5-Aza-4'-thio-2'-deoxycytidine, a New Orally Bioavailable Nontoxic "Best-in-Class": DNA Methyltransferase 1-Depleting Agent in Clinical Development.

DNA methyltransferase (DNMT) 1 is an enzyme that functions as a maintenance methyltransferase during DNA replication, and depletion of this enzyme from cells is considered to be a rational goal in DNA methylation-dependent disorders. Two DNMT1-depleting agents 5-aza-2'-deoxycytidine (aza-dCyd, decitabine) and 5-aza-cytidine (aza-Cyd, azacitidine) are currently used for the treatment of myelodysplastic syndromes and acute myeloid leukemia and have also been investigated for nononcology indications, such as sickle cell disease. However, these agents have several off-target activities leading to significant toxicities that limit dosing and duration of treatment. Development of more selective inhibitors of DNMT1 could therefore afford treatment of long durations at effective doses. We have discovered that 5-aza-4'-thio-2'-deoxycytidine (aza-T-dCyd) is as effective as aza-dCyd in depleting DNMT1 in mouse tumor models but with markedly low toxicity. In this review we describe the preclinical studies that led to the development of aza-T-dCyd as a superior DNMT1-depleting agent with respect to aza-dCyd and will describe its pharmacology, metabolism, and mechanism of action. In an effort to understand why aza-T-dCyd is a more selective DNMT1 depleting agent than aza-dCyd, we will also compare and contrast the activities of these two agents. SIGNIFICANCE STATEMENT: Aza-T-dCyd is a potent DNMT1-depleting agent. Although similar in structure to decitabine (aza-dCyd), its metabolism and mechanism of action is different than that of aza-dCyd, resulting in less off-target activity and less toxicity. The larger therapeutic index of aza-T-dCyd (DNMT1 depletion vs. toxicity) in mice suggests that it would be a better clinical candidate to selectively deplete DNMT1 from target cells and determine whether or not depletion of DNMT1 is an effective target for various diseases.

6-Methylpurine derived sugar modified nucleosides: Synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases.

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-(ß-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-ß-d-allofuranosyl)-6-methylpurine (3) and 9-(6-deoxy-5-C-methyl-ß-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-ß-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 ß-d-allo-furanosyl derivative 3 and the 5'-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.

PRE-CLINICAL AND CLINICAL VALIDATION OF AN ANTI-CANCER MODALITY THAT ABLATES REFRACTORY, LOW GROWTH FRACTION TUMORS.

Intratumoral expression of the E. coli purine nucleoside phosphorylase (PNP) gene was originally described by our laboratories as a means to inhibit growth of solid tumors in vivo. The approach generates purine bases that disrupt DNA, RNA, and protein synthesis, a unique mechanism when compared with all approved or experimental cancer therapeutics. Use of PNP has been validated by numerous laboratories worldwide against human tumor xenografts (lung, liver, pancreas, bladder, glioma, and prostate, among others). Data from a recently completed phase 1 clinical trial has indicated substantial anti-cancer activity in human subjects with no serious toxicities.

Role of methylthioadenosine/S-adenosylhomocysteine nucleosidase in Vibrio cholerae cellular communication and biofilm development.

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.

Deoxycytidine kinase regulates the G2/M checkpoint through interaction with cyclin-dependent kinase 1 in response to DNA damage.

Deoxycytidine kinase (dCK) is a rate limiting enzyme critical for phosphorylation of endogenous deoxynucleosides for DNA synthesis and exogenous nucleoside analogues for anticancer and antiviral drug actions. dCK is activated in response to DNA damage; however, how it functions in the DNA damage response is largely unknown. Here, we report that dCK is required for the G2/M checkpoint in response to DNA damage induced by ionizing radiation (IR). We demonstrate that the ataxia-telangiectasia-mutated (ATM) kinase phosphorylates dCK on Serine 74 to activate it in response to DNA damage. We further demonstrate that Serine 74 phosphorylation is required for initiation of the G2/M checkpoint. Using mass spectrometry, we identified a protein complex associated with dCK in response to DNA damage. We demonstrate that dCK interacts with cyclin-dependent kinase 1 (Cdk1) after IR and that the interaction inhibits Cdk1 activity both in vitro and in vivo. Together, our results highlight the novel function of dCK and provide molecular insights into the G2/M checkpoint regulation in response to DNA damage.

Effectiveness of disinfection therapies and promotion of osteoblast growth on osseotite and nanotite implant surfaces.

OBJECTIVES: To evaluate the effectiveness of 4 procedures to disinfect implant surfaces intentionally inoculated with bacteria and afterward to evaluate osteoblast viability to the disinfected implant surfaces. MATERIALS AND METHODS: Eighty-eight commercially pure Osseotite and Nanotite titanium implant discs were inoculated with Porphyromonas gingivalis. The implant surfaces were disinfected with EDTA, tetracycline, citric acid, or neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. The implant discs were then placed in cultures of osteoblast cells. RESULTS: Osseotite implant discs were easier to disinfect compared with the Nanotite implant discs. Citric acid and tetracycline were the most effective solutions for the disinfection of P. gingivalis from the Osseotite implant discs. CONCLUSION: The Nanotite implant discs were the most difficult to disinfect, likely because of their chemical and physical properties. Citric acid and tetracycline were most effective for disinfecting the Osseotite implant discs, and further clinical research is needed to verify these effects in vivo. The Nd:YAG laser was the weakest disinfection method, and it is not recommended for disinfecting implant surfaces until its effectiveness is improved.

Evaluating antitumor activity of Escherichia coli purine nucleoside phosphorylase against head and neck patient-derived xenografts.

BACKGROUND: Purine nucleoside phosphorylase (PNP) gene transfer represents a promising approach to treatment of head and neck malignancies. We tested recombinant adenovirus already in phase I/II clinical testing and leading-edge patient-derived xenografts (PDX) as a means to optimize this therapeutic strategy. METHODS: Our experiments investigated purine base cytotoxicity, PNP enzyme activity following treatment of malignant tissue, tumor mass regression, viral receptor studies, and transduction by tropism-modified adenovirus. RESULTS: Replication deficient vector efficiently transduced PDX cells and mediated significant anticancer effect following treatment with fludarabine phosphate in vivo. Either 6-methylpurine or 2-fluoroadenine (toxic molecules generated by the PNP approach) ablated head and neck cancer cell proliferation. High levels of adenovirus-3 specific receptors were detected in human tumor models, and vector was evaluated that utilizes this pathway. CONCLUSIONS: Our studies provide the scientific foundation necessary to improve PNP prodrug cleavage and advance a new treatment for head and neck cancer.

Identification and characterization of two adenosine phosphorylase activities in Mycobacterium smegmatis.

Purine nucleoside phosphorylase (PNP) is an important enzyme in purine metabolism and cleaves purine nucleosides to their respective bases. Mycobacterial PNP is specific for 6-oxopurines and cannot account for the adenosine (Ado) cleavage activity that has been detected in M. tuberculosis and M. smegmatis cultures. In the current work, two Ado cleavage activities were identified from M. smegmatis cell extracts. The first activity was biochemically determined to be a phosphorylase that could reversibly catalyze adenosine + phosphate ↔ adenine + alpha-D-ribose-1-phosphate. Our purification scheme led to a 30-fold purification of this activity, with the removal of more than 99.9% of total protein. While Ado was the preferred substrate, inosine and guanosine were also cleaved, with 43% and 32% of the Ado activity, respectively. Our data suggest that M. smegmatis expresses two PNPs: a previously described trimeric PNP that can cleave inosine and guanosine only and a second, novel PNP (Ado-PNP) that can cleave Ado, inosine, and guanosine. Ado-PNP had an apparent K(m) (K(m) ( app)) of 98 ± 6 µM (with Ado) and a native molecular mass of 125 ± 7 kDa. The second Ado cleavage activity was identified as 5'-methylthioadenosine phosphorylase (MTAP) based on its biochemical properties and mass spectrometry analysis. Our study marks the first report of the existence of MTAP in any bacterium. Since human cells do not readily convert Ado to Ade, an understanding of the substrate preferences of these enzymes could lead to the identification of Ado analogs that could be selectively activated to toxic products in mycobacteria.

The crystal structure of Streptococcus pyogenes uridine phosphorylase reveals a distinct subfamily of nucleoside phosphorylases.

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/ß 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 its substrate specificity is similar to that of EcUP, while EcUP is ∼7-fold more efficient than SpUP. Biochemical studies of SpUP mutants showed that mutations of His169 reduced activity, while mutation of Lys162 abolished all activity, suggesting that the 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.

Structure of grouper iridovirus purine nucleoside phosphorylase.

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 A resolution. The crystals belonged to space group R3, with unit-cell parameters a = 193.0, c = 105.6 A, and contained four protomers per asymmetric unit. The overall structure of givPNP shows high similarity to mammalian PNPs, having an alpha/beta structure with a nine-stranded mixed beta-barrel flanked by a total of nine alpha-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.

The use of Trichomonas vaginalis purine nucleoside phosphorylase to activate fludarabine in the treatment of solid tumors.

Treatment with fludarabine phosphate (9-ß-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.

Intratumoral generation of 2-fluoroadenine to treat solid malignancies of the head and neck.

This report describes treatment of locoregional head and neck squamous cell carcinoma (HNSCC) by an innovative, experimental strategy involving generation of a robust anti-cancer agent (2-fluoroadenine [F-Ade]) following transduction by Escherichia coli purine nucleoside phosphorylase (PNP) in a small number of tumor cells. F-Ade works by a unique mechanism of action (ablation of RNA and protein synthesis) and confers tumor regressions of otherwise refractory HNSCC in human subjects. Clinical studies have now advanced to a pivotal (registration-directed) trial involving locoregional HNSCC, with plans to begin subject enrollment late in 2018. The present review is the first to summarize use of PNP in the context of HNSCC, and provides background regarding this emerging anti-cancer approach.

Synthesis of 1-beta-D-ribofuranosyl-3-ethynyl-[1,2,4]triazole and its in vitro and in vivo efficacy against Hantavirus.

There are no FDA approved drugs for the treatment of hemorrhagic fever with renal syndrome (HFRS), a serious human illnesses caused by hantaviruses. Clinical studies using ribavirin (RBV) to treat HFRS patients suggest that it provides an improved prognosis when given early in the course of disease. Given the unique antiviral activity of RBV and the lack of other lead scaffolds, we prepared a diverse series of 3-substituted 1,2,4-triazole-beta-ribosides and identified one with antiviral activity, 1-beta-d-ribofuranosyl-3-ethynyl-[1,2,4]triazole (ETAR). ETAR showed an EC(50) value of 10 and 4.4 microM for Hantaan virus (HTNV) and Andes virus, respectively. ETAR had weak activity against Crimean Congo hemorrhagic fever virus, but had no activity against Rift Valley fever virus. Intraperitoneally delivered ETAR offered protection to suckling mice challenged with HTNV with a approximately 25% survival at 12.5 and 25mg/kg ETAR, and a MTD of 17.1+/-0.7 days. ETAR was phosphorylated in Vero E6 cells to its 5'-triphosphate and reduced cellular GTP levels. In contrast to RBV, ETAR did not increase mutation frequency of the HTNV genome, which suggests it has a different mechanism of action than RBV. ETAR is an exciting and promising lead compound that will be elaborated in further synthetic investigations as a framework for the rational design of new antivirals for treatment of HFRS.

c-Abl-independent p73 stabilization during gemcitabine- or 4'-thio-beta-D-arabinofuranosylcytosine-induced apoptosis in wild-type and p53-null colorectal cancer cells.

Nucleoside anticancer drugs like gemcitabine (2'-deoxy-2',2'-difluorocytidine) are potent inducers of p53, and ectopic expression of wild-type p53 sensitizes cells to these agents. However, it is also known that nucleosides are efficient activators of apoptosis in tumor cells that do not express a functional p53. To clarify this issue, we examined the effects of gemcitabine and 4'-thio-beta-d-arabinofuranosylcytosine (T-ara-C) on p73, a structural and functional homologue of p53, whose activation could also account for nucleoside-induced apoptosis because no functionally significant mutations of p73 have been reported in cancers. Acute treatment of HCT 116 colon carcinoma cells with gemcitabine or T-ara-C induced marked cytotoxicity and cleavage of caspase-3 and poly(ADP-ribose) polymerase. T-ara-C and gemcitabine markedly induced p53 accumulation as well as increased levels of phospho-p53 (Ser15/Ser20/Ser46) and induced its binding to a consensus p53 response element. Despite robust activation of p53 by T-ara-C and gemcitabine, we found that wild-type and p53-/- HCT 116 cells exhibited almost equivalent sensitivity towards these nucleosides. Examination of p73 revealed that T-ara-C and gemcitabine markedly increased p73 protein levels and p73 DNA-binding activities in both p53-/- and wild-type cells. Furthermore, T-ara-C- and gemcitabine-induced increases in p73 levels occur due to a decrease in p73 protein turnover. RNA interference studies show that nucleoside-induced p73 increases are independent of c-Abl, a nucleoside-activated kinase recently implicated in p73 stabilization. HCT 116 lines, wherein the downstream p53/p73 targets Bax and PUMA (p53 up-regulated modulator of apoptosis) were deleted, were less sensitive to T-ara-C and gemcitabine. Together, these studies indicate that c-Abl-independent p73 stabilization pathways could account for the p53-independent mechanisms in nucleoside-induced apoptosis.

Antiangiogenic activity of 4'-thio-beta-D-arabinofuranosylcytosine.

4'-Thio-beta-D-arabinofuranosylcytosine (T-araC), a new-generation deoxycytidine nucleoside analogue, showed significant efficacy against numerous solid tumors in preclinical studies and entered clinical development for cancer therapy. It is a structural analogue of cytarabine (araC), a clinically used drug in the treatment of acute myelogenous leukemia, which has no or very limited efficacy against solid tumors. In comparison with araC, the excellent in vivo activity of T-araC against solid tumors suggests that, in addition to inhibition of DNA synthesis, T-araC may target cellular signaling pathways, such as angiogenesis, in solid tumors. We studied T-araC and araC for their antiangiogenic activities in vitro and in vivo. Both compounds inhibited human endothelial cell proliferation with similar IC50s. However, only T-araC inhibited endothelial cell migration and differentiation into capillary tubules. T-araC also abrogated endothelial cell extracellular signal-regulated kinase (ERK) 1/2 phosphorylation, a key signaling molecule involved in cellular processes of angiogenesis. Results from chick chorioallantoic membrane angiogenesis assays revealed that T-araC significantly inhibited the development of new blood vessels in vivo, whereas araC showed much less effect. The findings of this study show a role of T-araC in antiangiogenesis and suggest that T-araC combines antiproliferative and antiangiogenic activity in one molecule for a dual mechanism of drug action to achieve the excellent in vivo efficacy against several solid tumors. This study also provides important information for optimizing dosage and sequence of T-araC administration in clinical investigations by considering T-araC as both an antiproliferative and an antiangiogenic agent.

Use of E. coli Purine Nucleoside Phosphorylase in the Treatment of Solid Tumors.

BACKGROUND: The selective expression of non-human genes in tumor tissue to activate non-toxic compounds (Gene Directed Prodrug Enzyme Therapy, GDEPT) is a novel strategy designed for killing tumor cells in patients with little or no systemic toxicity. Numerous non-human genes have been evaluated, but none have yet been successful in the clinic. METHODS: Unlike human purine nucleoside phosphorylase (PNP), E. coli PNP accepts adenine containing nucleosides as substrates, and is therefore able to selectively activate non-toxic purine analogs in tumor tissue. Various in vitro and in vivo assays have been utilized to evaluate E. coli PNP as a potential activating enzyme. RESULTS: We and others have demonstrated excellent in vitro and in vivo anti-tumor activity with various GDEPT strategies utilizing E. coli PNP to activate purine nucleoside analogs. A phase I clinical trial utilizing recombinant adenoviral vector for delivery of E. coli PNP to solid tumors followed by systemic administration of fludarabine phosphate (NCT01310179; IND# 14271) has recently been completed. In this trial, significant anti-tumor activity was demonstrated with negligible toxicity related to the therapy. The mechanism of cell kill (inhibition of RNA and protein synthesis) is distinct from all currently used anticancer drugs and all experimental compounds under development. The approach has demonstrated excellent ability to kill neighboring tumor cells that do not express E. coli PNP, is active against non-proliferating and proliferating tumors cells (as well as tumor stem cells, stroma), and is therefore very effective against solid tumors with a low growth fraction. CONCLUSION: The unique attributes distinguish this approach from other GDEPT strategies and are precisely those required to mediate significant improvements in antitumor therapy.

Structure-activity relationship for nucleoside analogs as inhibitors or substrates of adenosine kinase from Mycobacterium tuberculosis. I. Modifications to the adenine moiety.

Adenosine kinase (Ado kinase, EC 2.7.1.20) is a purine salvage enzyme that phosphorylates adenosine (Ado) to AMP. Ado kinase from Mycobacterium tuberculosis also catalyzes an essential step in the conversion of 2-methyl-Ado to a compound with selective antimycobacterial activity. In order to aid in the design of more potent and selective Ado analogs, eighty nucleoside analogs with modifications to the adenine (Ade) moiety of Ado were evaluated as both substrates and inhibitors of Ado kinase from M. tuberculosis, and a subset was further tested with human Ado kinase for the sake of comparison. The best substrates were 2-aza-Ado, 8-aza-9-deaza-Ado, and 2-fluoro-Ado and the most potent inhibitors were N1-benzyl-Ado (Ki=0.19 microM), 2-fluoro-Ado (Ki=0.5 microM), 6-cyclopentyloxy-purine riboside (Ki=0.15 microM), and 7-iodo-7-deaza-Ado (Ki=0.21 microM). These studies revealed the presence of a hydrophobic pocket near the N6- and N1-positions that can accommodate substitutions at least as large as a benzyl group. The ability to fit into this pocket increased the likelihood that a compound would be an inhibitor and not a substrate. The 2-position was able to accommodate exocyclic substitutions as large as a methoxy group, although substrate activity was low. Similarly, the 7-position could bind an exocyclic group as large as a carboxamido moiety. However, all of the compounds tested with modifications at the 7-position were much better inhibitors than substrates. MIC studies performed with selected compounds have yielded several Ado analogs with promising antitubercular activity. Future studies will utilize this information for the design of new analogs that may be selective antitubercular agents.

Long intracellular retention of 4'-thio-arabinofuranosylcytosine 5'-triphosphate as a critical factor for the anti-solid tumor activity of 4'-thio-arabinofuranosylcytosine.

4'-Thio-arabinofuranosylcytosine (T-araC) is a new cytosine analog, which exhibits excellent antitumor activity against various solid tumor xenografts in mice. T-araC is a structural analog of arabinofuranosylcytosine (araC), which is known to be marginally active against solid tumors. We have continued to study the biochemical pharmacology of T-araC in solid tumor cells to further characterize the mechanism of action of this new agent and to elucidate why these compounds show a profound difference in antitumor activity against solid tumors. AraC was a slightly more potent inhibitor of cell growth than T-araC when cells were continuously exposed to the drugs. However, T-araC was markedly more cytotoxic than araC when high concentrations of the compounds were given for short periods of time. Despite the fact that T-araC is a much poorer substrate, as compared to araC, for deoxycytidine kinase (the rate-limiting step in the formation of the triphosphates), similar intracellular concentrations of T-araC-5'-triphosphate (T-araCTP) and araCTP were formed in cells at these high, pharmacologically relevant concentrations due to similar Vmax's. The major difference in the metabolism of araC and T-araC was that the half-life of T-araCTP was tenfold longer than that of araCTP and much higher levels of T-araCTP were sustained in cells for long durations after exposure to T-araC. Inhibition of cytidine deaminase, deoxycytidylate deaminase, or DNA replication did not affect the half-life of either araCTP or T-araCTP. In addition, the rates of disappearance of the mono- and tri-phosphates of araC and T-araC in crude cell extracts were similar. These results indicated that these enzymes were not rate-limiting in the degradation of the respective triphosphates. However, the rate of phosphorylation of T-araC-5'-monophosphate (T-araCMP) in crude cell extracts was about tenfold greater than that of araCMP. The results of this work suggested that the longer intracellular retention of T-araCTP was responsible for the superior activity of T-araC against solid tumors in vivo, and that the greater activity of T-araCMP as a substrate of UMP/CMP kinase was responsible for the long intracellular half-life of T-araCTP.

Excellent in vivo bystander activity of fludarabine phosphate against human glioma xenografts that express the escherichia coli purine nucleoside phosphorylase gene.

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.