Biochemical Basis for the Anti-Herpes and Cytotoxic Activities of Nitrogen(4)-Derivatives of 2-Acetylpyridine Thiosemicarbazone (Antiviral,Ribonucleotide, Reductase).

Abstract

Previous studies in our laboratory have shown that N('4)-substituted derivatives of 2-acetylpyridine thiosemicarbazone are potent inhibitors of the replication of herpes simplex virus (HSV) types 1 and 2 in vitro and in vivo. This dissertation examines the mode of antiviral action of these compounds and the basis for their accompanying cytotoxicity. Studies in uninfected KB cells (an established cell line of human origin) indicated that the parent compound, 2-acetylpyridine thiosemicarbazone, reversibly inhibited DNA synthesis within 30 minutes after exposure to concentrations which had little effect on RNA and protein synthesis. Results obtained from deoxynucleoside antagonism experiments, an in situ assay for thymidylate synthetase, and nucleoside incorporation studies suggested that ribonucleoside diphosphate reductase was the site of drug inhibition. Viral DNA synthesis also has been shown by others in our laboratory to be inhibited by these compounds. Studies utilizing nuclei isolated from HSV-2-infected KB cells indicated that neither the herpes DNA polymerase nor the DNA template were sites of drug inhibition. Cellular and viral ribonucleoside diphosphate reductases were partially purified from uninfected and HSV-1-infected KB cells, respectively, by sequential salt fractionation with streptomycin sulfate and (NH(,4))(,2)SO(,4). The cellular reductase was further purified by affinity column chromatography. A reductase assay was developed which utilized thin-layer chromatography on polyethyleneimine-impregnated cellulose and development in a borate-containing solvent to separate reaction products converted to monophosphates by hydrolysis with HClO(,4). After characterization of the viral and cellular reductases, the effects of five derivatives of 2-acetylpyridine thiosemicarbazone on the two enzymes were examined. All five derivatives were potent inhibitors of the viral reductase. Furthermore, three of the derivatives were found to be more potent as inhibitors of the viral reductase by a factor of 2, 8, and >35, based on a comparison of 50% inhibitory concentrations. Kinetic studies indicated that inhibition of the viral reductase was not competitive with respect to either cytidine-5'-diphosphate or dithiotheitol.