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During the last few years, many gene therapy strategies have been developed for various disease targets. The development of anticancer gene therapy strategies to selectively generate cytotoxic nucleoside or nucleotide analogs is an attractive goal. One such approach involves the delivery of herpes simplex virus thymidine kinase followed by the acyclic nucleoside analog ganciclovir. We have developed another gene therapy methodology for the treatment of cancer that has several significant attributes. Specifically, our approach involves the delivery of E. coli purine nucleoside phosphorylase, followed by treatment with a relatively non-toxic nucleoside prodrug that is cleaved by the enzyme to a toxic compound. This presentation describes the concept, details our search for suitable prodrugs, and summarizes the current biological data.

In an effort to understand biochemical features that are important to the selective antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine [Cl-F( upward arrow)-dAdo], we evaluated the biochemical pharmacology of three structurally similar compounds that have quite different antitumor activities. Cl-F( upward arrow)-dAdo was 50-fold more potent as an inhibitor of CEM cell growth than were either 2-chloro-9-(2-deoxy-2-fluoro-beta-D-ribofuranosyl)adenine [Cl-F( downward arrow)-dAdo] or 2-chloro-9-(2-deoxy-2, 2-difluoro-beta-D-ribofuranosyl)adenine [Cl-diF( upward arrow downward arrow)-dAdo]. The compounds were similar as substrates of deoxycytidine kinase. Similar amounts of their respective triphosphates accumulated in CEM cells, and the rate of disappearance of these metabolites was also similar. Cl-F( upward arrow)-dAdo was 10- to 30-fold more potent in its ability to inhibit the incorporation of cytidine into deoxycytidine nucleotides than either Cl-F( downward arrow)-dAdo or Cl-diF( upward arrow downward arrow)-dAdo, respectively, which indicated that ribonucleotide reductase was differentially inhibited by these three compounds. Thus, the differences in the cytotoxicity of these agents toward CEM cells were not related to quantitative differences in the phosphorylation of these agents to active forms but can mostly be accounted for by differences in the inhibition of ribonucleotide reductase activity. Furthermore, the inhibition of RNA and protein synthesis by Cl-F( downward arrow)-dAdo and Cl-diF( upward arrow downward arrow)-dAdo at concentrations similar to those required for the inhibition of DNA synthesis can help explain the poor antitumor selectivity of these two agents because all cells require RNA and protein synthesis.

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.

The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.

We have developed a new strategy for the gene therapy of cancer based on the activation of purine nucleoside analogs by transduced E. coli purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). The approach is designed to generate antimetabolites intracellularly that would be too toxic for systemic administration. To determine whether this strategy could be used to kill tumor cells without host toxicity, nude mice bearing human malignant D54MG glioma tumors expressing E. coli PNP (D54-PNP) were treated with either 6-methylpurine-2'-deoxyriboside (MeP-dR) or arabinofuranosyl-2-fluoroadenine monophosphate (F-araAMP, fludarabine, a precursor of F-araA). Both prodrugs exhibited significant antitumor activity against established D54-PNP tumors at doses that produced no discernible systemic toxicity. Significantly, MeP-dR was curative against this slow growing solid tumor after only 3 doses. The antitumor effects showed a dose dependence on both the amount of prodrug given and the level of E. coli PNP expression within tumor xenografts. These results indicated that a strategy using E. coli PNP to create highly toxic, membrane permeant compounds that kill both replicating and nonreplicating cells is feasible in vivo, further supporting development of this cancer gene therapy approach.

Carbovir (CBV) is a guanine nucleoside analog with potent in vitro anti-HIV activity. A prodrug of CBV is currently being evaluated in clinical trials as a potential agent for the treatment of AIDS. The ability of CBV to inhibit mitochondrial DNA synthesis in intact CEM cells was evaluated in the present study, because most of the currently available anti-HIV nucleoside analogs have significant toxicities that result from their inhibition of mitochondrial DNA synthesis. No delayed cytotoxicity was observed in CEM cells treated with 50 microM CBV for 4 weeks. In addition, CBV at concentrations as high as 1 mM did not cause a decline in mitochondrial DNA levels and only minimally increased the concentration of lactic acid in the medium. In contrast to these results with CBV, treatment of CEM cells with 0.5 microM 2',3'-dideoxycytidine resulted in delayed cytotoxicity, a decrease in mitochondrial DNA content and increases in lactic acid levels in the medium. These results indicated that treatment of CEM cells with CBV did not result in the inhibition of mitochondrial DNA synthesis and suggested that treatment of AIDS patients with CBV, or a prodrug of CBV, would not result in some of the toxicities seen with the other anti-HIV nucleoside analogs.

4'-Thiothymidine (S-dThd) is a potent inhibitor of L1210 cell growth and is active against P388 leukemia in mice. Because of these activities and its novel structure, we have begun studies of its metabolism and metabolic actions in L1210 cells in order to understand its mechanism of cytotoxicity, S-dThd inhibited the incorporation of radiolabeled precursors into DNA, but did not inhibit the incorporation of either uridine or leucine into RNA or protein, respectively, which indicated that the mechanism of its toxicity was due to its inhibition of DNA synthesis. S-dThd did not decrease the concentration of any of the natural deoxynucleoside triphosphates, which indicated that its cytotoxicity was not due to the inhibition of ribonucleotide reductase. S-dThd was readily phosphorylated and used as a substrate for DNA synthesis. Because the rate of incorporation of S-dThd into DNA was 20% that of thymidine, it is likely that the mechanism of action of S-dThd is not due to inhibition of DNA polymerases by the 5'-triphosphate of S-dThd, but instead to its incorporation into the DNA and its subsequent disruption of some function of DNA.

Inefficiency of gene delivery, together with inadequate bystander killing, represent two major hurdles in the development of a toxin-mediated gene therapy for human malignancy. The product of the Escherischia coli DeoD gene (purine nucleoside phosphorylase, PNP) differs from the mammalian enzyme in its substrate specificity and is capable of catalyzing the conversion of several non-toxic deoxyadenosine analogs to highly toxic adenine analogs. We have found that expression of E. coli PNP in < 1% of a human colonic carcinoma cell line leads to the death of virtually all bystander cells after treatment with 6-methyl-purine-2'-deoxyribonucleoside, a deoxyadenosine analog that is a substrate for E. coli PNP but not human PNP. Minimal toxicity was observed in non-transfected or E. coli LacZ transfected cells that were treated with this compound. These results establish a rational approach to achieve significant bystander killing, even after gene transfer to only a small fraction of tumor cells.

6-Thio-2'-deoxyguanosine (T-dGuo) has been reported to be both phosphorylated by deoxycytidine kinase and converted to 6-thioguanine by purine nucleoside phosphorylase (PNP). Combination of T-dGuo with an inhibitor of PNP would be expected to generate the 5'-triphosphate of T-dGuo and limit or prevent the formation of 6-thioguanosine triphosphate. Because the incorporation of 6-thioguanine into DNA is believed to be primarily responsible for the antitumor activity of the thiopurines, this treatment might result in enhanced activity against certain tumors, particularly those of T-cell origin. We have evaluated the metabolic basis of this strategy by examining the effects of 9-benzyl-9-deazaguanine (BDG), a potent inhibitor of PNP, on the metabolism of T-dGuo in CEM cells. The concentration of T-dGuo required to inhibit cell growth by 50% was approximately 50-fold greater in the presence of 8.0 microM BDG than in its absence. As expected, the addition of BDG to cells treated with T-dGuo prevented the metabolism of T-dGuo to 6-thio-guanine-containing ribo-nucleotides, but, unexpectedly, no 6-thio-2'-deoxyguanosine 5'-triphosphate was detected. In cells treated with T-dGuo plus BDG, the major phosphorylated metabolite was T-dGMP. These results indicated that even in the absence of PNP activity, T-dGuo cannot be phosphorylated directly to 6-thio-2'-deoxyguanosine 5'-triphosphate due to the inability of guanylate kinase to utilize T-dGMP as a substrate.

Cyclopentadiene was converted in six steps to the key intermediate (+/-)-(1 alpha,2 beta,4 alpha)-4-amino-2-(benzyloxy)cyclopentanol (10), which in turn was converted to the carbocyclic nucleoside analogs 14 and 19 by standard procedures developed in these laboratories. Compounds 14 and 19 were then further converted to the target phosphonates 1b and 2b by modification of literature procedures. The phosphonate 1b was 40-fold more cytotoxic to HEp-2 cells than its parent, CDG, presumably after conversion to the diphosphoryl phosphonate.

CdG, the carbocyclic analog of 2'-deoxyguanosine, is active against herpes, hepatitis B, and human cytomegaloviruses. We have studied the interaction of the tritiated enantiomers of CdG with the herpes simplex virus type 1-specific thymidine kinase (HSV-1 TK) and have examined their metabolism in uninfected and HSV-1-infected cells. D- and L-CdG were equally effective competitive inhibitors of the phosphorylation of thymidine (dThd) by the partially purified HSV-1 TK (Ki values were 2.1 and 3.4 microM, respectively) and were also equal as substrates (Km values were 17 and 26 microM, respectively, and Vmax values of the enantiomers were equal and about 50% greater than the Vmax for dThd). The partially purified enzyme preparation, which contained cellular nucleotide kinase activities (pyruvate kinase also was present in the assay medium), converted D-CdG almost exclusively to the triphosphate and L-CdG almost exclusively to the monophosphate. Similarly, in virus-infected cells the D-enantiomer was converted predominantly to the triphosphate and the L-enantiomer predominantly to the monophosphate. In uninfected cells the results were qualitatively similar. In CEM cells deoxycytidine (dCyd) kinase (EC 2.7.1.74) seemed to be the enzyme principally responsible for the phosphorylation of both enantiomers, as shown by competition studies. Thus, both the HSV-1 TK and cellular dCyd kinase (of CEM cells) showed no selectivity for the enantiomers of CdG. This lack of enantiomeric specificity has obvious implications for the design of inhibitors of both viral proliferation and cellular metabolism.

Certain derivatives of 9-deazaguanine that contain arylmethyl, heteroarylmethyl or cycloalkylmethyl groups at the 9-position are potent inhibitors of purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1). To determine whether these agents can produce metabolically significant inhibition of PNP in cells and in animals, the authors performed pharmacological studies with a representative member of the series, 9-benzyl-9-deazaguanine (BzDAG). BzDAG was a potent inhibitor of PNP from calf spleen (Ki = 12 nM). It was also an effective inhibitor of PNP in cells and in animals as shown by the findings that it 1) inhibited the conversion of inosine to nucleotides in L1210 cells in culture at concentrations that had little effect on the utilization of hypoxanthine; 2) potentiated the toxicity of deoxyguanosine to CCRF-CEM cells in culture; 3) increased the pools of deoxy GTP in CCRF-CEM, Molt-3 and Molt-4 cells that had been treated with deoxyguanosine; 4) prevented the toxicity of 6-thioguanosine to HEp-2 cells in culture; 5) increased the plasma levels of endogenous inosine in rats; and 6) increased the plasma levels of 2',3'-dideoxyinosine in rats that had received BzDAG and dideoxyinosine in combination. Pharmacokinetic analysis of BzDAG in the rat showed it to be 48% orally bioavailable (at a dose of 5 mg/kg). About 95% of BzDAG was protein bound. After i.v. administration of BzDAG (5 mg/kg), more than 50% of the erythrocyte PNP was inhibited for 40 min. These results indicate that the 9-substituted-9-deazaguanines are potent orally active PNP inhibitors and are therefore of potential clinical interest as immunosuppressive and anti-inflammatory agents.

Carbovir (CBV) [the (--)-enantiomer of the carbocyclic analog of 2',3'-dideoxy-2',3'-didehydroguanosine] is a potent inhibitor of human immunodeficiency virus type 1 (HIV) replication in vitro. We have characterized the metabolism of CBV and its effect on cellular metabolism in an effort to better understand its mechanism of action. CBV was primarily metabolized to the 5'-triphosphate of CBV (CBV-TP) to concentrations sufficient to inhibit HIV reverse transcriptase. Infection of CEM cells with HIV did not affect the metabolism of CBV. In CEM cells, there was no evidence of the degradation of CBV by purine nucleoside phosphorylase. The half-life of CBV-TP in CEM cells was 2.5 h, similar to that of the 5'-triphosphate of zidovudine (AZT). However, unlike the levels of the 5'-triphosphate of AZT, CBV-TP levels declined without evidence of a plateau. CBV did not affect the metabolism of AZT, and AZT did not affect the metabolism of CBV. A small amount of CBV was incorporated into DNA in intact CEM cells, and this incorporation was increased by incubation with mycophenolic acid, an inhibitor of IMP dehydrogenase. CBV specifically inhibited the incorporation of nucleic acid precursors into DNA but had no effect on the incorporation of radiolabeled precursors into RNA or protein. CBV did not decrease the level of TTP, dGTP, dCTP, or dATP. These results suggested that the cytotoxicity of CBV was due to the inhibition of DNA synthesis. Further studies are necessary to identify the target(s) responsible for growth inhibition.

The carbocyclic analog of 2'-deoxyguanosine (CdG) is active against herpes simplex virus (HSV), human cytomegalovirus, and human hepatitis-B virus. In order to understand the mechanism of action of this compound against HSV, we have evaluated (a) the incorporation of [3H]CdG into viral and host DNA in HEp-2 cells infected with HSV and (b) the interaction of the 5'-triphosphate of CdG (CdG-TP) with the HSV DNA polymerase and human DNA polymerases alpha, beta, and gamma (EC 2.7.7.7). Incubation of HSV-1-infected HEp-2 cells with [3H]CdG resulted in the incorporation of CdG into both the HSV and the host cell DNA. These results indicated that CdG-TP was used as a substrate for HSV DNA polymerase and for at least one of the cellular DNA polymerases. Degradation of both viral and host DNA with micrococcal nuclease and spleen phosphodiesterase indicated that CdG was incorporated primarily into internal positions in both DNAs. The viral DNA containing CdG sedimented in neutral and alkaline sucrose gradients in the same way as did viral DNA labeled with [3H]thymidine, indicating that the HSV DNA containing CdG was similar in size to untreated HSV DNA. CdG-TP was a competitive inhibitor of the incorporation of dGTP into DNA by the HSV DNA polymerase (Ki of 0.35 microM) and the human DNA polymerase alpha (Ki of 1 microM). CdG-TP was not a potent inhibitor of either DNA polymerase beta or gamma. Using DNA-sequencing technology, CdG-TP was found to be an efficient substrate for HSV DNA polymerase. Incorporation of CdG monophosphate (CdG-MP) into the DNA by HSV DNA polymerase did not interfere with subsequent chain extension. These results suggested that the antiviral activity of CdG was due to its incorporation into the DNA and subsequent disruption of viral functions. In contrast, CdG-TP was not as good as dGTP as a substrate for DNA synthesis by DNA polymerase alpha, and incorporation of CdG-MP by DNA polymerase alpha inhibited further DNA chain elongation.

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine (Cl-F-ara-A) has activity against the P388 tumor in mice on several different schedules. Biochemical studies with a chronic myelogenous leukemia cell line (K562) grown in cell culture have been done in order to better understand its mechanism of action. Cl-F-ara-A was a potent inhibitor of K562 cell growth. Only 5 nM inhibited K562 cell growth by 50% after 72 h of continuous incubation. The 5'-triphosphate of Cl-F-ara-A was detected by strong anion exchange chromatography of the acid-soluble extract of K562 cells incubated with Cl-F-ara-A. Competition studies with natural nucleosides suggested that deoxycytidine kinase was the enzyme responsible for the metabolism to the monophosphate. Incubation of K562 cells for 4 h with 50 nM Cl-F-ara-A inhibited the incorporation of [3H]thymidine into the DNA by 50%. Incubation with 0.1, 1, or 10 microM Cl-F-ara-A for 4 h depressed dATP, dCTP, and dGTP pools but did not affect TTP pools. Similar inhibition of deoxyribonucleoside triphosphate pools was seen after incubation with 2-chloro-2'-deoxyadenosine. Both Cl-F-ara-ATP and Cl-dATP potently inhibited the reduction of ADP to dADP in crude extracts of K562 cells (concentration producing 50% inhibition, 65 nM). The effect of Cl-F-ara-ATP on human DNA polymerases alpha, beta, and gamma isolated from K562 cells grown in culture was determined and compared with those of Cl-dATP and 9-beta-D-arabinofuranosyl-2-fluoroadenine triphosphate (F-ara-ATP). Cl-F-ara-ATP was a potent inhibitor of DNA polymerase alpha. Inhibition of DNA polymerase alpha was competitive with respect to dATP (Ki of 1 microM). The three analogue triphosphates were incorporated into the DNA by DNA polymerase alpha as efficiently as dATP. The incorporation of Cl-F-ara-AMP inhibited the further elongation of the DNA chain, similarly to that seen after the incorporation of F-ara-AMP. Extension of the DNA chain after the incorporation of Cl-dAMP was not inhibited as much as it was with either Cl-F-ara-AMP or F-ara-AMP. Cl-F-ara-ATP was not a potent inhibitor of DNA polymerase beta, DNA polymerase gamma, or DNA primase.(ABSTRACT TRUNCATED AT 400 WORDS)

Two equilibrative (facilitated diffusion) nucleoside transport processes and a concentrative Na(+)-dependent co-transport process contribute to zero-trans inward fluxes of nucleosides in L1210 mouse leukemia cells. Na(+)-linked inward adenosine fluxes in L1210/AM cells (a clone deficient in adenosine, deoxyadenosine, and deoxycytidine kinase activities) were measured as initial rates of [3H]adenosine influx in medium containing Na+ salts and 10 microM dipyridamole. The Na(+)-linked transporter distinguished between the D- and L-enantiomers of adenosine, the latter being a virtual nonpermeant in the initial-rate assay. Adenine arabinoside, inosine, 2'-deoxyadenosine and 2'-deoxyadenosine derivatives with halogen atoms at the purine C-2 position were recognized as substrates of the Na(+)-linked system because of their inhibition of adenosine (10 microM) fluxes under the condition of Na(+)-dependence with IC50 values ranging between 25 and 183 microM; uridine, deoxycytidine, and cytosine arabinoside (each at 400 microM) inhibited adenosine fluxes by 10-40%. Inward Na(+)-linked adenosine fluxes were saturable with respect to extracellular adenosine and Na+ concentrations [( Na+]o); Km and Vmax values for adenosine influx were 9.4 +/- 2.6 microM and 1.67 +/- 0.2 pmol/microliter cell water/s when [Na+]o was 100 mM. The stoichiometry of Na+:adenosine co-transport, determined by Hill analysis of the dependence of adenosine fluxes on [Na+]o, was 1:1. The thiol-reactive agents, N-ethylmaleimide (NEM), showdomycin and p-chloromercuriphenylsulphonate (pCMPS), inhibited Na(+)-linked adenosine fluxes with IC50 values of 40, 10, and 2 microM, respectively. This inhibition was partially reversed by the presence of adenosine in incubation media containing pCMPS, but not NEM. Thiol groups accessible to pCMPS may be involved in substrate recognition by the transporter and in the permeation step.

Nucleoside permeation in L1210/AM cells is mediated by (a) equilibrative (facilitated diffusion) transporters of two types and by (b) a concentrative Na(+)-dependent transport system of low sensitivity to nitrobenzylthioinosine and dipyridamole, classical inhibitors of equilibrative nucleoside transport. In medium containing 10 microM dipyridamole and 20 microM adenosine, the equilibrative nucleoside transport systems of L1210/AM cells were substantially inhibited and the unimpaired activity of the Na(+)-dependent nucleoside transport system resulted in the cellular accumulation of free adenosine to 86 microM in 5 min, a concentration three times greater than the steady-state levels of adenosine achieved without dipyridamole. Uphill adenosine transport was not observed when extracellular Na+ was replaced by Li+, K+, Cs+, or N-methyl-D-glucammonium ions, or after treatment of the cells with nystatin, a Na+ ionophore. These findings show that concentrative nucleoside transport activity in L1210/AM cells required an inward transmembrane Na+ gradient. Treatment of cells in sodium medium with 2 mM furosemide in the absence or presence of 2 mM ouabain inhibited Na(+)-dependent adenosine transport by 50 and 75%, respectively. However, because treatment of cells with either agent in Na(+)-free medium decreased adenosine transport by only 25%, part of this inhibition may be secondary to the effects of furosemide and ouabain on the ionic content of the cells. Substitution of extracellular Cl- by SO4(-2) or SCN- had no effect on the concentrative influx of adenosine.

The carbocyclic analog of 2'-deoxyguanosine [(+-)-2-amino-1,9-dihydro-9-[(1 alpha,3 beta,4 alpha)-3-hydroxy-4-(hydroxymethyl)cyclopentyl]-6H-purine-6-one] (2'-CDG) is highly active in cell culture against strains S148 and E377 of herpes simplex virus type 1 (HSV-1), both of which code for thymidine kinase, and much less active against strain BW10168 which is deficient in this enzyme activity. Antiviral activity is associated primarily with the D-enantiomer; the L-enantiomer has much lower but significant activity. The metabolism of racemic 2'-CDG and its D- and L-enantiomers was studied in uninfected HEp-2 cells and in HEp-2 cells infected with the S148 or BW10168 strains of HSV-1. Nucleotides were separated by HPLC, and their elution was monitored by spectrophotometry. The chromatograms of extracts of cells infected with the S148 strain and treated with (+/-)-2'-CDG or D-2'-CDG included a new peak which appeared in the triphosphate region. This peak, the area of which exceeded that of the GTP peak, was shown to be due to the triphosphate of 2'-CDG. The new peak was not observed by HPLC of extracts of uninfected cells treated with (+/-)-2'-CDG or either of its enantiomers, cells infected with the S148 strain and treated with L-2'-CDG, or cells infected with the BW10168 strain and treated with (+/-)-2'-CDG or either of its enantiomers. The results were similar when these studies were performed with uninfected Vero cells and with Vero cells infected with strain S148 of HSV-1. In experiments with D-[8-3H]-2'-CDG, small amounts of phosphates of 2'-CDG could also be detected in uninfected HEp-2 cells and in cells infected with the BW10168 strain of HSV-1. Thus, 2'-CDG apparently is a good substrate for the virus-coded kinase and a very poor substrate for cellular phosphorylating enzymes. The selective phosphorylation of 2'-CDG by the virus-specific kinase presumably is critical for its antiviral activity as it is for that of acyclovir and other acyclic derivatives of guanine.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-ara-A), a derivative of 9-beta-D-arabinofuranosyladenine (ara-A) that is resistant to deamination, selectively inhibits DNA synthesis and has activity against mouse leukemia L1210 comparable to that of ara-A plus the adenosine deaminase inhibitor, 2'-deoxycoformycin. To determine if these two nucleosides have similar modes of action, comparisons were made of their effects and those of their triphosphates on enzymes known to be inhibited by ara-A or 9-beta-D-arabinofuranosyladenine 5'-triphosphate. 9-beta-D-Arabinofuranosyl-2-fluoroadenine 5'-triphosphate was more effective than 9-beta-D-arabinofuranosyladenine 5'-triphosphate in inhibiting the reduction of adenosine 5'-diphosphate and cytidine 5'-diphosphate by ribonucleotide reductase from HEp-2 cells or L1210 cells. DNA polymerase alpha from L1210 cells was equally sensitive to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate and 9-beta-D-arabinofuranosyladenine 5'-triphosphate, and DNA polymerase beta from L1210 cells was much less sensitive to both triphosphates. S-Adenosylhomocysteine hydrolase from L1210 cells was inactivated by 2-F-ara-A and ara-A, but higher concentrations of the fluoro derivative were required. These results are consistent with 2-F-ara-A and ara-A inhibition of DNA synthesis by inhibition of ribonucleotide reductase and DNA polymerase alpha.

2-Amino-6-chloro-1-deazapurine is of interest as a purine analog with demonstrated in vivo activity against mouse leukemia L1210. That the active form of this agent is a nucleotide and that the nucleotide is formed by the action of hypoxanthine (guanine) phosphoribosyltransferase were shown by the facts that (a) L1210 cells deficient in hypoxanthine phosphoribosyltransferase were insensitive to the analog; (b) hypoxanthine, but not adenine, prevented the formation of the analog nucleotide by enzyme preparations containing activities of both hypoxanthine and adenine phosphoribosyltransferases; and (c) the cytotoxicity of the analog was prevented by hypoxanthine. The ribonucleoside of this analog was not toxic to cell cultures and hence is not phosphorylated or cleaved to the base. In intact HEp-2 cells and L1210 cells, the analog was metabolized to the nucleoside 5'-phosphate which accumulated to concentrations as high as 1000 nmoles/10(9) cells; no di- or triphosphates were detected. In HEp-2 cells, the analog reduced the pools of purine nucleotides with some accumulation of IMP. The toxicity of minimal inhibitory concentrations of the analog to HEp-2 cells could be prevented or reversed by 4(5)-amino-5(4)-imidazolecarboxamide (AIC); the toxicity of higher concentrations could be prevented or reversed by a combination of adenine and guanosine but not by AIC. The analog inhibited the incorporation of formate into purine nucleotides and into macromolecules at concentrations that had no effect on utilization of hypoxanthine; at higher concentrations the incorporation of hypoxanthine was inhibited. Low concentrations also inhibited the utilization of uridine and thymidine. The incorporation of hypoxanthine and AIC into guanine nucleotides, but not adenine nucleotides, was inhibited. These results indicate two sites of inhibition of the biosynthesis of purine nucleotides, the more sensitive one being on an early step of the pathway and the less sensitive one on the IMP-GMP conversion. That the blockade of de novo synthesis probably was at the site of feedback inhibition was indicated by the fact that the analog inhibited the accumulation of formylglycinamide ribonucleotide in azaserine-treated cells but did not inhibit the synthesis of 5'-phosphoribosyl 1-pyrophosphate. Comparative studies were performed with the related analog, 2-amino-6-chloropurine, which has been reported to produce a similar dual blockade of the purine pathway. This purine was less toxic than its 1-deaza analog; it produced a modest decrease in adenine nucleotides but increased pools of guanine nucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)

Purified adenosine kinase from L1210 cells displayed substrate inhibition by high concentrations of adenosine (Ado), ATP, and MgCl2. When incubated with ATP and MgCl2, the enzyme was phosphorylated, and the phosphorylated kinase transferred phosphate to adenosine in the absence of ATP and MgCl2. Substrate binding, isotope exchange, and kinetic studies suggested that the enzyme catalyzes the reaction by means of a two-site ping-pong mechanism with the phosphorylated enzyme as an obligatory intermediate. Among many possible pathways within this mechanism probably a random-bi ordered-bi route is the preferred sequence in which the two substrates, adenosine and MgATP, bind in a random order to form the ternary complex MgATP . E . Ado followed by the sequential dissociation of MgADP and AMP. Dissociation constants of various enzyme-substrate and enzyme-product complexes and the first-order rate constant of the rate-limiting step were estimated.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-araA) inhibited the growth in vitro of HeLa cells by 50% at a concentration of 0.25 microM and depressed the replication of herpes simplex virus Types 1 and 2 by 99% at 25 microM. The analogue served as a substrate for cytoplasmic but not mitochondrial deoxycytidine (dCyd) kinase partially purified from human peripheral chronic lymphocytic leukemic blast cells. The Km values of dCyd and 2-F-araA for the cytoplasmic enzyme were 5 microM and 213 microM, respectively. However, at concentrations of 0.4 mM, the analogue was phosphorylated 2.9 times faster than dCyd. The 5'-triphosphate of 2-F-araA was examined for its biochemical effects on partially purified ribonucleotide reductase and highly purified DNA alpha- and beta-polymerases from HeLa cells. 2-F-araATP was a potent inhibitor of ribonucleotide reductase; the concentration required for 50% inhibition of ADP reduction (0.3 mM ADP; 5 mM GTP or dGTP) was 1 microM and for CDP reduction (0.15 mM CPD; 5 mM ATP) was 8.5 microM. Furthermore, 2-F-araATP was a competitive inhibition (Ki = 1.2 microM) with respect to dATP (Km = 3.8 microM) of DNA alpha-polymerase, whereas DNA beta-polymerase was relatively insensitive to the drug. The results suggest that the cytotoxic actions of 2-F-araA may be due, in part, to a "self-potentiating" inhibition of DNA synthesis. This is, by inhibiting the formation of competing dATP, 2-F-araATP may potentiate its inhibition of DNA synthesis.

Reaction of 5,6,7,8-tetrahydrofolic acid (THF,7) with phosgene, thiophosgene, and cyanogen bromide gave the bridged derivatives, 5,10-(CO)-THF (8), 5,10-(CS)-THF (9), and 5,10-(C = NH)-THF (11), respectively. Catalytic hydrogenation of 10-(chloroacetyl)folic acid (2) gave 5,10-(CH2CO)-THF (12). A similar reaction with 10-(3-chloropropionyl)folic acid (3) gave 10-(ClCH2CH2CO)-THF (14) rather than 5,10-(CH2CH2CO)-THF (13). In the catalytic hydrogenation of 10-ethoxalylfolic acid (5), the initial product 10-(EtO2CCO)-THF (22) rearranged readily to give 5-(EtO2CCO)-THF (21). Acylation of THF with chloroacetyl chloride gave a N5,N10-diacylated product (18 or 19), which could not be converted to 5,10-COCH2)-THF (17). Reductive alkylation of THF with glyoxylic acid and 5-hydroxypentanal, respectively, gave 5-(HO2CCH2)-THF (24) and 5-[HO(CH2)5]-THF (25). Reductive dialkylation of THF with formaldehyde gave 5,10-(CH3)2-THF (27), whereas glyoxal gave 5,10-CH2CH2)-THF (10). Also, both folic acid and 5-(CHO)-THF were reductively alkylated with formaldehyde to give 10-methylfolic acid (6) and 5-(CHO)-10-(CH3)-THF (28), respectively. These compounds were tested as inhibitors of the enzymes involved in folate metabolism and for activity against lymphocytic leukemia P388 in mice.

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)

In cell cultures treated with the carbocyclic analog of adenosine (C-Ado, (+/-)-aristeromycin), the utilization of hypoxanthine and guanine has been observed to be blocked. In an attempt to define the mechanism of this inhibition, we have reexamined the metabolism of C-Ado and its effects on the metabolism of guanine and hypoxanthine. In cultures of L1210 cells, C-Ado at a concentration of 25 microM inhibited the utilization of hypoxanthine and guanine for nucleotide synthesis by more than 90% but produced little or no inhibition of the utilization of these bases in cultures of L1210/MeMPR cells which lack adenosine kinase and cannot phosphorylate C-Ado. In cultures of mammalian cells (L1210, HEp-2, and colon-26 cells), C-Ado was converted to the triphosphate (as previously observed) and also to the triphosphate of the carbocyclic analog of guanosine. The presence of coformycin in the medium at a concentration sufficient to inhibit AMP deaminase almost completely prevented the formation of carbocyclic GTP; thus, the deamination of C-Ado monophosphate is essential for the formation of phosphates of carbocyclic guanosine. Since hypoxanthine (guanine) phosphoribosyltransferase is known to be subject to end product inhibition, it was considered likely that phosphates of carbocyclic guanosine or carbocyclic inosine, present in C-Ado-treated cells, were responsible for inhibition of utilization of hypoxanthine and guanine. The 5'-phosphates of the carbocyclic analogs of inosine and guanosine were synthesized and found to be effective inhibitors of the phosphoribosyltransferase. Carbocyclic GMP was a better inhibitor than carbocyclic IMP and was also superior to GMP and IMP; the concentration of C-GMP that produced a 50% inhibition of GMP formation was approximately 1 microM. It is probable that the presence of phosphates of carbocyclic guanosine accounts for the inhibition of utilization of hypoxanthine and guanine in C-Ado-treated cells.

The synthesis and characterization of 8-amino-6-fluoro-9-beta-D-ribofuranosyl-9H-purine (3a) are presented. This compound is a substrate for adenosine deaminase and adenosine kinase. In L1210 cells 3a is converted to 8-aminoinosine monophosphate (4b), apparently by the action of AMP deaminase on the monophosphate of 3a, as well as to the triphosphate derivative of 3a. Pentostatin was used to inhibit adenosine deaminase, and coformycin was used to inhibit AMP deaminase in experiments designed to delineate the metabolic fate of 3a. Pentostatin was without influence on the cytotoxicity of 3a, but coformycin potentiated the cytotoxicity. The potentiation was associated with an increased cellular concentration of phosphates of 3a and a decreased concentration of 4b.

We have developed a new assay for purine nucleoside phosphorylase which is based on the release of tritium when [2-3H]inosine is used as the substrate and the reaction is coupled with xanthine oxidase. After the reaction is terminated, residual [2-3H]inosine is adsorbed on charcoal and the supernatant solution is assayed for radioactivity by liquid scintillation spectrometry. The new method gave results indistinguishable from those obtained by spectrophotometric determination of uric acid produced by the phosphorylase-xanthine oxidase-coupled reaction or by radioassay of chromatographically isolated [8-14C]hypoxanthine when [8-14C]inosine was used as substrate. The new method is faster than those involving chromatographic isolation of products. In comparison with spectrophotometric methods, it not only requires less manual time, but it also has the advantage that it can be used to study inhibitors whose ultraviolet absorption might interfere with spectrophotometric determination of uric acid.

Previously, 8-deazafolic acid (17) was shown to be a potent inhibitor of the folate-dependent bacteria, Streptococcus faecium (ATCC 8043) and Lactobacillus casei (ATCC 7469), and to have activity against lymphoid leukemia L1210 in mice. To examine the 5,6,7,8-tetrahydro derivatives, a new synthesis of 17 was developed from 8-deaza-2,4-dichloro-6-methylpteridine. Treatment of the latter with aqueous base gave the corresponding pteridin-4(3H)-one, which was aminated with ammonia to give 8-deaza-6-methylpterin (9). Bromination of 9 gave mainly 8-deaza-6-(tribromomethyl)pterin, which on reaction with p-aminobenzoyl-L-glutamic acid resulted in the formation of the 9-oxo derivative of 17. In contrast, bromination of the 2-acetyl derivative of 9 gave mainly the corresponding 6-(bromomethyl)pterin, which was converted to 17 in 23% yield (from 9). Hydrogenation of 17 at atmospheric pressure and room temperature was unsuccessful either in a basic medium or formic acid. In trifluoroacetic acid, overreduction occurred to give a mixture containing 8-deaza-5,6,7,8-tetrahydro-6-methylpterin and the 5,6,7,8-tetrahydro derivative of 17. The latter was characterized by conversion to the methenyl analogue 21, which was also prepared by hydrogenation of the 10-formyl derivative of 17. Treatment of 21 with hydroxide gave 8-deaza-10-formyl-5,6,7,8-tetrahydrofolic acid. Compound 21 showed cytotoxicity to cultured H.Ep.-2 cells and was tested as an inhibitor of bovine dihydrofolic reductase. Lineweaver-Burk analysis indicated inhibition competitive with dihydrofolate.

The metabolism and metabolic effects of 2-azahypoxanthine and 2-azaadenosine were studied to elucidate the biochemical basis for their known cytotoxicities. 2-Azaadenosine is a known substrate for adenosine kinase. That 2-azahypoxanthine is a substrate for hypoxanthine (guanine) phosphoribosyltransferase is shown by the observations that, in cell-free fractions from HEp-2 cells supplemented with 5-phosphoribosyl-1-pyrophosphate, 2-azahypoxanthine inhibited the conversion of hypoxanthine to IMP but not the conversion of adenine to AMP, and hypoxanthine, but not adenine, inhibited the conversion of 2-azahypoxanthine to 2-azaIMP. [8-14C]2-Azahypoxanthine was synthesized from [8-14C]hypoxanthine via [2-14C]-4-amino-5-imidazolecarboxamide. In HEp-2 cells in culture, the principal metabolite of [8-14C]-2-azahypoxanthine was 2-azaATP; there was no detectable 14C in deoxynucleotides or in DNA or RNA fractions. 2-Azaadenosine was much more toxic than 2-azahypoxanthine, and, when used in the presence of an adenosine deaminase inhibitor, 2'-deoxycoformycin, was converted in HEp-2 cells to 2-azaATP in amounts that exceeded those of ATP in control cells. The pool of ATP was reduced by as much as 75% as 2-azaATP accumulated. In a short-term experiment (4 hr), 2-azaadenosine selectively reduced the pools of adenine nucleotides, whereas 2-azahypoxanthine reduced the pools of guanine nucleotides selectively. Both 2-azahypoxanthine and 2-azaadenosine inhibited the incorporation of formate into purine nucleotides and were without effect on the conversion of thymidine and uridine to nucleotides. 2-Azahypoxanthine inhibited the incorporation of thymidine into macro-molecules but not that of uridine or leucine; 2-azaadenosine inhibited the incorporation of all three of these precursors non-selectively. 2-AzaIMP inhibited IMP dehydrogenase competitively with IMP (Ki = 66 microM). The difference in effects of 2-azahypoxanthine and 2-azaadenosine perhaps may be due to the production, from 2-azahypoxanthine but not from 2-azaadenosine + 2'-deoxycoformycin, of 2-azaIMP, which inhibits synthesis of guanine nucleotides and thereby results in inhibition of DNA synthesis. Specific sites of action for 2-azaadenosine are yet undefined.

8-Azainosine (8-aza-HR) is of interest because of its activity against experimental tumors. Metabolic studies in cell cultures were performed with 8-aza-HR and with the structurally related nucleoside, 8-azaadenosine (9-beta-D-ribofuranosyl-8-azaadenine) (8-aza-AR), which has a lower degree of antitumor activity than does 8-aza-HR. In H. Ep. 2 cells and in Ca755 cells, both 14C-labeled nucleosides were metabolized to nucleotides of 8-azaadenine (8-aza-A) and 8-azaguanine (8-aza-G) and incorporated into polynucleotides as 8-aza-A and 8-aza-G. 8-Aza HR was incorporated primarily as 8-aza-G, whereas 8-aza-AR was incorporated about equally as 8-aza-A and 8-aza-G. In H. Ep. 2 cells, the extent of incorporation of 8-aza-HR as 8-aza-G was about one-half that found when [14C]-8-aza-G was the precursor. In the H. Ep. 2/FA/FAR cell line, 8-aza-AR and 8-aza-HR were metabolized similarly, in that both were incorporated into polynucleotides principally as 8-aza-G; apparently, in this cell line which is deficient in adenosine kinase and adenine phosphoribosyltransferase, 8-aza-AR is metabolized by conversion to 8-aza-HR. A cell line (H. Ep 2/8-aza HR), which was resistant to 8-aza-HR but sensitive to 8-aza-AR and which retained hypoxanthine (guanine)-phosphoribosyltransferase activity, metabolized 8-aza-HR to only a small extent. However, in this cell-line, 8-aza-AR was more extensively metabolized and was incorporated primarily as 8-aza-A. The failure of these cells to convert 8-aza-AR or 8-aza-HR to 8-aza-G indicates that the basis for resistance may be a change in the substrate specificities of the enzymes of guanosine monophosphate synthesis such that these cells no longer effectively convert 8-azainosine monophosphate to 8-azaguanosine monophosphate. 8-Aza-AR was a potent inhibitor of purine synthesis de novo, but 8-aza-HR, at concentrations much higher than the inhibitory concentration of 8-aza-AR, did not inhibit this process. In H. Ep. 2 cells, 8-aza-HR blocked the conversion of orotic acid to uridine nucleotides and caused an accumulation of orotidine. This inhibition of pyrimidine biosynthesis apparently does not contribute significantly to the cytotoxicity of 8-aza-HR because uridine provided no degree of reversal of its inhibition of the growth of cell cultures.

The synthesis and isolation of two derivatives of 2-amino-1,3,4-thialdiazole(aminothiadiazole) are described. The derivatives are a nicotinamide adenine dinucleotide (NAD) analog prepared by an exchange reaction with NAD in the presence of nicotineamide adenine dinucleotide glycohydrolase and a presumed aminothiadiazole mononucleotide prepared by treatment of the NAD analog with nucleotide pyrophosphatase. Both derivatives are potent inhibitors of inosine 5'-phosphate (IMP) dehydrogenase obtained from leukemia L1210 cells. The NAD analog is a pseudoir-reversible inhibitor of the enzyme, noncompetitive with either IMP or NAD. The aminothiadiazole mononucleotide has a K1 of about 0.1 muM, is competitive with IMP, and is uncompetitive with NAD: the inhibition appears to be reversible by Ackermann-Potter analysis. A metabolite of [5-14C]aminothiadiazole is formed in L1210 cells in vivo to a level of 0.3 nmole/10(9) cells. Retention volume of the metabolite on a high-pressure liquid chromatography system is the same as that of the aminothiadiazole mononucleotide prepared as described above. These results suggest that IMP dehydrogenase is the site of action for aminothiadiazole metabolites as was indicated by earlier observations. There is no evidence that the NAD analog is formed in vivo. Nicotinamide prevented formation of the mononucleotide in vivo. Therefore, since formation and cleavage of the NAD analog apparently are not the route to the thiadiazole nucleotide, some other pathway for the metabolism of nicotinamide may be involved such as the action of a phosphoribosyltransferase or the sequential action of a nucleoside phosphorylase and a nucleoside kinase.

Neplanocin A and aristeromycin are carbocyclic adenosine analogs that differ only in that neplanocin A contains a double bond in the carbocyclic ring, whereas this ring in aristeromycin is saturated. We have compared the metabolism and some of the metabolic effects of neplanocin A and synthetic (+/-)-aristeromycin (C-Ado) in murine leukemia L1210 cells in culture. C-Ado, as shown earlier, was not only converted to its own phosphates but also was metabolized to phosphates of carbocyclic guanosine. Both rapidly proliferating and slowly proliferating or resting cells phosphorylated C-Ado, but C-Ado was not converted to phosphates of carbocyclic guanosine in detectable amounts in cells whose growth had reached a plateau. When the metabolism of neplanocin and C-Ado was examined in the same experiment, both analogs were converted to the triphosphate analogs of ATP; no conversion of neplanocin A to the corresponding carbocyclic analogs of guanine nucleotides was detected, whereas C-Ado was converted to the carbocyclic analog of GTP in amounts that approximated the GTP pool. This difference in metabolism was associated with a marked difference in effects of the two analogs on the utilization of hypoxanthine and guanine which was inhibited by C-Ado but not by neplanocin. The failure of neplanocin A to be converted to analogs of guanine nucleotides apparently is the result of poor capacity of its monophosphate to serve as a substrate for AMP deaminase; the Vmax for deamination of neplanocin-5'-monophosphate by this enzyme was only 5% of that for C-Ado monophosphate. In contrast, neplanocin A was a better substrate than C-Ado for adenosine deaminase.