E. coli, which occurs predominantly as a dimer (4).…”

Section: Antiviral Activity and Tk Dependence In Orthopoxvirusesmentioning

confidence: 88%

“…An equal amount of 1% agarose was added to an equal volume of each drug dilution; this mixture was added to each well in 2-ml volumes, and the plates were incubated for three days. The cells were stained with a solution of 0.01% neutral red in phosphate-buffered saline (PBS) and incubated for 5 to 6 h. The stain was aspirated, and plaques were counted using a stereomicroscope at ϫ10 magnification and 50% effective concentration (EC 50 ) values were calculated by standard methods. The HSV plaque reduction assays were essentially the same as for VV and CV with the following changes.…”

Section: Methodsmentioning

confidence: 99%

“…To each well 35 l of CellTiter-Glo (Promega, Madison, WI) reagent was added, and luminescence was measured with a luminometer. Standard methods were used to determine the drug concentration which inhibited cell proliferation by 50% (IC 50 ).…”

Section: Methodsmentioning

confidence: 99%

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Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Prichard¹,

Keith²,

Johnson³

et al. 2007

Antimicrob Agents Chemother

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The antiviral activity of a new series of thymidine analogs was determined against vaccinia virus (VV), cowpox virus (CV), herpes simplex virus, and varicella-zoster virus. Several compounds were identified that had good activity against each of the viruses, including a set of novel 5-substituted deoxyuridine analogs. To investigate the possibility that these drugs might be phosphorylated preferentially by the viral thymidine kinase (TK) homologs, the antiviral activities of these compounds were also assessed using TK-deficient strains of some of these viruses. Some of these compounds were shown to be much less effective in the absence of a functional TK gene in CV, which was unexpected given the high degree of amino acid identity between this enzyme and its cellular homolog. This unanticipated result suggested that the CV TK was important in the mechanism of action of these compounds and also that it might phosphorylate a wider variety of substrates than other type II enzymes. To confirm these data, we expressed the VV TK and human TK1 in bacteria and isolated the purified enzymes. Enzymatic assays demonstrated that the viral TK could efficiently phosphorylate many of these compounds, whereas most of the compounds were very poor substrates for the cellular kinase, TK1. Thus, the specific phosphorylation of these compounds by the viral kinase may be sufficient to explain the TK dependence. This unexpected result suggests that selective phosphorylation by the viral kinase may be a promising new approach in the discovery of highly selective inhibitors of orthopoxvirus replication.

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Section: Antiviral Activity and Tk Dependence In Orthopoxvirusescontrasting

confidence: 91%

Section: Antiviral Activity and Tk Dependence In Orthopoxvirusesmentioning

confidence: 88%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Prichard¹,

Keith²,

Johnson³

et al. 2007

Antimicrob Agents Chemother

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“…The dotted line shows the 95% confidence interval of the best fit. stants (k cat ) were assessed using a continuous spectrophotometric assay described elsewhere (40). The data presented are the result of five independent series of measurements performed in triplicate.…”

Section: Fig 2 (S)-mct Kineticsmentioning

confidence: 99%

Biochemical and Structural Characterization of (South)-Methanocarbathymidine That Specifically Inhibits Growth of Herpes Simplex Virus Type 1 Thymidine Kinase-transduced Osteosarcoma Cells

Schelling¹,

Claus

Johner³

et al. 2004

Journal of Biological Chemistry

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Applications of Density Functional Theory-Based Methods in Medicinal Chemistry

Sulpizi

Folkers²,

Röthlisberger

et al. 2002

Quant. Struct.-Act.Relat.

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With the advances in genomics, proteomics and functional genomics new therapeutic targets to be tackled by medicinal chemistry are expected. This article reviews applications of first principle methods to address medicinal chemistry issue related to drug/target interactions. Two selected representative case studies involving therapeutically interesting targets are presented. The first case study presents how DFT can contribute to ameliorate scoring functions for drug screening in particular by enabling the discrimination between inhibitors and substrates. The second example shows the use of DFT within the framework of a QM/MM mixed approach for elucidating mechanisms of reaction. This approach allows defining the electronic state and structure of the reaction transition state whose knowledge is essential for designing potent and specific transition state analogs inhibitors. Finally, addressing the issue of other medicinal chemistry related application of DFT we suggest that DFT has indeed the potentiality of becoming very important for challenging new issues presented to medicinal chemistry in the post genomic era.

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A Spectrophotometric Assay for Quantitative Determination of kcat of Herpes Simplex Virus Type 1 Thymidine Kinase Substrates

Cited by 39 publications

References 27 publications

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Selective Phosphorylation of Antiviral Drugs by Vaccinia Virus Thymidine Kinase

Biochemical and Structural Characterization of (South)-Methanocarbathymidine That Specifically Inhibits Growth of Herpes Simplex Virus Type 1 Thymidine Kinase-transduced Osteosarcoma Cells

Applications of Density Functional Theory-Based Methods in Medicinal Chemistry

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