R2 Subunit Of Ribonucleotide Reductase Research Articles

Role of H-1 and H-2 Subunits of Soybean Seed Ferritin in Oxidative Deposition of Iron in Protein

Naturally occurring phytoferritin is a heteropolymer consisting of two different H-type subunits, H-1 and H-2. Prior to this study, however, the function of the two subunits in oxidative deposition of iron in ferritin was unknown. The data show that, upon aerobic addition of 48-200 Fe(2+)/shell to apoferritin, iron oxidation occurs only at the diiron ferroxidase center of recombinant H1 (rH-1). In addition to the diiron ferroxidase mechanism, such oxidation is catalyzed by the extension peptide (a specific domain found in phytoferritin) of rH-2, because the H-1 subunit is able to remove Fe(3+) from the center to the inner cavity better than the H-2 subunit. These findings support the idea that the H-1 and H-2 subunits play different roles in iron mineralization in protein. Interestingly, at medium iron loading (200 irons/shell), wild-type (WT) soybean seed ferritin (SSF) exhibits a stronger activity in catalyzing iron oxidation (1.10 ± 0.13 μm iron/subunit/s) than rH-1 (0.59 ± 0.07 μm iron/subunit/s) and rH-2 (0.48 ± 0.04 μm iron/subunit/s), demonstrating that a synergistic interaction exists between the H-1 and H-2 subunits in SSF during iron mineralization. Such synergistic interaction becomes considerably stronger at high iron loading (400 irons/shell) as indicated by the observation that the iron oxidation activity of WT SSF is ∼10 times larger than those of rH-1 and rH-2. This helps elucidate the widespread occurrence of heteropolymeric ferritins in plants.

Replication of the hepatitis B virus requires a calcium-dependent HBx-induced G1 phase arrest of hepatocytes

Chronic HBV infections cause hepatocellular carcinoma (HCC). Activities of the HBV HBx protein regulate HBV replication and may contribute to the development of HCC. We previously reported that HBx causes primary rat hepatocytes to exit G0 but stall in G1 phase of the cell cycle; entry into G1 stimulated HBV replication. We now report that the activity of the mitochondria permeability transition pore is required for HBx regulation of cell cycle proteins and HBV replication in primary rat hepatocytes, that progression from G0 to G1 stimulates HBV polymerase activity, and that HBV replication is facilitated by the HBx-induced G1 arrest. HBx stimulation of HBV replication was linked to elevation of the R2 subunit of ribonucleotide reductase. Our studies suggest that HBx uses mitochondrial-dependent calcium signaling to cause hepatocytes to exit G0 but stall in G1 and that this HBx activity alters the cellular environment and stimulates HBV replication.

A phase II study of the antisense oligonucleotide GTI-2040 plus docetaxel and prednisone as first-line treatment in castration-resistant prostate cancer

GTI-2040 is a novel antisense oligonucleotide to the R2 subunit of ribonucleotide reductase. This phase II trial was conducted to determine the efficacy and tolerability of GTI-2040 when combined with docetaxel and prednisone for the treatment of patients with castration-resistant prostate cancer (CRPC). Chemo-naïve CRPC patients with adequate performance status and organ function were treated with docetaxel 75 mg/m(2) IV on day 1 plus GTI-2040 5 mg/kg/day by continuous intravenous infusion day 1-14 on a 21 day cycle, with prednisone 5 mg orally twice daily. The primary endpoint was PSA response rate. Pharmacokinetic studies of GTI-2040 and pharmacodynamic studies on peripheral blood mononuclear cells (PBMC) were also performed. Twenty-two patients in total (19 from this study and 3 from a prior phase I/II study at this institution) were treated at the recommended phase II dose. A confirmed PSA response was seen in 9/22 patients (41%). Of 16 patients with measurable disease, there was 1 partial response (PR) and 12 stable disease (SD) lasting 3.6 months (median), as best response. The most common toxicities were anemia, fatigue, lymphopenia, leucopenia and neutropenia. Grade 3+ toxicities included neutropenia, lymphopenia, leucopenia, fatigue, febrile neutropenia and hypophosphatemia. The PSA response rate of GTI-2040 in combination with docetaxel and prednisone just met the minimum phase II criteria for further enrollment. However, after evaluation of all the clinical data, further study of this dose and schedule of GTI-2040 in CRPC was not recommended.

Cancer Treatment with Gemcitabine Does Not Increase Fetal Hemoglobin Levels.

Abstract 5112 BackgroundPharmacological induction of fetal hemoglobin (Hb F) can benefit patients with beta-hemoglobinopathies. Hydroxyurea (HU) is the drug most commonly used to induce Hb F. HU is converted to a free radical scavenger in vivo and inhibits the R2 subunit of ribonucleotide reductase. When used in the treatment of myeloproliferative syndromes, HU has been described to increase Hb F in people without hemoglobinopathies (Alter et al, Blood 66(2) 373-5). Gemcitabine is a nucleoside analog chemotherapeutic that inhibits the large R1 subunit of ribonucleotide reductase. We hypothesized that like HU, gemcitabine treatment would cause Hb F percentage to rise. Though oncology patients are a distinct population, gemcitabine induction of Hb F during the treatment of cancer would suggest a potential role for gemcitabine in the management of beta-hemoglobinopathies such as sickle cell disease and beta-thalassemia. MethodsParticipants for this prospective observational cohort study were recruited from the Moores UCSD Cancer Center upon documentation of a cancer diagnosis and oncologist-initiated plans for treatment with single agent gemcitabine. Enrolled subjects were gemcitabine naïve and had not received any chemotherapy for the preceding 4 weeks. Serum Hb F was quantified by electrophoresis prior to each of the first 4 gemcitabine treatments. All other laboratory evaluation was standard-of-care. The primary objective was to assess changes in Hb F among cancer patients in response to their initial 3 treatments with gemcitabine. Enrollment was halted early given the absence of a substantial treatment effect among the first 6 subjects. ResultsSix subjects were eligible. Three were men. Five were Caucasian and one was an Asian/Pacific Islander. All subjects received gemcitabine monotherapy for the treatment of adenocarcinoma of the pancreas or breast at a weekly dose of 1000 mg/m2. Baseline Hb F range was 0 – 1.0%. Four of the six subjects (three women, one man; all Caucasian) experienced no change in Hb F from baseline to the final measurement. Two subjects experienced a 0.4% increase in Hb F from baseline to the final measurement 4 weeks later. Final Hb F range was 0 – 1.4%. ConclusionsWe found that gemcitabine monotherapy for the treatment of cancer did not induce Hb F in this small study cohort. Gemcitabine may lack this ability. Alternative explanations for the absence of clinically meaningful Hb F induction include the small population studied, differences between oncology patients and the general population, short duration of treatment, and intermittent dosing of gemcitabine. DisclosuresNo relevant conflicts of interest to declare.

Targeted Delivery of Antisense Oligodeoxynucleotide by Transferrin Conjugated pH-Sensitive Lipopolyplex Nanoparticles: A Novel Oligonucleotide-Based Therapeutic Strategy in Acute Myeloid Leukemia

Therapeutic use of oligodeoxynucleotides (ODNs) that hybridize to and downregulate target mRNAs encoding proteins that contribute to malignant transformation has a sound rationale, but has had an overall limited clinical success in cancer due to insufficient intracellular delivery. Here we report a development of formulations capable of promoting targeted delivery and enhanced pharmacologic activity of ODNs in acute myeloid leukemia (AML) cell lines and patient primary cells. In this study, transferrin (Tf) conjugated pH-sensitive lipopolyplex nanoparticles (LPs) were prepared to deliver GTI-2040, an antisense ODN against the R2 subunit of ribonucleotide reductase that has been shown to contribute to chemoresistance in AML. LPs had an average particle size around 110 nm and a moderately positive zeta potential at approximately 10 mV. The ODN encapsulation efficiency of LPs was >90%. These nanoparticles could release ODNs at acidic endosomal pH and facilitate the cytoplasmic delivery of ODNs after endocytosis. In addition, Tf-mediated targeted delivery of GTI-2040 was achieved. R2 downregulation at both mRNA and protein levels was improved by 8-fold in Kasumi-1 cells and 2- to 20-fold in AML patient primary cells treated with GTI-2040-Tf-LPs, compared to free GTI-2040 treatment. Moreover, Tf-LPs were more effective than nontargeted LPs, with 10 to 100% improvement at various concentrations in Kasumi-1 cells and an average of 45% improvement at 3 microM concentration in AML patient primary cells. Treatment with 1 microM GTI-2040-Tf-LPs sensitized AML cells to the chemotherapy agent cytarabine, by decreasing its IC(50) value from 47.69 nM to 9.05 nM. This study suggests that the combination of pH sensitive LP formulation and Tf mediated targeting is a promising strategy for antisense ODN delivery in leukemia therapy.

A Phase I/II Study of GTI-2040 Plus Docetaxel as Second-Line Treatment in Advanced Non-small Cell Lung Cancer: A Study of the PMH Phase II Consortium

GTI-2040, an antisense oligonucleotide, targets the ribonucleotide reductase R2 subunit, critical for DNA synthesis. This study determined the recommended phase II dose (RP2D) of docetaxel plus GTI-2040, toxicity, and response rate in advanced non-small cell lung cancer (NSCLC). Advanced solid tumor patients, preferably with platinum-treated NSCLC, performance status 0 to 2, no symptomatic central nervous system metastases, adequate organ and bone marrow function, and >or=1 prior chemotherapy regimen were treated with escalating doses of GTI-2040 given by 14-day continuous intravenous infusion (CVI) plus docetaxel every 21 days. Twenty-nine patients were treated, (24 NSCLC, 3 hormone-refractory prostate cancer, 1 head and neck, and 1 small cell lung cancer). GTI-2040 5 mg/kg as CVI for 14 days plus docetaxel 75 mg/m(2) intravenously every 21days was determined as the RP2D. Dose-limiting toxicity was not seen. Two patients at RP2D developed grade 4/5 febrile neutropenia. One prostate specific antigen response was seen in phase I, but no objective tumor responses in the NSCLC patients. Median time to progression was 3.4 months, 3.2 months in the NSCLC patients treated at RP2D. Activity of the combination at RP2D, GTI-2040 5 mg/kg/d x 14 days by CVI plus docetaxel 75 mg/m(2) does not seem superior to docetaxel alone in previously treated NSCLC.

Antisense oligonucleotides targeting the large subunit (R1) of human ribonucleotide reductase synergistically increase the cytotoxicity of doxorubicin and paclitaxel to MCF-7 breast cancer cells

e14626 Ribonucleotide reductase (RR) is an essential enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides for use in DNA synthesis. Human RR consists of two subunits, a large subunit (R1) and small subunit (R2). RR provides an attractive target for anticancer therapy. In the present study, we synthesized phosphorothioated antisenses oligonucleotides (RR1AS1, RR1AS2, RR1AS3, RR1AS4 and RR1AS5) that target the R1 subunit of RR. We treated the human breast cancer cell line MCF-7 with antisenses, and doxorubicin or paclitaxel for 72 hours. Cell proliferation was measured by 3[H]- thymidine incorporation. The effects of the drug combinations were analyzed with Biosoft Calcusyn software. The levels of RR mRNA were measured by RT-PCR. We found two antisenses, RR1AS2 and RR1AS4, inhibited the proliferation of MCF-7 cells in a dose-dependent pattern (IC50s: 5.32+1.64μm and 2.57+2.72μm, respectively, 72 hours incubation). Also, RR1AS2 and RR1AS4 significantly suppressed the expression of RR1 mRNA. When MCF-7 cells were incubated in media with a mixture of antisense and doxorubicin or paclitaxel, both RR1AS2 and RR1AS4 synergistically increased the cytotoxicity of doxorubicin and paclitaxel. Calcusyn analysis showed that averaged combination index (CI) were 0.59+0.04, 0.66+0.22, 0.83+0.16 and 0.88+0.03, when MCF-7 cells were treated with the mixtures of RR1AS2 + doxorubicin, RR1AS2 + paclitaxel, RR1AS4 + doxorubicin and RR1AS4 + paclitaxel, respectively (CI<1 indicates synergism). These results suggest that the combination of RR antisenses and chemotherapeutic agents, such as doxorubicin or paclitaxel, may decrease both the dosages and side effects of both antisense oligonucleotides and chemotherapeutic agents (doxorubicin and paclitaxel) in cancer therapy. No significant financial relationships to disclose.

Molecular Mechanisms of Thioredoxin and Glutaredoxin as Hydrogen Donors for Mammalian S Phase Ribonucleotide Reductase

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in deoxyribonucleotide synthesis essential for DNA replication and repair. RNR in S phase mammalian cells comprises a weak cytosolic complex of the catalytic R1 protein containing redox active cysteine residues and the R2 protein harboring the tyrosine free radical. Each enzyme turnover generates a disulfide in the active site of R1, which is reduced by C-terminally located shuttle dithiols leaving a disulfide to be reduced. Electrons for reduction come ultimately from NADPH via thioredoxin reductase and thioredoxin (Trx) or glutathione reductase, glutathione, and glutaredoxin (Grx), but the mechanism has not been clarified for mammalian RNR. Using recombinant mouse RNR, we found that Trx1 and Grx1 had similar catalytic efficiency (k(cat)/K(m)). With 4 mm GSH, Grx1 showed a higher affinity (apparent K(m) value, 0.18 microm) compared with Trx1 which displayed a higher apparent k(cat), suggesting its major role in S phase DNA replication. Surprisingly, Grx activity was strongly dependent on GSH concentrations (apparent K(m) value, 3 mm) and a Grx2 C40S mutant was active despite only one cysteine residue in the active site. This demonstrates a GSH-mixed disulfide mechanism for glutaredoxin catalysis in contrast to the dithiol mechanism for thioredoxin. This may be an advantage with the low levels of RNR for DNA repair or in tumor cells with high RNR and no or low Trx expression. Our results demonstrate mechanistic differences between the mammalian and canonical Escherichia coli RNR enzymes, which may offer an explanation for the nonconserved shuttle dithiol sequences in the C terminus of the R1.

Iron-independent Phosphorylation of Iron Regulatory Protein 2 Regulates Ferritin during the Cell Cycle

Iron regulatory protein 2 (IRP2) is a key iron sensor that post-transcriptionally regulates mammalian iron homeostasis by binding to iron-responsive elements (IREs) in mRNAs that encode proteins involved in iron metabolism (e.g. ferritin and transferrin receptor 1). During iron deficiency, IRP2 binds IREs to regulate mRNA translation or stability, whereas during iron sufficiency IRP2 is degraded by the proteasome. Here, we identify an iron-independent IRP2 phosphorylation site that is regulated by the cell cycle. IRP2 Ser-157 is phosphorylated by Cdk1/cyclin B1 during G(2)/M and is dephosphorylated during mitotic exit by the phosphatase Cdc14A. Ser-157 phosphorylation during G(2)/M reduces IRP2 RNA-binding activity and increases ferritin synthesis, whereas Ser-157 dephosphorylation during mitotic exit restores IRP2 RNA-binding activity and represses ferritin synthesis. These data show that reversible phosphorylation of IRP2 during G(2)/M has a role in modulating the iron-independent expression of ferritin and other IRE-containing mRNAs during the cell cycle.

Targeting the Human Double Minute (HDM)-2 p53 Ubiquitin Ligase as a Strategy Against Non-Hodgkin Lymphoma.

Background: The ubiquitin-proteasome pathway has been validated as a target for non-Hodgkin lymphoma (NHL) with the recent approval of bortezomib for mantle cell lymphoma (MCL). In addition to anti-tumor activity, however, proteasome inhibitors have pleiotropic effects, including activation of an anti-apoptotic heat shock protein response, and their use clinically is complicated by toxicities such as peripheral neuropathy. By targeting E3 ubiquitin ligases, which are involved in ubiquitination of only a small subset of cellular proteins, it may be possible to achieve more specific anti-tumor effects with a better therapeutic index. One such attractive target is HDM-2, which is responsible for ubiquitination of the p53 tumor suppressor.Methods: To evaluate the therapeutic potential of agents targeting HDM-2, we studied the impact of the small molecule MI-63, an inhibitor of the HDM-2-p53 interaction, in both p53 wild-type and -mutant cell line models.Results: Treatment of wild-type p53 MCL, NHL, and acute lymphocytic leukemia (ALL) cell lines with MI-63 induced a dose- and time-dependent inhibition of proliferation, with an IC50 in the 1.0–5.0 μM range. This was associated with G1/S cell cycle arrest, and apoptosis mediated by caspases-3, 8 and 9. MI-63 induced accumulation and phosphorylation of p53 at serine 15 and 37, and also enhanced HDM-2 levels. Multiple p53 target genes were induced, including p21Cip1 and p53-upregulated modulator of apoptosis (PUMA), resulting in cleavage of poly-ADP-ribose-polymerase (PARP). MI-63 also decreased the levels of the ribonucleotide reductase subunit R2, and caused a corresponding increase in the R2p53 subunit. MI-63 also decreased the levels of E2F. Cell lines expressing certain p53 mutants were sensitive to the effects of MI-63, resulting in apoptosis. Cells without p53 expression were less sensitive to MI-63, but at higher drug concentrations proliferation was still inhibited, indicating a possible impact on HDM-2-mediated but p53-independent cell death pathways. Primary human umbilical cord vein endothelial cell growth was also inhibited and cells failed to recover after extended exposure to MI-63, whereas primary PBMC's were unaffected by MI-63. Combinations of MI-63 with the molecularly targeted chemotherapy agents bortezomib and rapamycin were synergistic, with mean CI values of 0.88 and 0.6 respectively. The conventional chemotherapy agents doxorubicin and cisplatin were less effective at inducing synergism, with mean CI values of 1.06 and 0.9 respectively. Pretreatment of cells with MI-63 followed by chemotherapy was antagonistic with all agents used, while treatment with a chemotherapeutic first followed by MI-63 was additive to synergistic, indicating a sequence-dependent interaction.Conclusions: Inhibition of the HDM-2-p53 interaction is a promising approach both by itself, and in combination with currently used chemotherapeutics, against lymphoid malignancies, providing a rationale for translation of such agents into the clinic.

Structure and Action of the N-oxygenase AurF from Streptomyces thioluteus

Nitro groups are found in a number of bioactive compounds. Most of them arise by a stepwise mono-oxygenation of amino groups. One of the involved enzymes is AurF participating in the biosynthesis of aureothin. Its structure was established at 2.1 Å resolution showing a homodimer with a binuclear manganese cluster. The enzyme preparation, which yielded the analyzed crystals, showed activity using in vitro and in vivo assays. Chain fold and cluster are homologous with ribonucleotide reductase subunit R2 and related enzymes. The two manganese ions and an iron content of about 15% were established by anomalous X-ray diffraction. A comparison of the cluster with more common di-iron clusters suggested an additional histidine in the coordination sphere to cause the preference for manganese over iron. There is no oxo-bridge. The substrate p-amino-benzoate was modeled into the active center. The model is supported by mutant activity measurements. It shows the geometry of the reaction and explains the established substrate spectrum.

RNA interference targeting the R2 subunit of ribonucleotide reductase inhibits growth of tumor cells in vitro and in vivo

RNA interference, a posttranscriptional gene-silencing mechanism, has received considerable attention for its potential as a new therapeutic strategy to treat human diseases and conditions including cancer. Various studies have supported a role for the R2 subunit of ribonucleotide reductase in cancer progression and metastasis. Short interfering siRNA 1284 was designed to target R2. In vitro studies, in which three different human tumor cell lines (A498, HT-29 and A2058) were transfected with short interfering siRNA 1284, demonstrate sequence-specific down-regulation of R2, which coincides with a decrease in cell proliferation, and cell cycle inhibition. In vivo studies with xenograft mouse models, generated from the same tumor cell lines, indicate that treatment with short interfering siRNA 1284 leads to inhibition of tumor growth and this effect was found to be dose dependent. Taken together, these results suggest that short interfering siRNA 1284, targeting R2, has great potential to serve as a therapeutic agent towards the treatment of human cancers.

(μ-1,2-Peroxo)diiron(III/III) Complex as a Precursor to the Diiron(III/IV) Intermediate X in the Assembly of the Iron-Radical Cofactor of Ribonucleotide Reductase from Mouse

Stopped-flow absorption and freeze-quench electron paramagnetic resonance (EPR) and Mössbauer spectroscopies have been used to obtain evidence for the intermediacy of a (mu-1,2-peroxo)diiron(III/III) complex on the pathway to the tyrosyl radical and (mu-oxo)diiron(III/III) cluster during assembly of the essential cofactor in the R2 subunit of ribonucleotide reductase from mouse. The complex accumulates to approximately 0.4 equiv in the first few milliseconds of the reaction and decays concomitantly with accumulation of the previously detected diiron(III/IV) cluster, X, which generates the tyrosyl radical and product (mu-oxo)diiron(III/III) cluster. Kinetic complexities in the reaction suggest the existence of an anti-cooperative interaction of the monomers of the R2 homodimer in Fe(II) binding and perhaps O2 activation. The detection of the (mu-1,2-peroxo)diiron(III/III) complex, which has spectroscopic properties similar to those of complexes previously characterized in the reactions of soluble methane monooxygenase, stearoyl acyl carrier protein Delta9 desaturase, and variants of Escherichia coli R2 with the iron ligand substitution, D84E, provides support for the hypothesis that the reactions of the diiron-carboxylate oxidases and oxygenases commence with the formation of this common intermediate.

Visualization of ribonucleotide reductase catalytic oxidation establishes thioredoxins as its major reductants in yeast

Thioredoxins and/or glutaredoxins assist ribonucleotide reductase, and other such enzymes that require disulfide bond reduction during their catalytic cycle. In Saccharomyces cerevisiae, the presence of either pathway is essential but which of these pathways operates in ribonucleotide reductase reduction and how this function contributes to the pathways’ essential nature have not been definitively established. We have identified two in vivo redox forms of the S. cerevisiae ribonucleotide reductase R1 subunit, which correspond to catalytically reduced or oxidized enzymes. Cells lacking thioredoxins, which exhibit an elongated S phase, accumulate R1 in its oxidized form and also contain significantly decreased deoxyribonucleotide levels during the S phase. Overexpressing R1 in these cells increases both the amount of the R1 reduced form and the concentrations of deoxyribonucleotides and accelerates DNA replication. These results establish thioredoxins as the major RNR reducing system in yeast and indicate that impaired RNR reduction accounts for the S phase defects of thioredoxin-deficient cells.

Theory of chemical bonds in metalloenzymes. IX. Theoretical study on the active site of the ribonucleotide reductase and the related species

AbstractThe radical harvesting state of a ribonucleotide reductase R2 subunit (RNR‐R2) is theoretically investigated at the broken‐symmetry DFT level. Recently, a high‐resolution X‐ray crystallography revealed a precise location of the active site structure of RNR‐R2 (Class 1b) from C. ammoniagenes, and found a linking water near the tyrosine (Tyr) radical site. In this study, the magnetic interaction between the Tyr radical and the diiron core JTyrFe is investigated for the first time. Used theoretical model for the active site is composed of diiron core, the Tyr residue, and surrounding side‐chain groups. After the geometrical structure optimization of the core structures, the magnetic interaction between the iron centers was calculated to be JFeFe = −77.82 cm−1, which was comparable to the experimental result of JFeFe = −84 cm−1. The natural orbital analysis clearly showed that the magnetic interaction between the two iron centers mainly interact through the p orbital of the bridging μ‐oxo. It was also calculated that the JTyrFe interaction is negligibly weak, and the Tyr radical is almost in an isolated or a free radical state. These results suggest that the linking water may be used for (1) a confinement of the reactive Tyr radical in the hydrogen bonds or (2) a hydrogen‐transfer coupled with electron transfer at the radical formation step of the Tyr. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007

Metabolism of GTI-2040, a phosphorothioate oligonucleotide antisense, using ion-pair reversed phase high performance liquid chromatography (HPLC) coupled with electrospray ion-trap mass spectrometry

GTI-2040 is a 20-mer phosphorothioate oligonucleotide, which is complementary to the messenger ribonucleic acid (mRNA) of the R2 subunit of ribonucleotide reductase. This study characterized both the in vivo and in vitro metabolism of GTI-2040. A highly specific ion-pair reversed-phase electrospray ionization (IP-RP-ESI) liquid chromatography-mass spectrometry (LC-MS) method was used for the identification of GTI-2040 and metabolites from a variety of biological samples including exonuclease enzyme solutions, plasma, urine, mouse liver/kidney homogenates, and human liver microsomes. Progressively chain-shortened metabolites truncated from the 3' terminal of GTI-2040 were detected in all of the evaluated biological samples. GTI-2040 was found to be a good substrate for 3' but not 5' exonuclease. While the pattern of n-1 chain-shortened 3'-exonucleolytic degradation was similar in the mouse liver and kidney homogenates, the latter was found to contain a larger number of shortenmers, the kidneys appeared to possess higher enzymatic reactivity toward GTI-2040. Thus, metabolism of GTI-2040 was found to occur in a variety of biological samples, mainly mediated by the 3' exonuclease.

Dose escalation phase of a phase I/II study of GTI-2501, an antisense to the R1 subunit of ribonucleotide reductase (RNR) and docetaxel in patients with metastatic hormone-refractory prostate cancer

2078 Background: GTI-2501 (GTI) is a 20-mer oligonucleotide that is complementary to the R1 subunit of the RNR mRNA. The R1 protein is overexpressed in multiple tumour cell lines. GTI displays anti-tumour activity against prostate cancer xenografts in mice as a single agent and in combination with mitoxantrone. GTI also adds to the anti-tumour efficacy of taxanes in breast cancer xenografts in mice. A Phase 1 study of a 14 day continuous infusion of GTI in patients with solid tumours showed no dose limiting toxicities at doses up to 210.9 mg/m2/day. The objective of this dose escalation phase of a phase I/II study was to define a safe phase II dose of GTI in combination with docetaxel (D) in men with metastatic hormone refractory prostate cancer (HRPC). Methods: Men with metastatic HRPC were enrolled at 3 centres in Canada. GTI was given as a 14d continuous IV infusion every 21d with D IV infusion started 2 hrs prior to the end of the GTI infusion. Planned dose escalation cohorts are summarized in table . Results: 13 men were enrolled to the 3 cohorts. All patients are evaluable for toxicity. There was one possible DLT - an episode of grade 4 neutropenia reported at cycle 2 day 1 in the highest dose cohort - but the duration of neutropenia could not be confirmed. 3 additional patients were accrued to that cohort with no DLTs. The most common gr 3/4 toxicity was attributable to D (10 pts with Gr 3/4 neutropenia). The observed incidence of Gr 3/4 neutropenia was expected since patients had weekly CBCs. Only 1 patient had febrile neutropenia. 11 pts had fatigue (4 Gr 3) related to D and /or GTI. Other GTI attributable adverse events were Gr 1/2 including transient rises in transaminases and PTT. The pharmacokinetic data which is summarized in the table will be presented in full at the meeting. Conclusions: GTI can be given safely at its highest planned dose with standard doses of D. A Phase II evaluation of the GTI + D combination is planned for men with HRPC. [Table: see text] [Table: see text]

Comparative immunogenicity of M. hyopneumoniae NrdF encoded in different expression systems delivered orally via attenuated S. typhimurium aroA in mice

The Mycoplasma hyopneumoniae ribonucleotide reductase R2 subunit (NrdF) gene fragment was cloned into eukaryotic and prokaryotic expression vectors and its immunogenicity evaluated in mice immunized orally with attenuated Salmonella typhimurium aroA CS332 harboring either of the recombinant expression plasmids. We found that NrdF is highly conserved among M. hyopneumoniae strains. The immunogenicity of NrdF was examined by analyzing antibody responses in sera and lung washes, and the cell-mediated immune (CMI) response was assessed by determining the INF-γ level produced by splenocytes upon in vitro stimulation with NrdF antigen. S. typhimurium expressing NrdF encoded by the prokaryotic expression plasmid (pTrcNrdF) failed to elicit an NrdF-specific serum or secretory antibody response, and IFN-γ was not produced. Similarly, S. typhimurium carrying the eukaryotic recombinant plasmid encoding NrdF (pcNrdF) did not induce a serum or secretory antibody response, but did elicit significant NrdF-specific IFN-γ production, indicating induction of a CMI response. However, analysis of immune responses against the live vector S. typhimurium aroA CS332 showed a serum IgG response but no mucosal IgA response in spite of its efficient invasiveness in vitro. In the present study we show that the DNA vaccine encoding the M. hyopneumoniae antigen delivered orally via a live attenuated S. typhimurium aroA can induce a cell-mediated immune response. We also indicate that different live bacterial vaccine carriers may have an influence on the type of the immune response induced.

Mono-, Di-, Tri-, and Tetra-Substituted Fluorotyrosines: New Probes for Enzymes That Use Tyrosyl Radicals in Catalysis

A set of N-acylated, carboxyamide fluorotyrosine (F(n)()Y) analogues [Ac-3-FY-NH(2), Ac-3,5-F(2)Y-NH(2), Ac-2,3-F(2)Y-NH(2), Ac-2,3,5-F(3)Y-NH(2), Ac-2,3,6-F(3)Y-NH(2) and Ac-2,3,5,6-F(4)Y-NH(2)] have been synthesized from their corresponding amino acids to interrogate the detailed reaction mechanism(s) accessible to F(n)()Y*s in small molecules and in proteins. These Ac-F(n)()Y-NH(2) derivatives span a pK(a) range from 5.6 to 8.4 and a reduction potential range of 320 mV in the pH region accessible to most proteins (6-9). DFT electronic-structure calculations capture the observed trends for both the reduction potentials and pK(a)s. Dipeptides of the methyl ester of 4-benzoyl-l-phenylalanyl-F(n)()Ys at pH 4 were examined with a nanosecond laser pulse and transient absorption spectroscopy to provide absorption spectra of F(n)()Y*s. The EPR spectrum of each F(n)()Y* has also been determined by UV photolysis of solutions at pH 11 and 77 K. The ability to vary systematically both pK(a) and radical reduction potential, together with the facility to monitor radical formation with distinct absorption and EPR features, establishes that F(n)()Ys will be useful in the study of biological charge-transport mechanisms involving tyrosine. To demonstrate the efficacy of the fluorotyrosine method in unraveling charge transport in complex biological systems, we report the global substitution of tyrosine by 3-fluorotyrosine (3-FY) in the R2 subunit of ribonucleotide reductase (RNR) and present the EPR spectrum along with its simulation of 3-FY122*. In the companion paper, we demonstrate the utility of F(n)()Ys in providing insight into the mechanism of tyrosine oxidation in biological systems by incorporating them site-specifically at position 356 in the R2 subunit of Escherichia coli RNR.

Determination of GTI-2040, a novel antisense oligonucleotide, in human plasma by using HPLC combined with solid phase and liquid–liquid extractions

GTI-2040 is a 20-mer phosphorothioate oligonucleotide complementary to the mRNA of the R2 subunit of ribonucleotide reductase (RNR). It is under clinical development as an anti-cancer agent. A reverse phase high-performance liquid chromatograph (HPLC) method was established for the quantitative analysis of GTI-2040 in human plasma. Plasma samples were prepared with an initial solid-phase extraction (SPE) followed by a liquid-liquid extraction step. HPLC analysis was performed with a gradient system on a Waters XTerraMS C18 column. The mobile phase consisted of acetonitrile-tetrabutyl ammonium hydrogen sulfate (TBAS) buffer (pH 9.0, 20 mM) at a flow rate of 1.0 ml/min, and the detector was set at a wavelength of 260 nm. A cationic pairing reagent, tetrabutyl ammonium hydrogen sulfate was added during plasma sample clean-up with solid-phase extraction, resulting in significant improvement in extraction recovery. In addition, TBAS addition to the mobile phase improved the peak symmetry of GTI-2040. This method was successfully used in the analysis of GTI-2040 in clinical plasma samples.