Ming S. Chen

[45] Thymidine kinase from Escherichia coli

Publisher Summary This chapter describes the purification procedure of thymidine kinase from Escherichia coli. Thymidine kinase from E. coli is an unusual allosteric enzyme whose most striking characteristic is its regulation not only by the end-product deoxythymidine triphosphate (dTTP) but also by a number of nucleoside di and triphosphates. Dimerization of the enzyme molecule accounts for the regulatory properties, and this dimer molecule has either an active or an inactive conformation, depending on the specific nature of the effector nucleotide with which it interacts. Frozen E. coli B cells that had been grown in Kornbergs medium and harvested in late log phase, were purchased from General Biochemicals and stored at -70°. Enzymic activity is assayed by the conversion of labeled deoxythymidine to deoxythymidine monophosphate, with separation of the substrate and product by high-voltage paper electrophoresis, column chromatography, thin-layer chromatography, paper chromatography, or disc diethylaminoethyl (DEAE)-cellulose.

In vitro characterization of the anti-human cytomegalovirus activity of PMEA (Adefovir)

PMEA [9-[2-(phosphonomethoxy)ethyl]adenine; adefovir] has shown anti-cytomegalovirus activity in animal models and in preliminary human trials. PMEA diphosphate (PMEApp), the active antiviral metabolite of PMEA, is a potent inhibitor of human cytomegalovirus (HCMV) DNA polymerase. PMEA is efficiently taken up and phosphorylated to PMEApp in numerous human cell lines. In vitro replication of wild type and drug resistant HCMV clinical isolates is effectively inhibited by PMEA. PMEA in combination with other anti-HCMV agents shows additive inhibition of HCMV replication.

MOLECULAR BASIS FOR SERENDIPITOUS DEVELOPMENT OF ANTIVIRAL AND ANTICANCER AMINONUCLEOSIDES

Publisher Summary This chapter discusses molecular basis for serendipitous development of antiviral and anticancer aminonucleosides. The development of antiviral nucleosides may be considered to have gone through three phases. During the first period, it is generally accepted that a nontoxic antiviral agent could not be found, because a virus not only must replicate within a host cell but also in so doing utilizes much of the host cells metabolic processes. The inhibition of viral replication is assumed to be a consequence of inhibition of the host cell and hence, any antiviral agent would be too toxic for practical use.