Biosynthetic Ganciclovir Triphosphate: Its Isolation and Characterization from Ganciclovir-Treated Herpes Simplex Thymidine Kinase-Transduced Murine Cells
| Autor: | Harry Ford, R. Michael Blaese, David A. Cooney, Riad Agbaria, James A. Kelley, Zhang Hao, David G. Johns, Fabio Candotti |
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| Rok vydání: | 2001 |
| Předmět: |
Anions
Ganciclovir Time Factors DNA polymerase viruses Biophysics DNA-Directed DNA Polymerase Adenocarcinoma Antiviral Agents Binding Competitive Thymidine Kinase Biochemistry Mass Spectrometry Mice chemistry.chemical_compound Biosynthesis Transduction Genetic Tumor Cells Cultured medicine Animals Humans Molecular Biology Cells Cultured Chromatography High Pressure Liquid Polymerase chemistry.chemical_classification Dose-Response Relationship Drug biology Kinase Herpes Simplex Cell Biology Chromatography Ion Exchange DNA Polymerase I Molecular biology Kinetics Enzyme Models Chemical chemistry Thymidine kinase Colonic Neoplasms biology.protein DNA polymerase I medicine.drug |
| Zdroj: | Biochemical and Biophysical Research Communications. 289:525-530 |
| ISSN: | 0006-291X |
| Popis: | A method is described for the preparation of ganciclovir triphosphate (GCV-TP) using murine colon cancer cells (MC38) transduced with the herpes simplex virus-thymidine kinase (MC38/HSV-tk). Murine cells transduced with viral-tk contain required viral and host enzymes needed for complete cellular synthesis of this potent antiviral metabolite. Dose response studies showed optimal intracellular levels of GCV-TP occurred after exposure of MC38/HSV-tk cells to 300 microM ganciclovir for 24 h producing 7.5 nmol GCV-TP/10(6) cells. This reflects cellular accumulation of GCV-TP to levels 25-fold greater than the medium concentration of parent drug. A simple isolation scheme included methanolic extraction and anion-exchange chromatography to recover the target triphosphate. Mass spectral analysis and selective enzyme degradation provided structural confirmation of the purified product. Biological activity of the purified GCV-TP was demonstrated by competitive inhibition experiments using human DNA polymerase alpha and HSV DNA polymerase that showed substantially greater sensitivity for the viral polymerase in agreement with previous reports. The GCV-TP obtained was further used to enzymatically prepare GCV mono- and diphosphate in high yield. This method provides an easily scalable means of preparing milligram amounts of the triphosphates of pharmacologically active acyclic nucleosides like ganciclovir. |
| Databáze: | OpenAIRE |
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