Behavior of thymidylate kinase toward monophosphate metabolites and its role in the metabolism of 1-(2'-deoxy-2'-fluoro-beta-L-arabinofuranosyl)-5-methyluracil (Clevudine) and 2',3'-didehydro-2',3'-dideoxythymidine in cells.

L-nucleoside analogs are a new class of antiviral and anticancer agents, several of which are currently used in the clinic. The phosphorylation of these agents to the triphosphate form is thought to be important for exertion of their pharmacological activities. 1-(2'-deoxy-2'-fluoro-beta-L-arabinofuranosyl)-5-methyluracil (L-FMAU; Clevudine) is a thymidine analog that is currently under phase III clinical trials as an anti-human hepatitis B virus agent. We examined the behavior of its monophosphate metabolite with human recombinant thymidylate kinase (TMPK) and showed that L-FMAU monophosphate (L-FMAUMP) is a poorer substrate than its D-configuration anomer (D-FMAUMP). The phosphorylation efficiency of l-FMAUMP is similar to that of the monophosphate of 2',3'-didehydro-2',3'-dideoxythymidine (d4T), an anti-human immunodeficiency virus analog, both of which are approximately 1% TMP. To clarify the role of human TMPK in the phosphorylation of L-FMAUMP to the diphosphate metabolite in cells, a Tet-On inducible human TMPK cell line system was established. In this system, the expression of TMPK is closely regulated in response to various concentrations of doxycycline. When the cells were treated with L-FMAU or d4T, the amounts of the diphosphate and triphosphate metabolites of these analogs were increased, in accordance with an increase in human TMPK activity in cells. In conclusion, this is the first demonstration of the behavior of TMPK toward L-FMAUMP. This study indicates that human TMPK can phosphorylate L-FMAUMP and play a critical role in L-FMAU metabolism in cells.

Better preservation of the peritoneum in rats exposed to amino acid-based peritoneal dialysis fluid.

BACKGROUND: Glucose-containing peritoneal dialysis fluids (PDF) show impaired biocompatibility, which is related partly to their high glucose content, presence of glucose degradation products, low pH, and lactate buffer, or a combination of these factors. In a rat chronic peritoneal exposure model, we compared effects of an amino acid-based PDF (AA-PDF) with a glucose-containing PDF on the peritoneal microcirculation and morphology. METHOD: Two groups of rats received 10 mL of either fluid daily for 5 weeks via peritoneal catheters connected to implanted subcutaneous mini vascular access ports. Leukocyte-endothelium interactions in the mesenteric venules were investigated by intravital microscopy. Quantification of angiogenesis and fibrosis and inspection of the mesothelial cell layer were performed by light and electron microscopy. RESULTS: Daily exposure to glucose-containing PDF resulted in a significant increase in the number of rolling leukocytes in mesenteric venules, whereas instillation of AA-PDF did not change the level of leukocyte rolling. Glucose-containing PDF evoked a significantly higher number of milky spots in the omentum, whereas this response was significantly reduced in animals exposed to the AA-PDF (p < 0.02). Chronic instillation of glucose-containing PDF induced angiogenesis in various peritoneal tissues, accompanied by fibrosis in the mesentery and parietal peritoneum. Quantitative morphometric evaluation of omentum and mesentery showed a clear trend toward less angiogenesis after treatment with the AA-PDF compared to the glucose-containing PDF, which reached statistical significance in the parietal peritoneum (p < 0.04). Instillation of AA-PDF resulted in approximately 50% reduction of fibrosis in the mesentery (p < 0.04) and approximately 25% reduction in the parietal peritoneum (p < 0.009) compared to glucose-containing PDF. Glucose-containing PDF damaged the mesothelial cell layer, whereas the mesotheium was intact after AA-PDF treatment, as evidenced by electron microscopy. CONCLUSION: Our data in a rat chronic peritoneal exposure model clearly demonstrate reduced immune activation (evidenced by decreased number of rolling leukocytes and decreased induction of omental milky spots) and reduced neoangiogenesis, fibrosis, and mesothelial damage of the peritoneal membrane after treatment with AA-PDF compared to glucose-containing PDF.

Crystal structure of varicella zoster virus thymidine kinase.

Herpes virus thymidine kinases are responsible for the activation of nucleoside antiviral drugs including (E)-5-(2-bromovinyl)-2'-deoxyuridine. Such viral thymidine kinases (tk), beside having a broader substrate specificity compared with host cell enzymes, also show significant variation in nucleoside phosphorylation among themselves. We have determined the crystal structure of Varicella zoster virus (VZV, human herpes virus 3) thymidine kinase complexed with (E)-5-(2-bromovinyl)-2'-deoxyuridine 5'-monophosphate and ADP. Differences in the conformation of a loop region (residues 55-61) and the position of two alpha-helices at the subunit interface of VZV-tk compared with the herpes simplex virus type 1 (human herpes virus 1) enzyme give rise to changes in the positioning of residues such as tyrosine 66 and glutamine 90, which hydrogen bond to the substrate in the active site. Such changes in combination with the substitution in VZV-tk of two phenylalanine residues (in place of a tyrosine and methionine), which sandwich the substrate pyrimidine ring, cause an alteration in the positioning of the base. The interaction of the (E)-5-(2-bromovinyl)-2'-deoxyuridine deoxyribose ring with the protein is altered by substitution of tyrosine 21 and phenylalanine 139 (analagous to herpes simplex virus type 1 histidine 58 and tyrosine 172), which may explain some of the differences in nucleoside sugar selectivity between both enzymes. The altered active site architecture may also account for the differences in the substrate activity of ganciclovir for the two thymidine kinases. These data should be of use in the design of novel antiherpes and antitumor drugs.