Tiazofurin is phosphorylated by three enzymes from Chinese hamster ovary cells.

The growth inhibitory activity of tiazofurin toward adenosine kinase deficient Chinese hamster ovary (CHO) cells was partially reversed by the presence of nicotinamide riboside. Similarly, the formation of tiazofurin 5'-monophosphate and the active metabolite, tiazofurin 5'-adenine dinucleotide could be partially inhibited by 100 microM nicotinamide riboside in CHO cells and substantially inhibited (80-90%) in adenosine kinase deficient cells. Tiazofurin phosphorylating activity from CHO cell extracts was resolved into two peaks by DEAE-cellulose chromatography. The first peak of activity was identified as adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20). The second peak of activity correlated with a previously described 3-deazaguanosine phosphorylating activity that was identified as a nicotinamide ribonucleoside kinase. Contaminating purine nucleoside phosphorylase was removed by sedimentation through a sucrose density gradient which also resolved the tiazofurin phosphorylating activity into two peaks, one requiring just ATP and the other requiring both ATP and IMP. Of the substrates tested with the lower density peak, nicotinamide riboside was most efficient and was the only natural substance that competed well with tiazofurin for phosphorylation, substantiating its suggested identity as a nicotinamide ribonucleoside kinase. The apparent Km value for nicotinamide riboside (2 microM) was significantly less than that for tiazofurin (13.6 microM). ATP was the best phosphate donor; CTP and UTP were utilized less efficiently and IMP did not support the reaction. The best substrate for the higher density peak of tiazofurin phosphorylation was inosine and both ATP and IMP were required for the reaction, suggesting its identity as a 5'-nucleotidase. In summary, it appears that adenosine kinase, nicotinamide ribonucleoside kinase, and 5'-nucleotidase may all contribute to the phosphorylation of tiazofurin in CHO cells.

Phosphorylation of 3-deazaguanosine by nicotinamide riboside kinase in Chinese hamster ovary cells.

The growth inhibitory activity of 3-deazaguanosine toward a mutant line (TGR-3) of Chinese hamster ovary cells deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was substantially reversed by the simultaneous addition of nicotinamide riboside. The activities of most other ribonucleoside analogues tested were unaffected. The formation of cellular 3-deazaGMP and 3-deazaGTP from the ribonucleoside analogue, as measured by high-pressure liquid chromatography, was inhibited by the presence of nicotinamide riboside. The inhibition was dependent on concentration of 3-deazaguanosine and could also be demonstrated by following the metabolism of 3-deazaguanosine, labeled with 14C in the ribose moiety, to [14C]3-deazaGTP. In the presence of 100 microM nicotinamide riboside formation of the labeled triphosphate derivative of 3-deazaguanosine was undetectable. A 3-deazaguanosine phosphorylating activity was separated from other cellular kinases by DEAE-cellulose chromatography. Contaminating purine nucleoside phosphorylase (EC 2.4.2.1) was subsequently removed by sucrose density gradient centrifugation. The resulting enzyme preparation demonstrated the greatest activities with nicotinamide riboside and 3-deazaguanosine and, in addition, could also phosphorylate tiazofurin and guanosine to lesser, but significant, degrees. These and other observations suggest that 3-deazaguanosine, and perhaps other agents such as tiazofurin, may, at least in part, be phosphorylated by a nicotinamide ribonucleoside kinase in these cells. If so, it is possible that the activity of this agent in other types of cells in vivo could be dependent upon the presence of this enzyme and that it could be influenced by cellular concentrations of the natural pyridine nucleoside.

Use of tiazofurin to enhance the metabolism and cytotoxic activities of analogues of guanine, guanosine, and deoxyguanosine.

An effective modulator of cellular guanine nucleotide pools, 2-beta-D-ribofuranosylthiazole-4-carboxamide (tiazofurin) was tested for its ability to affect utilization of certain guanine, guanosine, and deoxyguanosine analogues by Chinese hamster ovary cells and hypoxanthine guanine phosphoribosyltransferase (HGPRTase)-deficient variants. The nucleoside analogues investigated were chosen for their potential to be metabolized to the nucleotide level by pathways other than those requiring the action of HGPRTase. Exposure of tiazofurin-treated (500 microM for 3 h) cells to 3-deazaguanosine (200 microM for 3 h) resulted in enhanced 3-deazaGTP formation and an increase (5-10-fold) in the ratio 3-deazaGTP/GTP. Tiazofurin treatment also stimulated [3H]deoxyguanosine utilization (8-fold) by HGPRTase-deficient cells, and accordingly, greatly increased the cytotoxicity of 2'-deoxy-3-deazaguanosine and arabinosylguanine. This study emphasizes the potential usefulness of tiazofurin in sequential combination with appropriate analogues of guanosine and deoxyguanosine in a clinical setting and as a tool in studying the metabolism of these agents.

3-Deazaguanosine is metabolized to the triphosphate derivative in Chinese hamster cells deficient in hypoxanthine-guanine phosphoribosyltransferase.

3-Deazaguanosine containing a 14C label in the ribose moiety was prepared using [U-14C]inosine as the [14C] ribose donor and commercial purine-nucleoside phosphorylase (EC 2.4.2.1) both to degrade the inosine, in the presence of phosphate, and to synthesize [14C-ribosyl]3-deazaguanosine in reduced phosphate and an excess of 3-deazaguanine. Purification was by high-pressure liquid chromatography (HPLC). [14C-ribosyl]3-Deazaguanosine was metabolized by Chinese hamster ovary cells to two metabolites, one major and one minor, eluting in the triphosphate region after HPLC analysis, and appeared to be incorporated into perchloric acid-insoluble material. Cell line TGR-3, deficient in hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) and resistant to 3-deazaguanine, also formed both metabolites. Line TGR-1/DGRR-9, deficient in hypoxanthine-guanine phosphoribosyltransferase and resistant to both 3-deazaguanine and 3-deazaguanosine, formed greatly reduced levels of the major metabolite. 3-Deazaguanosine 5'-triphosphate, prepared enzymically from authentic 3-deazaguanosine 5'-monophosphate, co-eluted with the major metabolite peak during HPLC analysis. Treatment of a metabolite-containing extract with bacterial alkaline phosphatase (EC 3.1.3.1) resulted in the formation of 3-deazaguanosine. These observations indicate that 3-deazaguanosine can be metabolized, in Chinese hamster ovary cells, to the triphosphate derivative in lieu of the action of hypoxanthine-guanine phosphoribosyltransferase.

Characteristics of magnesium-adenosine triphosphate-dependent inactivators of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The inhibition of homogeneous 3-hydroxy-3-methyl-glutaryl coenzyme A reductase by MgATP-dependent inactivators isolated from rat liver cytosol and microsomes was examined. The inhibition was independent of incubation time. The inhibition was readily reversed by dilution or dialysis, by the addition of EDTA, and by incubating the inhibited enzyme with glycerol and glycerol kinase to convert the ATP to ADP. The inactivated enzyme was not reactivated by various phosphatases. When inactivated in the presence of [gamma-32P]ATP, no radioactivity was incorporated into the reductase. These observations indicate that the inactivators do not exert their effects through covalent modification of the reductase.