Peptidomimetic-based identification of FDA-approved compounds inhibiting IRE1 activity.

Inositol-requiring enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease that is a major mediator of the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. Tumour cells experience ER stress due to adverse environmental cues such as hypoxia or nutrient shortage and high metabolic/protein-folding demand. To cope with those stresses, cancer cells utilise IRE1 signalling as an adaptive mechanism. Here, we report the discovery of the FDA-approved compounds methotrexate, cefoperazone, folinic acid and fludarabine phosphate as IRE1 inhibitors. These were identified through a structural exploration of the IRE1 kinase domain using IRE1 peptide fragment docking and further optimisation and pharmacophore development. The inhibitors were verified to have an impact on IRE1 activity in vitro and were tested for their ability to sensitise human cell models of glioblastoma multiforme (GBM) to chemotherapy. We show that all molecules identified sensitise glioblastoma cells to the standard-of-care chemotherapy temozolomide (TMZ).

Effect of expression of adenine phosphoribosyltransferase on the in vivo anti-tumor activity of prodrugs activated by E. coli purine nucleoside phosphorylase.

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-ß-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.

Pharmacokinetics, bioavailability and effects on electrocardiographic parameters of oral fludarabine phosphate.

The pharmacokinetics, bioavailability and effects on electrocardiographic (ECG) parameters of fludarabine phosphate (2F-ara-AMP) were evaluated in adult patients with B-cell chronic lymphocytic leukemia. Patients received single doses of intravenous (IV) (25 mg/m(2), n=14) or oral (40 mg/m(2), n=42) 2F-ara-AMP. Plasma concentrations of drug and metabolites and digital 12-lead ECGs were monitored for 23 h after dosing. The dephosphorylated product fludarabine (2F-ara-A) was the principal metabolite present in the systemic circulation. Mean (+/-SD) elimination half-life did not differ significantly between IV and oral dosage groups (11.3+/-4.0 vs 9.7+/-2.0 h, p=0.053). Renal excretion was a major clearance pathway, along with transformation to a hypoxanthine metabolite 2F-ara-Hx. Estimated mean oral bioavailability of 2F-ara-A was 58%. Compared to the time-matched drug-free baseline Fridericia correction of the QT interval (QTcF), the mean QTcF change following 2F-ara-AMP did not differ from zero, and a treatment effect of >+10 and >+15 ms could be excluded following oral and IV 2F-ara-AMP, respectively. Similarly, heart rate, PR interval and QRS duration did not change following 2F-ara-AMP treatment. Thus the 25 mg/m(2) IV and 40 mg/m(2) oral doses of 2F-ara-AMP produce similar systemic exposure, and do not prolong QTcF, indicating low risk of drug induced Torsades de Pointes.

Collision induced dissociation studies of alkali metal adducts of tetracyclines and antiviral agents by electrospray ionization, hydrogen/deuterium exchange and multiple stage mass spectrometry.

The collision induced dissociation (CID) mass spectra were obtained for the X(+)-adducts (X=Na(+) or Li(+)) of five tetracyclines, four pyrimidine and three purine derivatives and their fully D-exchanged species in which the labile hydrogens were replaced by deuterium by either gas phase or liquid phase exchange. The CID spectra were obtained for [M + Na](+) and [M + Li](+) and the exchanged analogs, [M(D) + Na](+) and [M(D) + Li](+), and compositions of product ions and mechanisms of decomposition were determined by comparison of the MS(n) spectra of the undeuterated and deuterated species. Metal ions are bound to the base of purine and pyrimidine antiviral agents and dissociate primarily to give the metal complexes of the base [B + X](+). For vidarabine monophosphate, however, the metal ions are bound to the phosphate group, resulting in unique and characteristic cleavage reactions not observed in the uncomplexed system, and dissociate through the loss of phosphate and/or phosphate metal ion complex. The [B + X](+) of these antiviral agents are relatively stable and show no or little fragmentation compared to [B + H](+). The CID of [B + X](+) of guanine derivative occurs mainly through elimination of NH(3) and that of trifluoromethyl uracil dissociates primarily through the loss of HF. For tetracyclines, metal ions are bound to ring A at the tricarbonylmethyl group and dissociate initially by the loss of NH(3)/ND(3) from [M(H) + X](+) and [M(D) + X](+). The CID spectra of [M + X](+) of tetracyclines are somewhat similar to those of [M + H](+). The dominant fragments from the metal complexes of these compounds are charge remote decompositions involving molecular rearrangements and the loss of small stable molecules. Additionally, tetracyclines and the antiviral agents show more selectivity towards Li+ ion than the corresponding complexes with Na(+) or K(+).

Delivery of replication-competent retrovirus expressing Escherichia coli purine nucleoside phosphorylase increases the metabolism of the prodrug, fludarabine phosphate and suppresses the growth of bladder tumor xenografts.

We have developed unique replication-competent retroviral (RCR) vectors based on murine leukemia virus that provide improved efficiency of viral delivery, allow for long-term transgene expression and demonstrate an intrinsic selectivity for transduction of rapidly dividing tumor cells. The purpose of this study was to evaluate the in vivo transduction efficiency and the therapeutic efficacy of the RCR vector mediated delivery of Escherichia coli purine nucleoside phosphorylase (PNP) in combination with fludarabine phosphate for bladder cancer. We constructed vectors containing green fluorescent protein (GFP) gene (ACE)-GFP) or PNP gene (ACE-PNP). KU-19-19 bladder tumors exhibited 28.3+/-16.1, 46.6+/-5.8 and 93.7+/-7.8% of GFP expression on 14, 18 and 26 days after intratumoral injection of ACE-GFP, respectively. GFP expression could not be observed in normal tissues surrounding the injected tumors. No detectable polymerase chain reaction products of GFP gene could be observed in any distant organs. Intratumoral injection of ACE-PNP, followed by systemically administered fludarabine phosphate, significantly inhibited the growth of pre-established KU-19-19 tumors. Our results indicate that RCR vectors are a potentially efficient gene delivery method and that the RCR vector mediated PNP gene transfer and fludarabine phosphate treatment might be a novel and potentially therapeutic modality for bladder cancer.

F-ara-AMP is a substrate of cytoplasmic 5'-nucleotidase II (cN-II): HPLC and NMR studies of enzymatic dephosphorylation.

Intracellular accumulation of triphosphorylated derivatives is essential for the cytotoxic activity of nucleoside analogues. Different mechanisms opposing this accumulation have been described. We have investigated the dephosphorylation of monophosphorylated fludarabine (F-ara-AMP) by the purified cytoplasmic 5'-nucleotidase cN-II using HPLC and NMR. These studies clearly showed that cN-II was able to convert F-ara-AMP into its non phosphorylated form, F-ara-A, with a Km in the millimolar range and Vmax = 35 nmol/min/mg, with both methods. Cytoplasmic 5'-nucleotidase cN-II can degrade this clinically useful cytotoxic nucleoside analogue and its overexpression is thus likely to be involved in resistance to this compound.

Preclinical evaluation of a prostate-targeted gene-directed enzyme prodrug therapy delivered by ovine atadenovirus.

Gene-directed enzyme prodrug therapy (GDEPT) based on the Escherichia coli enzyme, purine nucleoside phosphorylase (PNP), provides a novel strategy for treating slowly growing tumors like prostate cancer (CaP). PNP converts systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine, that kills PNP-expressing and nearby cells by inhibiting DNA, RNA and protein synthesis. Reporter gene expression directed by a hybrid prostate-directed promoter and enhancer, PSMEPb, was assayed after plasmid transfection or viral transduction of prostate and non-CaP cell lines. Androgen-sensitive (AS) LNCaP-LN3 and androgen-independent (AI) PC3 human CaP xenografts in nude mice were injected intratumorally with an ovine atadenovirus vector, OAdV623, that carries the PNP gene under PSMEPb, formulated with cationic lipid for enhanced infectivity. Fludarabine phosphate was then given intraperitoneally for 5 days at 75 mg/m2/day. PNP expression was evaluated by enzymic conversion of its substrate using reverse phase HPLC. OAdV623 showed excellent in vitro transcriptional specificity for CaP cells. In vivo, expression of PNP persisted for > 6 days after OAdV623 injection and a single treatment provided 100% increase in tumor doubling time and > 50% inhibition of tumor growth for both LNCaP-LN3 and PC3 lines, with increased tumor necrosis and apoptosis and decreased tumor cell proliferation. OAdV623 significantly suppressed the growth of AS and AI human CaP xenografts in mice.

The histone deacetylase inhibitor MS-275 interacts synergistically with fludarabine to induce apoptosis in human leukemia cells.

Interactions between the novel benzamide histone deacetylase (HDAC) inhibitor MS-275 and fludarabine were examined in lymphoid and myeloid human leukemia cells in relation to mitochondrial injury, signal transduction events, and apoptosis. Prior exposure of Jurkat lymphoblastic leukemia cells to a marginally toxic concentration of MS-275 (e.g., 500 nM) for 24 h sharply increased mitochondrial injury, caspase activation, and apoptosis in response to a minimally toxic concentration of fludarabine (500 nM), resulting in highly synergistic antileukemic interactions and loss of clonogenic survival. Simultaneous exposure to MS-275 and fludarabine also led to synergistic effects, but these were not as pronounced as observed with sequential treatment. Similar interactions were noted in the case of (a) other human leukemia cell lines (e.g., U937, CCRF-CEM); (b) other HDAC inhibitors (e.g., sodium butyrate); and (c) other nucleoside analogues (e.g., 1-beta-D-arabinofuranosylcytosine, gemcitabine). Potentiation of fludarabine lethality by MS-275 was associated with acetylation of histones H3 and H4, down-regulation of the antiapoptotic proteins XIAP and Mcl-1, enhanced cytosolic release of proapoptotic mitochondrial proteins (e.g., cytochrome c, Smac/DIABLO, and apoptosis-inducing factor), and caspase activation. It was also accompanied by the caspase-dependent down-regulation of p27(KIP1), cyclins A, E, and D(1), and cleavage and diminished phosphorylation of retinoblastoma protein. However, increased lethality of the combination was not associated with enhanced fludarabine triphosphate formation or DNA incorporation and occurred despite a slight reduction in the S-phase fraction. Prior exposure to MS-275 attenuated fludarabine-mediated activation of MEK1/2, extracellular signal-regulated kinase, and Akt, and enhanced c-Jun NH(2)-terminal kinase phosphorylation; furthermore, inducible expression of constitutively active MEK1/2 or Akt significantly diminished MS-275/fludarabine-induced lethality. Combined exposure of cells to MS-275 and fludarabine was associated with a significant increase in generation of reactive oxygen species; moreover, both the increase in reactive oxygen species and apoptosis were largely attenuated by coadministration of the free radical scavenger L-N-acetylcysteine. Finally, prior administration of MS-275 markedly potentiated fludarabine-mediated generation of the proapoptotic lipid second messenger ceramide. Taken together, these findings indicate that the HDAC inhibitor MS-275 induces multiple perturbations in signal transduction, survival, and cell cycle regulatory pathways that lower the threshold for fludarabine-mediated mitochondrial injury and apoptosis in human leukemia cells. They also provide insights into possible mechanisms by which novel, clinically relevant HDAC inhibitors might be used to enhance the antileukemic activity of established nucleoside analogues such as fludarabine.

Gene-directed enzyme prodrug therapy for prostate cancer in a mouse model that imitates the development of human disease.

BACKGROUND: Gene-directed enzyme prodrug therapy (GDEPT) based on the E. coli enzyme purine nucleoside phosphorylase (PNP) represents a new approach for treating slow growing tumours like prostate cancer (PCa). Expressed enzyme converts a systemically administered prodrug, fludarabine phosphate, to a toxic metabolite, 2-fluoroadenine. Infected and neighbouring cells are killed by a bystander effect that results from the inhibition of DNA and RNA synthesis. METHODS: These studies were carried out using the transgenic adenocarcinoma of the prostate (TRAMP) model that mimics human PCa development and progression. Control TRAMP mice were injected intraprostatically with vector vehicle and thereafter intraperitoneally with saline or fludarabine phosphate ( approximately 600 mg/m(2)/day) once daily for 5 consecutive days. Treated mice received a single intraprostatic injection containing 10(10) particles of OAdV220, an ovine atadenovirus which expresses the E. coli PNP gene under the control of the Rous sarcoma virus promoter, followed by systemic fludarabine treatment. The weight of the genitourinary tract, seminal vesicles and the prostate as well as animal survival were monitored. Tumours were also analysed histologically. RESULTS: Preliminary studies showed that fludarabine alone caused no significant change in genitourinary (GU) tract weight in TRAMP mice. Animals injected with vector and prodrug showed a significant reduction (36-47%) in GU tract weight (ANOVA p = 0.0002) and a 35-50% reduction in seminal vesicle weight (ANOVA p = 0.0007). In particular, the target organ showed a significant 57% reduction in prostate weight (ANOVA p = 0.0007). PNP-GDEPT mice also showed a survival advantage over control mice. Histological analysis suggested that the cancer progression was slowed in GDEPT-treated animals. CONCLUSION: A single course of GDEPT based on OAdV-delivered PNP and fludarabine produced highly significant suppression of PCa progression in immune-competent TRAMP mice.

Cellular and clinical pharmacology of fludarabine.

In the past decade, fludarabine has had a major impact in increasing the effectiveness of treatment of patients with indolent B-cell malignancies. This has come about in a variety of clinical circumstances, including use of fludarabine alone as well as in combinations with DNA-damaging agents or membrane-targeted antibodies. Other strategies have used fludarabine to reduce immunological function, thus facilitating non-myeloablative stem cell transplants. Fludarabine is a prodrug that is converted to the free nucleoside 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) which enters cells and accumulates mainly as the 5'-triphosphate, F-ara-ATP. The rate-limiting step in the formation of triphosphate is conversion of F-ara-A to its monophosphate, which is catalyzed by deoxycytidine kinase. Although F-ara-A is not a good substrate for this enzyme, the high specific activity of this protein results in efficient phosphorylation of F-ara-A in certain tissues. F-ara-ATP has multiple mechanisms of action, which are mostly directed toward DNA. These include inhibition of ribonucleotide reductase, incorporation into DNA resulting in repression of further DNA polymerisation, and inhibition of DNA ligase and DNA primase. Collectively these actions affect DNA synthesis, which is the major mechanism of F-ara-A-induced cytotoxicity. Secondarily, incorporation into RNA and inhibition of transcription has been shown in cell lines. With the standard dose of fludarabine (25 to 30 mg/m(2)/day given over 30 minutes for 5 days), plasma concentrations of about 3 micromol/L F-ara-A are achieved at the end of each infusion. Serial sampling of leukaemia cells from patients receiving these standard doses of fludarabine has demonstrated that the peak concentrations of F-ara-ATP are achieved 4 hours after start of fludarabine infusion. Although there is heterogeneity among individuals with respect to rate of F-ara-ATP accumulation, the peak concentrations are generally proportional to the dose of the drug. Knowledge of the plasma pharmacokinetics of its principal nucleoside metabolite F-ara-A, and the cellular pharmacology of the proximal active metabolite, F-ara-ATP, has provided some understanding of the activity of fludarabine when used as a single agent. Preclinical studies directed toward learning the mechanisms of action of this agent have formed the basis for several mechanism-based strategies for its combination and scheduling with other agents. As a single agent fludarabine has been effective for the indolent leukaemias. Biochemical modulation strategies resulted in enhanced accumulation of cytarabine triphosphate and led to the use of fludarabine for the treatment of acute leukaemias. Combination of fludarabine with DNA damaging agents to inhibit DNA repair processes has been highly effective for indolent leukaemias and lymphomas. The current review brings together knowledge of the mechanisms of fludarabine, the state of understanding of the plasma pharmacokinetics, and cellular pharmacodynamics of fludarabine nucleotides. This may be useful in the design of future therapeutic approaches.

The role of c-Jun kinase in the apoptotic response to nucleoside analogue-induced DNA damage.

Activation of the c-Jun NH2-terminal kinase type 1 (JNK1) signaling pathway is often associated with apoptosis. In this report, we elucidated the role of this kinase in the programmed cell death induced by the nucleoside analogue 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A). Treatment of ML-1 cells with 3 or 10 microM F-ara-A specifically killed cells in the S-phase of the population. Incorporation of F-ara-ATP, the nucleoside triphosphate of F-ara-A, into DNA resulted in the activation of JNK1 in a time- and dose-dependent fashion. Activation of JNK1 temporally preceded DNA fragmentation. When incorporation of F-ara-A into DNA was blocked by pretreatment of the cells with aphidicolin to inhibit DNA synthesis, neither JNK1 signaling nor apoptosis was evident. Furthermore, inhibition of JNK1 by treatment of the cells with forskolin or by pretreatment with an antisense oligonucleotide directed against JNK1 mRNA resulted in a decrease in F-ara-A-induced apoptosis. Finally, the JNK1 signaling pathway appeared to be upstream to that of the effector caspases in nucleoside analogue-induced apoptosis. Thus, our data strongly suggest that JNK1 is involved in transduction of F-ara-A-induced distress signals into an apoptotic response.

Incorporation of fludarabine and 1-beta-D-arabinofuranosylcytosine 5'-triphosphates by DNA polymerase alpha: affinity, interaction, and consequences.

Fludarabine and 1-beta-D-arabinofuranosylcytosine (ara-C) are effective nucleoside analogues for the treatment of leukemias when used as single agents or together. Recent trials of the fludarabine and ara-C therapy with or without growth factors suggested an improved clinical response by combining fludarabine and ara-C. The activity of these antimetabolites depends on their phosphorylation to the respective triphosphates, F-ara-ATP and ara-CTP. The principal mechanism through which these triphosphates cause cytotoxicity is incorporation into DNA and inhibition of further DNA synthesis. A model system of DNA primer extension on a defined template sequence was used to quantitate the consequences of incorporation of one or two analogues by human DNA polymerase alpha (pol alpha). The template (31-mer) was designed so that DNA pol alpha incorporated six deoxynucleotides (alternately G and T) on the 17-mer primer, followed by insertion of an A and then a C. The primer was then elongated with G and T to the full-length product. The apparent Kms of DNA pol alpha to incorporate these analogues (0. 053 and 0.077 microM, respectively) were similar to the Km for dCTP (0.037 microM) and dATP (0.044 microM), suggesting that the enzyme recognized these analogues and incorporated them efficiently on the growing DNA primer. The velocity of extension (Vmax) of these primers ranged between 0.53 and 0.77%/min when normal nucleotides were present. Once inserted at the 3'-terminus, F-ara-AMP or ara-CMP were poor substrates for extension. However, in reactions lacking dCTP and dATP and with high concentrations of ara-CTP, ara-CMP was inserted by pol alpha after incorporation of the F-ara-AMP residue. This tandem incorporation of the two analogues resulted in almost complete inhibition (99.3%) of further extension of the primer. In the presence of competing deoxynucleotides, each analogue resulted in a dose-dependent inhibition of DNA synthesis. When present together, inhibition of the primer elongation was more than additive at low concentrations of analogue triphosphates. Based on these results and the intracellular pharmacokinetics of ara-CTP and F-ara-ATP in leukemia blasts, we propose a pharmacodynamic model to explain interactions between these analogues during combination chemotherapy.

Liver targeting of antiviral nucleoside analogues through the asialoglycoprotein receptor.

In order to reduce the extrahepatic side-effects of antiviral nucleoside analogues in the treatment of chronic viral hepatitis, these drugs are conjugated with galactosyl-terminating macromolecules. The conjugates selectively enter hepatocytes after interaction of the carrier galactose residues with the asialoglycoprotein receptor present in large amounts and high affinity only on these cells. Within hepatocytes the conjugates are delivered to lysosomes where enzymes split the bond between the carrier and the drug, allowing the latter to become concentrated in the liver. The validity of this chemotherapeutic strategy has been endorsed by a clinical study. Adenine arabinoside monophosphate (ara-AMP), conjugated with lactosaminated human serum albumin (L-HSA) and administered to hepatitis B virus (HBV)-infected patients for 28 days, exerted an antiviral activity to the same extent as the free drug without producing any clinical side-effects, including the severe neurotoxicity caused by the free drug. Preclinical studies are now underway with conjugates obtained using lactosaminated poly-L-lysine (Lac-poly(Lys)) as the hepatotropic carrier. These new conjugates have some advantages over those prepared with L-HSA: they can be administered by the intramuscular route; they are obtained entirely by chemical synthesis, thus eliminating the problems involved in the use of haemoderivatives; they have a heavy drug load, which permits administration of smaller quantities of conjugate that are more easily digested in lysosomes; and they enable higher quantities of drug to be introduced into hepatocytes. The results of the experiments with two Lac-poly(Lys) conjugates, one with ara-AMP and one with ribavirin, are reported in this review.

Encapsulation, metabolism and release of 2-fluoro-ara-AMP from human erythrocytes.

2-Fluoro-ara-AMP (fludarabine phosphate) is a purine analogue with anti-neoplastic activity in lymphoproliferative malignancies. Fludarabine phosphate activity and toxicity is schedule-dependent; multiple daily administrations (for five days) are more effective than single dose. We have encapsulated fludarabine phosphate in human erythrocytes and found that it is slowly released as fludarabine for more than four days. Encapsulated fludarabine phosphate does not affect erythrocyte metabolism and is rapidly converted by erythrocyte enzymes both to fludarabine with a Km of 0.4 mM and a Vmax of 20 nmol/min per g hemoglobin and to fludarabine diphosphate and triphosphate. The apparent Km for fludarabine monophosphate in the phosphorylation reaction was 0.4 mM and the Vmax 40 nmol/min per g hemoglobin. In the phosphorylation of 2-fluoro-ara-AMP to the di- and triphosphate derivatives, ATP was the phosphate donor with apparent Km of 0.12 and 1.0 mM, respectively. During incubations of 2-fluoro-ara-AMP-loaded erythrocytes at 37 degrees C fludarabine was found in equilibrium between the erythrocyte and the culture medium suggesting that permeation of the erythrocyte membrane is not rate-limiting. Thus, fludarabine phosphate-loaded erythrocytes might be used as a slow-delivery system for fludarabine administration in the treatment of lymphoid malignancies.

Arabinofuranosyl nucleotides are not chain-terminators during initiation of new strands of DNA by DNA polymerase alpha-primase.

Polymerization of NTPs and arabinofuranosyladenosine triphosphate (araATP) during DNA polymerase alpha catalyzed elongation of primase-synthesized primers was examined. After primase synthesizes a primer, pol alpha normally polymerizes multiple dNTPs onto this primer. In the absence of a required dNTP, however, primers were still elongated by up to 35 nucleotides via polymerization of the corresponding NTP in place of the missing dNTP. During the elongation of exogenously added primer/templates, however, NTPs were not readily polymerized. AraATP was readily incorporated into products during elongation of primase-synthesized primers. Importantly, polymerization of araATP did not result in chain termination; rather, the next correct nucleotide was added such that araATP was simply an alternate substrate. In contrast, polymerization of araATP during elongation of exogenously added primer/templates resulted in strong chain termination. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer/templates with respect to interactions with dNTP analogs. Furthermore, these data provide a rationale for how araNMPs are efficiently incorporated into internucleotide linkages of DNA in whole cells and suggest that the initiation of new strands of DNA by pol alpha-primase may be a unique target for inhibiting replication.

Primer RNA chain termination induced by 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate. A mechanism of DNA synthesis inhibition.

The studies described herein were aimed at defining the mechanism by which 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (FaraATP), the active intracellular metabolite of fludarabine phosphate, inhibits the synthesis of primer RNA and RNA-primed DNA by the polymerase alpha-primase complex. Incubation of the purified DNA polymerase alpha-primase complex with a poly(dT) template, 500 microM ATP, and increasing concentrations of FaraATP from 2.5 to 50 microM resulted in the progressive accumulation of smaller oligoribonucleotides (2-6 nucleotides) at the expense of the full-length products of DNA primase (7-10 nucleotides). Comparison of the kcat/KM values for incorporation of FaraATP and ATP into oligoribonucleotides revealed that DNA primase incorporated FaraATP 30-fold more efficiently than ATP. FaraAMP was present exclusively at the 3'-termini of the growing primer RNA chains, which prevented further elongation of the primers by DNA primase (primer RNA chain termination). At all FaraATP concentrations tested, inhibition of RNA-primed DNA synthesis was accompanied by primer chain termination. In contrast, DNA polymerase alpha added FaraATP onto full-length primer RNAs about 8-fold less efficiently than dATP, and the incorporation of FaraAMP at the 3'-termini of the primers did not prevent further elongation of these primers by DNA polymerase alpha. These results indicate that primer RNA chain termination is the major effect responsible for the inhibition of RNA-primed DNA synthesis by fludarabine phosphate.

1-beta-D-arabinofuranosylcytosine-diphosphate-choline is formed by the reversal of cholinephosphotransferase and not via cytidylyltransferase.

In an effort to identify the pathway leading to the formation of 1-beta-D-arabinofuranosylcytosine-diphosphate (ara-CDP)-choline from 1-beta-D-arabinofuranosylcytosine (ara-C) treatment of cultured cells, as well as of cells obtained from leukemia patients, we probed the enzymatic steps involved in the CDP-choline pathway for phosphatidylcholine biosynthesis. Ara-C-triphosphate was not a substrate for CTP:phosphocholine cytidylyltransferase activity under the conditions employed, whereas CTP and dCTP were utilized to form CDP-choline and dCDP-choline, respectively. When presented together, ara-C-triphosphate and CTP inhibited the enzymatic conversion of CTP to CDP-choline in the presence of phosphocholine, with a Ki of 6 mM. Since CTP:phosphocholine cytidylyltransferase did not appear to be responsible for the increased levels of ara-CDP-choline, we next studied the other enzyme in the pathway for phosphatidylcholine synthesis that could form ara-CDP-choline, CDP-choline:1,2-diacylglycerol cholinephosphotransferase. CDP-choline:1,2-diacylglycerol cholinephosphotransferase activity present in microsomes isolated from L5178Y murine leukemia cells exhibited a reversal of its normal catalytic activity, using CMP and 1-beta-D-arabinofuranosylcytosine-monophosphate (ara-CMP) along with phosphatidylcholine to produce either CDP-choline or ara-CDP-choline, plus diradylglycerol. The Vmax and Km values for CMP were 0.78 +/- 0.04 nmol/min/mg and 340 +/- 20 microM, respectively, whereas the Vmax and Km for ara-CMP were 0.22 +/- 0.06 nmol/min/mg and 1410 +/- 540 microM, respectively. A Ki value of 3 mM was obtained for ara-CMP under the cell-free assay conditions used. These results indicate that ara-CDP-choline most likely arises from a reversal of the CDP-choline:1,2-diacylglycerol cholinephosphotransferase utilizing ara-CMP, rather than from the catalysis of ara-C-triphosphate plus phosphocholine to ara-CDP-choline by CTP:phosphocholine cytidylyltransferase. It is speculated that this mechanism may explain, in part, the rapid cellular lysis observed with high dose ara-C therapy.

Selective anabolism of 6-methoxypurine arabinoside in varicella-zoster virus-infected cells.

6-Methoxypurine arabinoside (ara-M) is a highly selective inhibitor of varicella-zoster virus (VZV). It belongs to a class of purine arabinosides whose anti-VZV activity in vitro correlates with substrate utilization by the VZV-encoded thymidine kinase (TK) (D. R. Averett, G. W. Koszalka, J. A. Fyfe, G. B. Roberts, D. J. M. Purifoy, and T. A. Krenitsky, Antimicrob Agents Chemother. 35:851-857, 1991). In this study, the mechanism of action of ara-M was explored. VZV-infected human fibroblasts selectively accumulated ara-M and its phosphorylated metabolites, whereas in uninfected fibroblasts or in those infected with a TK-deficient strain of VZV, there was virtually no cellular uptake of ara-M. The major intracellular metabolite of ara-M in VZV-infected cells was identified as the triphosphate of adenine arabinoside (ara-ATP). Appreciable levels of ara-ADP, ara-AMP, and ara-MMP were also detected. However, di- or triphosphorylated forms of ara-M were not detected. Moreover, in VZV-infected cells, the concentrations of ara-ATP which accumulated in the presence of ara-M were up to eightfold higher than those generated with ara-A itself. In contrast, in uninfected cells, the levels of ara-ATP which accumulated in the presence of ara-M were barely detectable. Clearly, Ara-M activation was dependent on the activity of the virus-encoded TK, while ara-A anabolism resulted primarily from the activity of host cell enzymes. Therefore, ara-M selectively generates the DNA polymerase inhibitor ara-ATP in the VZV-infected cell.

9-Beta-D-arabinofuranosyladenine 5'-monophosphate (araAMP) is converted directly to its antivirally active 5'-triphosphate form by 5-phosphoribosyl-1-pyrophosphate (PRPP) synthetase.

The antiherpetic agent 9-beta-D-arabinofuranosyladenine (araA) needs to be phosphorylated to its 5'-triphosphate to be effective as an inhibitor of herpes simplex virus replication. Adenosine kinase and deoxycytidine kinase are assumed to convert araA to its 5'-monophosphate. We now found that araAMP is converted to its 5'-triphosphate through a direct pyrophosphate transfer from 5-phosphoribosyl-1-pyrophosphate (PRPP) by PRPP synthetase. The efficiency of phosphorylation of araAMP to araATP is about 5% of that of AMP, as estimated from their Vmax/Km ratios for PRPP synthetase. AraATP is converted to araAMP by PRPP synthetase at a 4-fold higher Km but similar Vmax as ATP.