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Treatment with fludarabine phosphate (9-β-D-arabinofuranosyl-2-F-adenine 5'-phosphate, F-araAMP) leads to regressions and cures of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (EcPNP). This occurs despite the fact that fludarabine (F-araA) is a relatively poor substrate for EcPNP, and is cleaved to liberate 2-fluoroadenine at a rate only 0.3% that of the natural E. coli PNP substrate, adenosine. In this study, we investigated a panel of naturally occurring PNPs to identify more efficient enzymes that may be suitable for metabolizing F-araA as part of experimental cancer therapy. We show that Trichomonas vaginalis PNP (TvPNP) cleaves F-araA with a catalytic efficiency 25-fold greater than the prototypic E. coli enzyme. Cellular extracts from human glioma cells (D54) transduced with lentivirus stably expressing TvPNP (D54/TvPNP) were found to cleave F-araA at a rate similar to extracts from D54 cells expressing EcPNP, although much less enzyme was expressed per cell in the TvPNP transduced condition. As a test of safety and efficacy using TvPNP, human head and neck squamous cell carcinoma (FaDu) xenografts expressing TvPNP were studied in nude mice and shown to exhibit robust tumor regressions, albeit with partial weight loss that resolved post-therapy. F-araAMP was also a very effective treatment for mice bearing D54/TvPNP xenografts in which approximately 10% of tumor cells expressed the enzyme, indicating pronounced ability to kill non-transduced tumor cells (high bystander activity). Moreover, F-araAMP demonstrated activity against D54 tumors injected with an E1, E3 deleted adenoviral vector encoding TvPNP. In that setting, despite higher F-araA cleavage activity using TvPNP, tumor responses were similar to those obtained with EcPNP, indicating factors other than F-Ade production may limit regressions of the D54 murine xenograft model. Our results establish that TvPNP is a favorable enzyme for activating F-araA, and support further studies in combination with F-araAMP for difficult-to-treat human cancers.

6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5'-C-methylated-6-methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

In Vibrio cholerae, the genes required for biofilm development are repressed by quorum sensing at high cell density due to the accumulation in the medium of two signaling molecules, cholera autoinducer 1 (CAI-1) and autoinducer 2 (AI-2). A significant fraction of toxigenic V. cholerae isolates, however, exhibit dysfunctional quorum sensing pathways. It was reported that transition state analogs of the enzyme methylthioadenosine/S-adenosylhomocysteine nucleosidase (MtnN) required to make AI-2 inhibited biofilm formation in the prototype quorum sensing-deficient strain N16961. This finding prompted us to examine the role of both autoinducers and MtnN in biofilm development and virulence gene expression in a quorum sensing-deficient genetic background. Here we show that deletion of mtnN encoding methylthioadenosine/S-adenosylhomocysteine nucleosidase, cqsA (CAI-1), and/or luxS (AI-2) do not prevent biofilm development. However, two independent mtnN mutants exhibited diminished growth rate and motility in swarm agar plates suggesting that, under certain conditions, MtnN could influence biofilm formation indirectly. Nevertheless, MtnN is not required for the development of a mature biofilm.

The use of E. coli purine nucleoside phosphorylase (PNP) to activate prodrugs has demonstrated excellent activity in the treatment of various human tumor xenografts in mice. E. coli PNP cleaves purine nucleoside analogs to generate toxic adenine analogs, which are activated by adenine phosphoribosyl transferase (APRT) to metabolites that inhibit RNA and protein synthesis. We created tumor cell lines that encode both E. coli PNP and excess levels of human APRT, and have used these new cell models to test the hypothesis that treatment of otherwise refractory human tumors could be enhanced by overexpression of APRT. In vivo studies with 6-methylpurine-2′-deoxyriboside (MeP-dR), 2-F-2′-deoxyadenosine (F-dAdo) or 9-β-D-arabinofuranosyl-2-fluoroadenine 5′-monophosphate (F-araAMP) indicated that increased APRT in human tumor cells coexpressing E. coli PNP did not enhance either the activation or the anti-tumor activity of any of the three prodrugs. Interestingly, expression of excess APRT in bystander cells improved the activity of MeP-dR, but diminished the activity of F-araAMP. In vitro studies indicated that increasing the expression of APRT in the cells did not significantly increase the activation of MeP. These results provide insight into the mechanism of bystander killing of the E. coli PNP strategy, and suggest ways to enhance the approach that are independent of APRT.

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides to the corresponding free bases and ribose 1-phosphate. The crystal structure of grouper iridovirus PNP (givPNP), corresponding to the first PNP gene to be found in a virus, was determined at 2.4 Å resolution. The crystals belonged to space groupR3, with unit-cell parametersa= 193.0,c= 105.6 Å, and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an α/β structure with a nine-stranded mixed β-barrel flanked by a total of nine α-helices. The predicted phosphate-binding and ribose-binding sites are occupied by a phosphate ion and a Tris molecule, respectively. The geometrical arrangement and hydrogen-bonding patterns of the phosphate-binding site are similar to those found in the human and bovine PNP structures. The enzymatic activity assay of givPNP on various substrates revealed that givPNP can only accept 6-oxopurine nucleosides as substrates, which is also suggested by its amino-acid composition and active-site architecture. All these results suggest that givPNP is a homologue of mammalian PNPs in terms of amino-acid sequence, molecular mass, substrate specificity and overall structure, as well as in the composition of the active site.

Metalation of 6-methyl-9-(tetrahydro-2H-pyran-2-yl)purine (10) with lithiating agents of varying basicities such as n-BuLi and LiHMDS in THF at -78 degrees C resulted in metalation at both of the 6-CH(3) moiety and the 8-CH position, irrespective of the molar equivalence of the base. On the other hand, a regioselective metalation at the 6-CH(3) moiety of 10 was observed with NaHMDS or KHMDS, under similar conditions. Treatment of the potassium salts of 10 and of the protected riboside derivative 6-methyl-9-(beta-D-2,3,5-tri-O-tert-butyldimethylsilylribofuranosyl)purine (22) with N-fluorobenzenesulfonamide (NFSI) at -78 degrees C gave the corresponding 6-fluoromethylpurine derivatives 11 and 23, respectively, in good yields. Deprotection of 11 and 23 under standard conditions gave 6-fluoromethylpurine (6-FMeP, 3) and 6-fluoromethyl-9-(beta-D-ribofuranosyl)purine (6-FMePR, 4), respectively, in high yield. Both 3 and 4 demonstrated cytotoxic activity against CCRF-CEM cells in culture. 6-FMePR is a good substrate for E. coli purine nucleoside phosphorylase (E. coli PNP) with a comparable substrate activity to that of the parent nucleoside, 6-methyl-9-(beta-D-ribofuranosyl)purine (6-MePR, 21). The cytotoxic activity of 6-FMeP along with the substrate activity of 6-FMePR with E. coli PNP meet the fundamental requirements for using 6-FMeP as a potential toxin in PNP/prodrug based cancer gene therapy.

Impressive antitumor activity has been observed with fludarabine phosphate against tumors that express Escherichia coli purine nucleoside phosphorylase (PNP) due to the liberation of 2-fluoroadenine in the tumor tissue. 6-Methylpurine (MeP) is another cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving E. coli PNP. The prototype MeP releasing prodrug 9-(2-deoxy-β-d-ribofuranosyl)-6-methylpurine (1) [MeP-dR] has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify a combination of non-toxic MeP prodrugs and non-human adenosine glycosidic bond cleaving enzymes. The two best MeP-based substrates with M64V-E coli PNP, a mutant which was engineered to tolerate modification at the 5'-position of adenosine and its analogs, were 9-(6-deoxy-α-l-talofuranosyl)-6-methylpurine (3) [methyl(talo)-MeP-R] and 9-(α-l-lyxofuranosyl)6-methylpurine (4) [lyxo-MeP-R]. The detailed synthesis methyl(talo)-MeP-R and lyxo-MeP-R, and the evaluation of their substrate activity with 4 enzymes not normally associated with cancer patients is described. In addition, we have determined the intraperitoneal pharmacokinetic (ip-PK) properties of methyl(talo)-MeP-R and have determined its in vivo bystander activity in mice bearing D54 tumors that express M64V PNP. The observed good in vivo bystander activity of [methyl(talo)-MeP-R/M64V-E coli PNP combination suggests that these agents could be useful for the treatment of cancer.

The use of Escherichia coli purine nucleoside phosphorylase (PNP) to activate fludarabine has demonstrated safety and antitumor activity during preclinical analysis and has been approved for clinical investigation.A first-in-human phase I clinical trial (NCT 01310179; IND 14271) was initiated to evaluate safety and efficacy of an intratumoral injection of adenoviral vector expressing E. coli PNP in combination with intravenous fludarabine for the treatment of solid tumors. The study was designed with escalating doses of fludarabine in the first three cohorts (15, 45, and 75 mg/m(2)) and escalating virus in the fourth (10(11)-10(12) viral particles, VP).All 12 study subjects completed therapy without dose-limiting toxicity. Tumor size change from baseline to final measurement demonstrated a dose-dependent response, with 5 of 6 patients in cohorts 3 and 4 achieving significant tumor regression compared with 0 responsive subjects in cohorts 1 and 2. The overall adverse event rate was not dose-dependent. Most common adverse events included pain at the viral injection site (92%), drainage/itching/burning (50%), fatigue (50%), and fever/chills/influenza-like symptoms (42%). Analysis of serum confirmed the lack of systemic exposure to fluoroadenine. Antibody response to adenovirus was detected in two patients, suggesting that neutralizing immune response is not a barrier to efficacy.This first-in-human clinical trial found that localized generation of fluoroadenine within tumor tissues using E. coli PNP and fludarabine is safe and effective. The pronounced effect on tumor volume after a single treatment cycle suggests that phase II studies are warranted.NCT01310179.

Our studies have led to the identification of an E. coli PNP mutant (M64V) that is able to cleave numerous 5'-modified nucleoside analogs with much greater efficiency than the wild-type enzyme. The biological activity of the three best substrates of this mutant (9-[6-deoxy-alpha-L-talofuranosyl]-6-methylpurine (methyl(talo)-MeP-R), 9-[6-deoxy-alpha-L-talofuranosyl]-2-F-adenine, and 9-[alpha-L-lyxofuranosyl]-2-F-adenine) were evaluated so that we can optimally utilize these compounds. Our results indicated that the mechanism of toxicity of methyl(talo)-MeP-R to mice was due to its cleavage to MeP by a bacterial enzyme, and that the toxicity of the two F-Ade analogs was due to their cleavage to F-Ade by mammalian methylthioadenosine phosphorylase.

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.

Purine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2'-deoxypurine nucleosides to the free base and ribose (or 2'-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4'- and 5'-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency.

We have demonstrated antitumor activity against refractory human glioma and pancreatic tumors with 6-methylpurine (MeP) using either a suicide gene therapy strategy to selectively release 6-methylpurine in tumor cells or direct intratumoral injection of 6-methylpurine itself. A single i.p. injection in mice of the prodrug 9-beta-D-[2-deoxyribofuranosyl]-6-methylpurine (MeP-dR; 134 mg/kg) caused sustained regression lasting over 70 days of D54 (human glioma) tumors transduced with the Escherichia coli purine nucleoside phosphorylase (PNP), and a single intratumoral injection of 6-methylpurine (5-10 mg/kg) elicited prolonged delays of the growth of D54 tumors and CFPAC human pancreatic carcinoma. Because the D54 tumor doubling time is >15 days, the experiments indicate that prodrug activation by E. coli PNP engenders destruction of both dividing and nondividing tumor compartments in vivo and, therefore, address a fundamental barrier that has limited the development of suicide gene strategies in the past. A prolonged retention time of 6-methylpurine metabolites in tumors was noted in vivo (T(1/2) >24 h compared with a serum half-life of

2-Methyladenosine (methyl-ado) has demonstrated selective activity against Mycobacterium tuberculosis, which indicates that differences in the substrate preferences between mycobacterial and human purine metabolic enzymes can be exploited to develop novel drugs for the treatment of mycobacterial diseases. Therefore, in an effort to better understand the reasons for the anti-mycobacterial activity of methyl-ado, its metabolism has been characterized in Mycobacterium smegmatis. In a wild-type strain, methyl-ado was phosphorylated by adenosine kinase to methyl-AMP, which was further converted to methyl-ATP and incorporated into RNA. In contrast, a mutant strain of M. smegmatis was isolated that was resistant to methyl-ado, deficient in adenosine kinase activity and was not able to generate methyl-ado metabolites in cells treated with methyl-ado. These results indicated that phosphorylated metabolites of methyl-ado were responsible for the cytotoxic activity of this compound. Methyl-ado was not a substrate for either adenosine deaminase or purine-nucleoside phosphorylase from M. smegmatis. Treatment of M. smegmatis with methyl-ado resulted in the inhibition of ATP synthesis, which indicated that a metabolite of methyl-ado inhibited one of the enzymes involved in de novo purine synthesis. These studies demonstrated the importance of adenosine kinase in the activation of methyl-ado to toxic metabolites in M. smegmatis.

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.

Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from non-expressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2'-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

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.

Currently approved DNA hypomethylating nucleosides elicit their effects in part by depleting DNA methyltransferase I (DNMT1). However, their low response rates and adverse effects continue to drive the discovery of newer DNMT1 depleting agents. Herein, we identified two novel 2'-deoxycytidine (dCyd) analogs, 4'-thio-2'-deoxycytidine (T-dCyd) and 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) that potently deplete DNMT1 in both in vitro and in vivo models of cancer and concomitantly inhibit tumor growth.DNMT1 protein levels in in vitro and in vivo cancer models were determined by Western blotting and antitumor efficacy was evaluated using xenografts. Effects on CpG methylation were evaluated using methylation-specific PCR. T-dCyd metabolism was evaluated using radiolabeled substrate.T-dCyd markedly depleted DNMT1 in CCRF-CEM and KG1a leukemia and NCI-H23 lung carcinoma cell lines, while it was ineffective in the HCT-116 colon or IGROV-1 ovarian tumor lines. On the other hand, aza-T-dCyd potently depleted DNMT1 in all of these lines indicating that dCyd analogs with minor structural dissimilarities induce different DNMT1 turnover mechanisms. Although T-dCyd was deaminated to 4'-thio-2'-deoxyuridine, very little was converted to 4'-thio-thymidine nucleotides, suggesting that inhibition of thymidylate synthase would be minimal with 4'-thio dCyd analogs. Both T-dCyd and aza-T-dCyd also depleted DNMT1 in human tumor xenografts and markedly reduced in vivo tumor growth. Interestingly, the selectivity index of aza-T-dCyd was at least tenfold greater than that of decitabine.Collectively, these data show that 4'-thio modified dCyd analogs, such as T-dCyd or aza-T-dCyd, could be a new source of clinically effective DNMT1 depleting anticancer compounds with less toxicity.

The chemistry required for covalent biotinylation of drugs, radiopharmaceuticals and other ligands is highly developed, and a large number of biotinylated reagents can be readily synthesized. In order to investigate whether expression of avidin cDNA in mammalian cells might be useful as part of a drug targeting strategy, we transiently expressed the avidin gene in two human tumor cell lines (the cervical carcinoma cell line, HeLa, and the liver derived line, Hep G2). Avidin protein as detected by either immunohistochemistry or binding of streptavidin-biotin complexes was present and functional following transient expression. This result indicated that the mechanisms underlying avidin oligomerization which are necessary for proper protein folding are present within mammalian carcinoma cell lines. Next, we generated a producer cell line (derived from psi2) capable of releasing a recombinant retrovirus encoding chicken avidin, and a tumorigenic murine breast cancer cell line (16/C) with stable avidin expression. We show that these cell lines are suitable for conferring functional expression of avidin in vitro. These experiments establish a means by which avidin gene expression can be explored as a mechanism for targeted gene delivery of biotin-derivitized drugs in vitro, and have important implications for utilization of this strategy in vivo.

A selective metalation at the 6-CH3 over C-8 of 6-methylpurine derivative 6 was observed with softer counter cation (Na+ or K+) of the base, while the harder Li+ showed no selectivity. In the presence of N-fluorobenzenesulfonamide (NFSI), this property was utilized for the synthesis of 6-fluoromethylpurine derivatives 4 and 5 as potential toxins for suicide gene therapy.

Systemically administered fludarabine phosphate (F-araAMP) slows growth of human tumor xenografts that express Escherichia coli purine nucleoside phosphorylase (PNP). However, this treatment has been limited by the amount of F-araAMP that can be administered in vivo. The current study was designed to (1) determine whether efficacy of this overall strategy could be improved by intratumoral administration of F-araAMP, (2) test enhancement of the approach with external beam radiation, and (3) optimize recombinant adenovirus as a means to augment PNP delivery and bystander killing in vivo. The effects of systemic or intratumoral F-araAMP in mice were investigated with human tumor xenografts (300 mg), in which 10 % of the cells expressed E. coli PNP from a lentiviral promoter. Tumors injected with an adenoviral vector expressing E. coli PNP (Ad/PNP; 2 × 1011 viral particles, 2 times per day × 3 days) and the impact of radiotherapy on tumors treated by this approach were also studied. Radiolabeled F-araAMP was used to monitor prodrug activation in vivo. Intratumoral administration of F-araAMP in human tumor xenografts expressing E. coli PNP resulted in complete regressions and/or prolonged tumor inhibition. External beam radiation significantly augmented this effect. Injection of large human tumor xenografts (human glioma, nonsmall cell lung cancer, or malignant prostate tumors) with Ad/PNP followed by intratumoral F-araAMP resulted in excellent antitumor activity superior to that observed following systemic administration of prodrug. Activation of F-araAMP by E. coli PNP results in destruction of large tumor xenografts in vivo, augments radiotherapy, and promotes robust bystander killing. Our results indicate that intratumoral injection of F-araAMP leads to ablation of tumors in vivo with minimal toxicity.

Pd(IV)-Mediated cross-coupling reactions of ribofuranosylpurine (I) with organozinc halides (II) and subsequent deprotection afford the C-6 substituted purine ribosides (III) in satisfactory to high yields.

A series of C-6 alkyl, cycloalkyl, and aryl-9-(β- d -ribofuranosyl)purines were synthesized and their substrate activities with Escherichia coli purine nucleoside phosphorylase (E. coli PNP) were evaluated. (Ph3P)4Pd-mediated cross-coupling reactions of 6-chloro-9-(2,3,5-tri-O-acetyl-β- d -ribofuranosyl)-purine (6) with primary alkyl (Me, Et, n-Pr, n-Bu, isoBu) zinc halides followed by treatment with NH3/MeOH gave the corresponding 6-alkyl-9-(β- d -ribofuranosyl)purine derivatives 7–11, respectively, in good yields. Reactions of 6 with cycloalkyl(propyl, butyl, pentyl)zinc halides and aryl (phenyl, 2-thienyl)zinc halides gave under similar conditions the corresponding 6-cyclopropyl, cyclobutyl, cyclopentyl, phenyl, and thienyl -9-(β- d -ribofuranosyl)purine derivatives 12–16, respectively in high yields. E. coli PNP showed a high tolerance to the steric and hydrophobic environment at the 6-position of the synthesized purine ribonucleosides. Significant cytotoxic activity was observed for 8, 12, 15, and 16. Evaluation of 12 and 16 against human tumor xenografts in mice did not demonstrate any selective antitumor activity. In addition, 6-methyl-9-(β- d -arabinofuranosyl)purine (18) was prepared and evaluated.

Uridine phosphorylase (UP), a key enzyme in the pyrimidine salvage pathway, catalyzes the reversible phosphorolysis of uridine or 2′-deoxyuridine to uracil and ribose 1-phosphate or 2′-deoxyribose 1-phosphate. This enzyme belongs to the nucleoside phosphorylase I superfamily whose members show diverse specificity for nucleoside substrates. Phylogenetic analysis shows Streptococcus pyogenes uridine phosphorylase (SpUP) is found in a distinct branch of the pyrimidine subfamily of nucleoside phosphorylases. To further characterize SpUP, we determined the crystal structure in complex with the products, ribose 1-phosphate and uracil, at 1.8 Å resolution. Like Escherichia coli UP (EcUP), the biological unit of SpUP is a hexamer with an α/β monomeric fold. A novel feature of the active site is the presence of His169, which structurally aligns with Arg168 of the EcUP structure. A second active site residue, Lys162, is not present in previously determined UP structures and interacts with O2 of uracil. Biochemical studies of wild type SpUP showed that substrate specificity is similar to that of EcUP, while EcUP is about sevenfold more efficient than SpUP. Biochemical studies on active site mutant SpUP showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that negative charge in the transition state resides mostly on uracil O2. This is in contrast to EcUP for which transition state stabilization occurs mostly at O4.

Metalation of 6-methyl-9-(tetrahydro-2H-pyran-2-yl)purine (10) with lithiating agents of varying basicities such as n-BuLi and LiHMDS in THF at -78 degrees C resulted in metalation at both of the 6-CH(3) moiety and the 8-CH position, irrespective of the molar equivalence of the base. On the other hand, a regioselective metalation at the 6-CH(3) moiety of 10 was observed with NaHMDS or KHMDS, under similar conditions. Treatment of the potassium salts of 10 and of the protected riboside derivative 6-methyl-9-(beta-D-2,3,5-tri-O-tert-butyldimethylsilylribofuranosyl)purine (22) with N-fluorobenzenesulfonamide (NFSI) at -78 degrees C gave the corresponding 6-fluoromethylpurine derivatives 11 and 23, respectively, in good yields. Deprotection of 11 and 23 under standard conditions gave 6-fluoromethylpurine (6-FMeP, 3) and 6-fluoromethyl-9-(beta-D-ribofuranosyl)purine (6-FMePR, 4), respectively, in high yield. Both 3 and 4 demonstrated cytotoxic activity against CCRF-CEM cells in culture. 6-FMePR is a good substrate for E. coli purine nucleoside phosphorylase (E. coli PNP) with a comparable substrate activity to that of the parent nucleoside, 6-methyl-9-(beta-D-ribofuranosyl)purine (6-MePR, 21). The cytotoxic activity of 6-FMeP along with the substrate activity of 6-FMePR with E. coli PNP meet the fundamental requirements for using 6-FMeP as a potential toxin in PNP/prodrug based cancer gene therapy.

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.

3084 Background: The use of E. coli purine nucleoside phosphorylase (PNP) to activate fludarabine was safe and demonstrated robust antitumor activity in preclinical studies. The regimen demonstrate...

The polyamines putrescine, spermine, and spermidine, present in all living cells, have been implicated in the replication of some herpesviruses and retroviruses, and elevated levels of these polyamines have been found in the lymphocytes of patients infected with HIV-1. We have examined the effect of HIV-1 infection on polyamine pools in cell culture. HIV-1 did not significantly affect the polyamine pools in CEM cells. Consistent with this observation, inhibitors of the two key enzymes of this pathway, ornithine decarboxylase and S-adenosylmethionine decarboxylase, did not prevent viral-induced cytopathic effects (CPE) in this cell line. Our results indicate that inhibitors of this pathway will not be therapeutically useful in the treatment of AIDS.

Objectives: Analyse a series of halogenated 3-deaza-adenosine analogues for efficacy against Mycobacterium tuberculosis H37Ra and determine if adenosine (Ado) kinase plays a role in the mechanism of action of these compounds. Methods: The MIC as determined by microdilution broth assay provided a measure of antitubercular efficacy. MIC values were measured in M. tuberculosis strains H37Ra, SRICK1 (an Ado kinase-deficient strain of M. tuberculosis derived from H37Ra) and SRICK1 complemented with adoK, the gene which codes for Ado kinase in M. tuberculosis, in order to determine if Ado kinase played a role in the mechanism of action of these compounds. Furthermore, each compound was analysed as both a substrate and inhibitor for purified Ado kinases from M. tuberculosis and human sources. Results: 2-Fluoro-3-deaza-adenosine, 3-fluoro-3-deaza-adenosine and 2,3-difluoro-3-deaza-adenosine exhibited antitubercular activity that was Ado kinase-dependent. Furthermore, these compounds were at least 10-fold better substrates for M. tuberculosis Ado kinase than the human homologue. Conclusions: The Ado kinase-dependent antitubercular activity exhibited by several of the halogenated 3-deaza-adenosine analogues investigated in this study warrants further investigation of these compounds as antitubercular agents. Furthermore, substrate and inhibition studies provided insight into the Ado-binding domain of Ado kinase, indicating that steric hindrance may limit the size of exocyclic modifications at the 3-position of Ado.

A novel series of 6-methylpurine nucleoside derivatives with substitutions at 5-position have been synthesised These compounds bear a 5'-heterocycle such as triazole or a imidazole with a two carbon chain, and an ether, thio ether or amine. To extend the SAR study of 2-fluoroadenine and 6-methyl purine nucleosides, their corresponding alpha-linker nucleosides with L-xylose and L-lyxose were also synthesized. All of these compounds have been evaluated for their substrate activity with E. coli PNP.

The E. coli PNP suicide gene sensitizes solid tumors to nucleoside prodrugs, such as 6-methylpurine-2'-deoxyriboside (MeP-dR). In this study using lentiviral, MuLv, and HSV-based gene transfer, we quantified thresholds for inhibition of tumor growth and bystander killing by E. coli PNP and tested the role of intestinal flora in this process. Regressions of human glioma tumors following retroviral transduction exhibited dose dependence on both the level of PNP expression and the dose of MeP-dR administered, including strong tumor inhibition when 90-99% bystander cells comprised the tumor mass. A replication competent, non-neurovirulent herpes simplex virus (HSV) deficient in both copies of the gamma-1 34.5 gene was next engineered to express E. coli PNP under the egr-1 promoter (HSV-PNP). HSV-PNP injected intratumorally (17 million pfu/0.05 ml) in nude mice bearing 300 mg human glioma flank tumors produced a delay in tumor growth (approximately 24 days delay to one doubling). MeP-dR treatment after antibiotic therapy (to eliminate enteric flora encoding PNP enzymes) resulted in antitumor enhancement, with arrest of tumor growth (delay to doubling50 days). Bystander killing of the magnitude described here has been difficult to accomplish with other suicide genes, such as HSV-tk or cytosine deaminase. The results establish a model for applying E. coli PNP to HSV treatment of glioma.

Escherichia coli purine nucleoside phosphorylase (PNP) expressed in tumors converts relatively nontoxic prodrugs into membrane-permeant cytotoxic compounds with high bystander activity. In the present study, we examined tumor regressions resulting from treatment with E. coli PNP and fludarabine phosphate (F-araAMP), a clinically approved compound used in the treatment of hematologic malignancies. We tested bystander killing with an adenoviral construct expressing E. coli PNP and then more formally examined thresholds for the bystander effect, using both MuLv and lentiviral vectoring. Because of the importance of understanding the mechanism of bystander action and the limits to this anticancer strategy, we also evaluated in vivo variables related to the expression of E. coli PNP (level of E. coli PNP activity in tumors, ectopic expression in liver, percentage of tumor cells transduced in situ, and accumulation of active metabolites in tumors). Our results indicate that F-araAMP confers excellent in vivo dose-dependent inhibition of bystander tumor cells, including strong responses in subcutaneous human glioma xenografts when 95 to 97.5% of the tumor mass is composed of bystander cells. These findings define levels of E. coli PNP expression necessary for antitumor activity with F-araAMP and demonstrate new potential for a clinically approved compound in solid tumor therapy.

The selective expression of Escherichia coli purine nucleoside phosphorylase (PNP) in solid tumors has been successfully used to activate two purine nucleoside analogs [9-(2-deoxy-beta-D-ribofuranosyl)-6-methylpurine (MeP-dR) and 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-araA)] resulting in lasting tumor regressions and cures. E. coli PNP also cleaves 2-fluoro-2'-deoxyadenosine (F-dAdo) to 2-F-adenine, which is the toxic purine analog liberated from F-araA that has high bystander activity and is active against nonproliferating tumor cells. As F-dAdo is 3000 times better than F-araA as a substrate for E. coli PNP, we have evaluated its antitumor activity against D54 gliomas that express E. coli PNP and have characterized its in vivo metabolism in order to better understand its mechanism of action with respect to the other two agents. Like MeP-dR and F-araA-5'-monophosphate (F-araAMP, a prodrug of F-araA), treatment of mice bearing D54 tumors that express E. coli PNP with F-dAdo resulted in excellent antitumor activity. Although F-dAdo was as active as MeP-dR and better than F-araAMP, it was not dramatically better than either compound because of its short plasma half-life and the limited activation of F-adenine to toxic metabolites. Regardless, these results indicated that F-dAdo was also an excellent prodrug for use with gene vectors that deliver E. coli PNP to tumor cells.

This report examines a major barrier to suicide gene therapy in cancer and other diseases: namely, bystander cell killing. Existing vectors for in vivo gene delivery are inefficient and often transduce or transfect less than 1% of target cells. The E. coli PNP gene brings about cellular necrosis under conditions when 1 in 100 to 1 in 1000 cells express the gene product in vitro. In vivo bystander killing at or near this magnitude has not been reported previously. In the present experiments, transfection of cells with the E. coli PNP gene controlled by a SV40 promoter resulted in 30 nmol 6-methyl purine deoxyriboside (MeP-dR) converted per milligram tumor cell extract per hour (or conversion units (CU)). This level of expression led to elimination of entire populations of tumor cells in vitro after treatment with MeP-dR. Much earlier killing was observed using a tat transactivated E. coli PNP vector (approximately seven-fold higher activity, 230 CU). In vivo effects on tumor growth were next examined. Human ovarian tumors transfected with E. coli PNP were excised 5 days after i.p. implantation from the peritoneal cavities of mice in order to determine both E. coli PNP enzymatic activity and the fraction of cells expressing the gene. PNP activity at 5 days after gene transfer was approximately 170 CU and was expressed in approximately 0.1% of the tumor cells as judged by in situ hybridization. The expression of E. coli PNP at this level produced a 30% increase in life span (P < 0.001) and 49% reduction in tumor size (P < 0.005) after MeP-dR treatment, as compared with control tumors. Our observations lead to the conclusion that pronounced bystander killing by E. coli PNP is conferred in vivo, and that vectors capable of transgene expression in as few as one in 1000 cells can produce substantial antitumor effects if expression on a per cell basis is very high.

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.

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.

For 3 consecutive days, the nucleoside cordycepin (3'-deoxyadenosine) was administered as 1-hr iv infusions (0, 1, 4, 8, 10, or 20 mg/kg/day) to dogs. These doses were given 1 hr after a bolus iv injection (0.25 mg/kg/day) of 2'-deoxycoformycin (dCF), a potent inhibitor of adenosine deaminase. The hypothesis was that dCF would affect the toxicity of cordycepin. Plasma adenosine deaminase activity was strongly inhibited during the dose period and for 5 days following the final dose of dCF. Dogs given cordycepin alone showed no drug-related toxicities. In dogs given only dCF, drug-related toxicity to lymphoid tissue (lymphopenia and thymus lymphoid depletion), thrombocytopenia, and decreases in food consumption were observed. Cordycepin in combination with dCF produced symptoms associated with severe gastrointestinal toxicity (decreased body weights, emesis, diarrhea, decreased food consumption, and necrosis of the gastrointestinal tract) and bone marrow toxicity (lymphopenia, thrombocytopenia, and depletion of hematopoietic cells). The gastrointestinal tract and bone marrow were sites associated with dose-limiting toxicities. In surviving dogs, most of the effects were reversible by Day 30. The maximum tolerated dose of cordycepin administered in combination with dCF was 8 mg/kg/day (160 mg/m2/day) given daily for 3 days.

Within a series of halo derivatives of adenosine, deoxyadenosine and arabinosyladenine attempts have been made to correlate structure with cytotoxicity, substrate activity for adenosine deaminase and nucleoside kinases, and efficacy of the corresponding nucleotides against target enzymes.

From H. Ep.#2 cells in culture, a subline was selected for high resistance to 4-aminopyrazolo (3,4-d)pyrimidine (APP). Upon culture in the absence of APP, the selected line reverted partially to sensitivity. The APP-resistant cells were also highly resistant to APP-ribonucleoside and 6-methylthiopurine ribonucleoside, but showed a much smaller degree of resistance to 2-fluoroadenine, 2-fluoroadenosine, tubercidin and certain other analogs of purines and purine nucleosides. There was generally good correlation between concentrations of purine and nucleoside analogs required to produce inhibition of de novo synthesis of purines and those required to produce growth inhibition. Metabolic and other studies showed that: (1) sensitive and resistant cells were equally permeable to APP; (2) extracts of sensitive and resistant cells had about the same activities of AMP and IMP-pyrophosphorylases and some capacity to convert APP to its nucleotide; (3) extracts of resistant cells had adenosine kinase activity, but it was less than in extracts of sensitive cells; (4) intact resistant cells grown in the presence of APP- 14 C or 6-methylthiopurine- 35 S ribonucleoside contained little or no radioactive nucleotides, whereas they metabolized 2-fluoroadenosine- 3 H to phosphate derivatives to about the same extent as did sensitive cells; (5) extracts of resistant cells were more active than extracts of sensitive cells in dephosphorylating nucleotides (5'-AMP, 5'-IMP, APP-ribonucleotide, and 6-methylthiopurine ribonucleotide); (6) when treated with azaserine, sensitive cells incorporated formate- 14 C predominantly into formylglycinamide ribonucleotide, whereas in resistant cells the corresponding ribonucleoside was the principal product; (7) resistant cells were more active than sensitive cells in metabolizing adenine (inosine and hypoxanthine were the major soluble products). Resistance to APP thus appears to be associated with incapacity of the cells to accumulate APP-ribonucleotides and this incapacity may be related to the higher rate of degradation of APP-ribonucleotides in resistant cells. Sensitive cells grown in the presence of APP- 14 C contained, in addition to APP-ribonucleotides, small amounts of APP-ribonucleoside and its deamination product, 4-hydroxypyrazolo (3,4- d )pyrimidine ribonucleoside. APP-ribonucleoside was a substrate for calf intestinal adenosine deaminase; the K m value was 2.5 ×1O −4 M and the V max was 15 μmoles/min/mg protein.

SYNOPSIS Eighty-four purine and pyrimidine analogs were evaluated for growth inhibition of Tetrahymena pyriformis. The most toxic were 2-fluoroadenine, 2-fluoroadenosine, 6-methylpurine, a series of 5-fluoropyrimidines, and a series of adenine derivatives substituted in the 9-position. 2-Fluoroadenine was metabolized to the ribonucleoside triphosphate and was incorporated into nucleic acids; its inhibition of growth was reversed by high levels of adenine. 6-Methylthiopurine ribonucleoside was phosphorylated, but only to the monophosphate derivative. Contrasting T. pyriformis with mammalian cells gave clues to the mechanism of action of some of the agents. 6-Mercaptopurine, 6-methylthiopurine ribonucleoside, and 6-thioguanine, all potent pseudofeedback inhibitors of de novo purine biosynthesis in mammalian cells, are not toxic to T. pyriformis, which lacks the de novo purine pathway; this implies that inhibition of de novo purine biosynthesis by them underlies their growth inhibition of mammalian cells.

A simple procedure is described for improving the reproducibility of two-dimensional papergrams made by the solvent systems: phenol—water followed by butanol—propionic acid—water. The procedure involves control of the water content of the chromatographic paper prior to the addition of solvent. Some effects noted upon the variation of the water content of paper are described for some chromatographic patters of biochemical interest.

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)

Studies on 8-aza-6-thioinosine (azaMPR) have revealed a very complex picture of its chemistry and metabolism. Under physiologic conditions, azaMPR rearranges at a significant rate to N-β- d -ribofuranosyl[1,2,3]thiadiazolo [5,4-d] pyrimidine-7-amine (TPR). This ribosylamine (TPR) is, in turn, unstable under these conditions undergoing isomerization, via opening of the ribofuranose ring, to give the four possible ribosylamines — the α- and β-ribofuranoses and the α- and β-ribopyranoses and, after a longer period, loss of the sugar moiety giving the biologically inactive [1,2,3]thiadiazolo[5,4-d]pyrimidin-7-amine. An examination of extracts of cells treated with azaMPR showed the presence of three nucleotides — 8-aza-6-thioinosinic acid and N-α- and β- d -ribofuranosyl[1,2,3]thiadiazolo [5,4-d] pyrimidin-7-amine 5′-phosphates (TPRP and α-TPRP). TPR, which is more cytotoxic than azaMPR, gives rise in treated cells to TPRP and its α-anomer only. AzaMPR and TPR are both excellent substrates for adenosine kinase and selectively reduce guanine nucleotide pools and inhibit the synthesis of RNA and DNA without affecting protein synthesis. It is possible that TPRP is the biologically active metabolite, but this has not been established. 8-Aza-S-methyl-6-thioinosine and 8-aza-O6-methylinosine are also highly cytotoxic nucleosides, as are the corresponding ethyl compounds. The thio compounds, poor substrates for adenosine deaminase and excellent substrates for adenosine kinase, owe their biologic activity to their conversion to the alkylthio ribonucleotides. On the other hand, the O6-alkyl compounds are substrates for both the deaminase and the kinase, and conversion to 8-azainosinic acid probably contributes to their activity. The conversion of 8-aza-O6-methylguanosine to 8-azaguanosine is entirely responsible for its activity since it is a good substrate for the deaminase, is not a substrate for the kinase, and its activity is blocked by 2′-deoxycoformycin, a potent inhibitor of the deaminase. 2-Amino-6-chloro-1-deazapurine (ACD), a highly cytotoxic purine analog with activity against leukemia L1210, is converted to its ribonucleotide in cells, presumably by hypoxanthine-guanine phosphoribosyl transferase. Since the ribonucleoside of ACD is inactive, it would appear not to be a substrate for either purine nucleoside phosphorylase or any cellular kinase. The mechanism of action of ACD ribonucleotide is currently under investigation.

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.

This study was undertaken to attempt to rationalize previously obtained and apparently conflicting findings that although adenosine kinase (EC 2.7.1.20) from H.Ep. # 2 cells did not accept inosine as a substrate, these cells became resistant to an inosine analog, 8-azainosine, only when activities of both hypoxanthine (guanine) phosphoribosyltransferase [H(G)PRTase] and adenosine kinase were lost. No evidence could be found for the presence of inosine kinase in H.Ep. # 2 cells: crude supernatants from these cells converted ring-labeled inosine to IMP, but the conversion was prevented by the addition of hypoxanthine and therefore apparently was achieved by the alternative pathway involving the action of H(G)PRTase on hypoxanthine. An investigation of inosine and inosine analogs as substrates for adenosine kinase revealed that certain inosine analogs were substrates and that the substrate activity could be correlated with the degree of ionization of the N-1 proton. Of four 6-oxo and 6-thio nucleosides studied. 8-aza-6-thioinosine had the lowest pKa (6.75) and was the only one that was a good substrate at pH 7.0; the Km was 210μM and the Vmax was about 1.5 times that of adenosine. The rate of phosphorylation of 8-aza-6-thioinosine increased markedly as the pH of the reaction mixture was increased in the pH range 6.0 to 7.0. Phosphorylation of 8-azainosine (pKa 7.45) and 6-thioinosine (pKa 7.60) was much poorer and could be demonstrated at pH 7.0 only after overnight incubation with the kinase. The fact that 8-azainosine and 8-aza-6-thioinosine are substrates whereas ionsine is not, can be rationalized by the facts that (a) the substitution of an N-atom for the C-8 atom of the nucleoside lowers the pKa so that the N-1 proton is more strongly ionized at physiological pH; and (b) the ionized form of a 6-oxo or 6-thio nucleoside resembles adenosine with respect to the bond structure at the 1- and 6-positions of the purine ring whereas the unionized form does not.