Sue C. Shaddix

Metabolism and Metabolic Actions of 6-Methylpurine and 2-Fluoroadenine in Human Cells

Activation of purine nucleoside analogs by Escherichia coli purine nucleoside phosphorylase (PNP) is being evaluated as a suicide gene therapy strategy for the treatment of cancer. Because the mechanisms of action of two toxic purine bases, 6-methylpurine (MeP) and 2-fluoroadenine (F-Ade), that are generated by this approach are poorly understood, mechanistic studies were initiated to learn how these compounds differ from agents that are being used currently. The concentration of F-Ade, MeP, or 5-fluorouracil required to inhibit CEM cell growth by 50% after a 4-hr incubation was 0.15, 9, or 120 microM, respectively. F-Ade and MeP were also toxic to quiescent MRC-5, CEM, and Balb 3T3 cells. Treatment of CEM, MRC-5, or Balb 3T3 cells with either F-Ade or MeP resulted in the inhibition of protein, RNA, and DNA syntheses. CEM cells converted F-Ade and MeP to F-ATP and MeP-ribonucleoside triphosphate (MeP-R-TP), respectively. The half-life for disappearance of HeP-ribonucleoside triphosphate from CEM cells was approximately 48 hr, whereas the half-lives of F-ATP and ATP were approximately 5 hr. Both MeP and F-Ade were incorporated into the RNA and DNA of CEM cells. These studies indicated that the mechanisms of action of F-Ade and MeP were quite different from those of other anticancer agents, and suggested that the generation of these agents in tumor cells by E. coli PNP could result in significant advantages over those generated by either herpes simplex virus thymidine kinase or E. coli cytosine deaminase. These advantages include a novel mechanism of action resulting in toxicity to nonproliferating and proliferating tumor cells and the high potency of these agents during short-term treatment.

Heterocyclic thiosemicarbazones: correlation between structure, inhibition of ribonucleotide reductase, and inhibition of DNA viruses.

Summary The thiosemicarbazones of 2-formylpyridine, 3-formylpyridine, 4-for-mylpyridine, 5-hydroxy-2-formylpyridine, 1-formylisoquinoline, 5-hydroxy-1-formylisoquinoline, 6-formylpurine, isatin, and 1-methylisatin were examined for activity against herpes simplex virus in H.Ep.−2 cells and human cytomegalovirus in WI-38 cells, and also for inhibition of ribonucleotide reductase activity in H.Ep.−2 cells. A correlation was seen between inhibition of reductase and antiviral activity, with those compounds having the -CH=N-NH-C(=S) -NH2 moiety affixed to the heterocyclic ring system in the position alpha to the ring nitrogen being active. The suggestion is made that the activity of ribonucleotide reductase may be a limiting factor in the replication of certain members of the herpesvirus group.

Metabolism of 4′-thio-β-d-arabinofuranosylcytosine in CEM cells

Because of the excellent in vivo activity of 4′-thio-β-d-arabinofuranosylcytosine (T-araC) against a variety of human solid tumors, we have studied its metabolism in CEM cells to determine how the biochemical pharmacology of this compound differs from that of β-d-arabinofuranosylcytosine (araC). Although there were many quantitative differences in the metabolism of T-araC and araC, the basic mechanism of action of T-araC was similar to that of araC: it was phosphorylated to T-araC-5′-triphosphate (T-araCTP) and inhibited DNA synthesis. The major differences between these two compounds were: (i) T-araC was phosphorylated to active metabolites at 1% the rate of araC; (ii) T-araCTP was 10- to 20-fold more potent as an inhibitor of DNA synthesis than was the 5′-triphosphate of araC (araCTP); (iii) the half-life of T-araCTP was twice that of araCTP; (iv) the catalytic efficiency of T-araC with cytidine deaminase was 10% that of araC; and (v) the 5′-monophosphate of araC was a better substrate for deoxycytidine 5′-monophosphate deaminase than was the 5′-monophosphate of T-araC. Of these differences in the metabolism of these two compounds, we propose that the prolonged retention of T-araCTP is a major factor contributing to the activity of T-araC against solid tumors. The data in this study represent another example of how relatively small structural changes in nucleoside analogs can profoundly affect the biochemical activity.

The Synthesis and Biological Activity of Certain 4′-Thionucleosides

Abstract Results are presented on the synthesis and biological activity of several types of 4′-thionucleosides as potential anticancer agents. Detailed studies on the mechanism of action of 4′-thiothymidine are also presented.

Biochemical aspects of chemotherapy of mouse colon carcinoma. Fluoropyrimidines and pyrazofurin

Fluorouracil was metabolized to nucleotides and incorporated into RNA of mouse colon carcinomas and normal tissues. No significant difference was observed in three mouse colon tumors, but the extent of incorporation of the analog into RNA of normal colon tissue was lower than that in colon tumors. Fluorouracil phosphoribosyltransferase activity, although low, was observed to be about five times higher in mouse colon carcinomas than it was in normal colon tissue. Fluorouracil and 5‐fluoro‐2′‐deoxyuridine, as anticipated, inhibited the incorporation of [6‐3H]‐2′‐deoxyuridine into DNA of colon carcinomas and normal tissues. Colon and spleen tissue made a more rapid recovery of capacity for DNA synthesis than did colon tumors. In normal tissues examined the recovery from inhibition of DNA synthesis by fluorodeoxyuridine appeared to be more rapid than was recovery from fluorouracil inhibition. Effects of pyrazofurin, an inhibitor of orotidylic acid decarboxylase, on pyrimidine synthesis in mouse colon carcinomas and in normal tissues was analyzed by means of high‐pressure liquid chromatography. Consequences of pyrazofurin‐induced inhibition of pyrimidine biosynthesis in mouse colon carcinomas were evident in decreased pools of pyrimidine ribonucleotides. Orotidylic acid did not accumulate behind this block but elevated levels of orotic acid and orotidine were observed in acid‐soluble extracts. The maximum reduction in uracil ribonucleotide pools was observed 24 hours after pyrazofurin treatment. Recovery of uracil ribonucleotide pools was evident within 48 hours and was complete 72 hours after treatment. The maximum levels of orotic acid and orotidine in colon carcinomas were attained 24 hours after treatment; these levels remained elevated above control levels for 72 hours after pyrazofurin treatment. The pool of uracil ribonucleotides was not depressed in colon and spleen tissue from pyrazofurin‐treated animals; nevertheless, pronounced elevation of orotic acid and orotidine levels in these normal tissues was observed. These results reveal differences in effects of pyrazofurin on pyrimidine ribonucleotide pools in mouse colon carcinoma and in colon tissue. These differences may be due in part to availability or extent of utilization of exogenous pyrimidines or precursors of pyrimidines. Such differences may be exploited by means of scheduled combination chemotherapy with inhibitors of pyrimidine synthesis and pyrimidine analogs.

Use of enzyme-deficient cell culture lines as a biochemical screen for study of purines, purine nucleosides and related compounds.

Abstract From H.Ep. No. 2 cells in culture, two new sublines have been selected for resistance to certain cytotoxic purines and purine nucleosides. These new sublines, together with five resistant sublines previously described, constitute a series of cell lines deficient in the following enzymes, either singly or in combinations: adenine phosphoribosyltransferase (EC 2.4.2.7), hypoxanthine (guanine) phosphoribosyltransferase (EC 2.4.2.8) and adenosine kinase (EC 2.7.1.20). Data are presented illustrative of the usefulness of these cell lines as a convenient biochemical screen that provides an indication of the pathways of anabolism of cytotoxic purines, purine nucleosides or analogs of purines and purine nucleosides.

Alterations in nucleotide pools induced by 3-deazaadenosine and related compounds role of adenylate deaminase

3-Deazaadenine, 3-deazaadenosine, and the carbocyclic analog of 3-deazaadenosine produced similar effects on nucleotide pools of L1210 cells in culture: each caused an increase in IMP and a decrease in adenine nucleotides and had no effect on nucleotides of uracil and cytosine. Concentrations of 50-100 microM were required to produce these effects. Although 3-deazaadenosine and carbocyclic 3-deazaadenosine are known to be potent inhibitors of adenosylhomocysteine hydrolase, the effects on nucleotide pools apparently are not mediated via this inhibition because they are also produced by the base, 3-deazaadenine, and because the concentrations required are higher than those required to inhibit the hydrolase. Cells grown in the presence of 3-deazaadenine or 3-deazaadenosine contained phosphates of 3-deazaadenosine (the mono- and triphosphates were isolated); from cells grown in the presence of the carbocyclic analog of 3-deazaadenosine, the monophosphate was isolated, but evidence for the presence of the triphosphate was not obtained. A cell-free supernatant fraction from L1210 cells supplemented with ATP catalyzed the formation of monophosphates from 3-deazaadenosine or carbocyclic 3-deazaadenosine, and a cell-free supernatant fraction supplemented with 5-phosphoribosyl 1-pyrophosphate (PRPP) catalyzed the formation of 3-deaza-AMP from 3-deazaadenine. Adenosine kinase apparently was not solely responsible for the phosphorylation of the nucleosides because a cell line that lacked this enzyme converted 3-deazaadenosine to phosphates. No evidence was obtained that the effects on nucleotide pools resulted from a block of the IMP-AMP conversion, but the results could be rationalized as a consequence of increased AMP deaminase activity. This explanation is supported by two observations: (a) coformycin, an inhibitor of AMP deaminase, prevented the effects on nucleotide pools, and (b) 3-deazaadenine decreased the conversion of carbocyclic adenosine to carbocyclic ATP and increased its conversion to carbocyclic GTP. The latter conversion requires the action of AMP deaminase and the observed effects can be rationalized by a nucleoside analog-mediated increase in AMP deaminase activity. Because these effects on nucleotide pools are produced only by concentrations higher than those required to inhibit adenosylhomocysteine hydrolase, they may not contribute significantly to the biological effects of 3-deazaadenosine or carbocyclic 3-deazaadenosine.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological activities and modes of action of 9-α-d-arabinofuranosyladenine and 9-α-d-arabinofuranosyl-8-azaadenine

Abstract From earlier studies it is known that 9-α- d -arabinofuranosyladenine (α-araA) and 9-α- d -arabinofuranosyl-8-azaadenine (α-ara-8-azaA) bave in vitro antiviral activity, are cytotoxic, and are metabolized in mammalian cells to the triphosphates. This study was designed to compare the in vivo antiviral activities of these compounds and their loci of action with those of 9-β- d -arabinofura-nosyladenine (β-araA). the latter compound selectively inhibits DNA synthesis in intact cells, and its triphosphate is a known inhibitor of DNA polymerases and ribonucleotide reductase. Whereas β-araA was significantly effective in the treatment of systemic herpes simplex virus type 1 (HSV-1) infections in mice, α-araA and α-ara-8-azaA were therapeutically ineffective. α-AraATP at a concentration of ~1 mM did not inhibit (1) DNA polymerases present in crude extracts of cultured H.Ep.-2 cells; (2) DNA polymerases present in extracts of KB cells; (3) partially purified DNA polymerase-α from mouse embryo cells; or (4) DNA polymerases induced by HSV-1 and HSV-2. DNA polymerase-β from mouse embryo cells was inhibited to a small extent by 10−4 M α-araATP. In contrast, all of these enzymes were inhibited by β-araATP at a concentration of 10−5M (as shown in these or in earlier studies). the reductions of CDP and UDP by ribonucleotide reductase from L1210 cells were not inhibited by αaraATP (~10−3M), whereas β-araATP produced 70–80 per cent inhibition at this concentration. In cultured H.Ep.-2 cells, α-ara-8-azaA inhibited the incorporation of thymidine, uridine, and formate into macromolecules, but it was without effect on the incorporation of adenine and hypoxanthine, and produced marginal inhibition of the incorporation of leucine. α-Ara-8-azaA produced a dose-dependent inhibition of the accumulation of [14C] formyl-glycinamide ribonucleotide in H.Ep.-2 cells treated with azaserine and [14C] formate. These results indicate that the α-nucleosides inhibit nucleic acid synthesis by mechanisms different from those of β-araA.

Metabolism of O6-Propyl and N6-Propyl-carbovir in CEM Cells

Abstract The metabolism of O6-propyl-carbovir and N6-propyl-carbovir, two selective inhibitors of HIV replication, has been evaluated in CEM cells. Both compounds were phosphorylated in intact cells to carbovir-5′-triphosphate. The metabolism of these two agents was inhibited by deoxycoformycin and mycophenolic acid, but not erythro-9-(2-hydroxy-3-nonyl)adenine. No evidence of the 5′-triphosphate of either compound was detected in CEM cells.