thioguanine-anhydrous and 6-thioguanosine

The chromatography of several thiopurines is investigated using aqueous normal phase (ANP) conditions in conjunction with a silica hydride-based column. Both isocratic and gradient elution modes are tested. Detection of higher concentration samples is done by UV to demonstrate feasibility in this format while lower concentration samples utilize mass spectrometry (MS). Repeatability of successive runs is also tested with particular attention to gradient methods where the equilibration time of the stationary phase can be evaluated.

Topics: Chromatography, High Pressure Liquid; Guanine Nucleotides; Guanosine; Thioguanine; Thionucleosides; Thionucleotides

Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis.

Topics: Alanine; Animals; Bacillus anthracis; Cell Line; Guanosine; Macrophages; Mice; Spores, Bacterial; Structure-Activity Relationship; Thionucleosides

Depletion of guanine and deoxyguanine nucleotides by inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) or introduction of 6-thioguanine nucleotide antimetabolites are two principles of retarding cell proliferation by interference with the cellular purine nucleotide pool. IMPDH activity may be a promising pharmacodynamic biomarker during immunosuppressive and anticancer pharmacotherapy. The aim of the study was to investigate the impact of mycophenolic acid (MPA) and 6-thioguanosine (tGuO) on IMPDH basal activity.. We studied the IMPDH basal activity (i.e. the enzyme activity following inhibitor exposure, but measured in absence of the inhibitor) in response to increasing concentrations of the IMPDH inhibitor MPA and the antimetabolite tGuO in MOLT-4 human leukaemia cells. In parallel, IMPDH gene expression and cellular purine nucleotide concentrations were examined.. A biphasic concentration-dependent influence of MPA on the IMPDH basal activity was observed. At concentrations < or =IC50, MPA increased the IMPDH basal activity. The increase was associated with elevated expression of IMPDH2. Despite increased expression, the basal enzyme activity decreased following exposure to high MPA concentrations. The IMPDH2 expression increased modestly in response to tGuO exposure. However, the IMPDH basal activity decreased when the cells were exposed to a proliferation-blocking tGuO concentration.. These findings demonstrate that IMPDH basal activity is influenced by MPA and tGuO, and suggest that reduced IMPDH basal activity is related to the proliferation-blocking effects of these agents.

Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Expression Regulation, Leukemic; Guanosine; Humans; IMP Dehydrogenase; Intracellular Space; Leukemia; Mycophenolic Acid; Purines; Thionucleosides

This study describes approaches for stacking a large volume of sample solutions containing a mixture of mercaptopurine monohydrate, 6-methylmercaptopurine, thioguanine, thioguanosine, and thioxanthine in capillary electrophoresis (CE). After filling the run buffer (60 mM borate buffer, pH 8.5), a large sample volume was loaded by hydrodynamic injection (2.5 psi, 99.9 s), followed by the removal of the large plug of sample matrix from the capillary using polarity switching (-15 kV). Monitoring the current and reversing the polarity when 95% of current recovered, the separation of anionic analytes was performed in a run buffer < 20 kV. Around 44- to 90-fold improvement of sensitivity for five analytes was achieved by large-volume stacking with polarity switching when compared with CE without stacking. This method was feasible for determination of the analytes spiked in plasma. Removing most of electrolytes from plasma is a key step for performing large-volume sample stacking. Solid-phase extraction was used for pretreatment of biological samples. To our knowledge, this study is one of few applications showing the possibilities of this stacking procedure to analyze biological samples by large-volume sample stacking with polarity switching (LVSSPS) in CE.

Topics: Electrophoresis, Capillary; Guanosine; Humans; Mercaptopurine; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Sensitivity and Specificity; Thioguanine; Thionucleosides; Xanthines

There are two well-described syndromes caused by tumor production of parathyroid hormone-related peptide (PTHrP), namely osteolytic bone disease associated with breast cancer and humoral hypercalcemia of malignancy (HHM) that occurs with or without bone metastasis. Both syndromes have been shown experimentally to be inhibited by neutralizing antibodies to PTHrP. In a search for small-molecule inhibitors of PTHrP production or effects, we have identified guanine-nucleotide analogs as compounds that inhibit PTHrP expression by human tumor cells associated with these syndromes. We show in nude athymic murine models that these compounds reduce PTHrP-mediated osteolytic lesions associated with metastatic human breast-cancer cells as well as the degree of hypercalcemia caused by excessive PTHrP production by a squamous-cell carcinoma of the lung. These results suggest that the PTHrP gene promoter may be a suitable target for treating the skeletal effects of malignancy.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Cytokines; Female; Gene Expression; Guanine Nucleotides; Guanosine; Humans; Hypercalcemia; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms; Osteolysis; Paraneoplastic Endocrine Syndromes; Parathyroid Hormone-Related Protein; Peptide Hormones; Promoter Regions, Genetic; Thioguanine; Thionucleosides; Transplantation, Heterologous; Tumor Cells, Cultured

The principal products of the reaction of 6-thio-2'-deoxyguanosine (6-TdGuo) with dimethylsulfate are S6-methyl-6-thiodeoxyguanosine and 7-methyl-6-thiodeoxyguanosine, identified by ultraviolet and mass spectrometry. To study the reactions of 6-TdGuo in DNA, cells were treated with 6-thioguanine, which is incorporated into DNA during S-phase; DNA was purified from cell lysates and reacted with 3H-methylnitrosourea. In addition to the expected methylated purines (O6-methylguanine, 7-methylguanine and 3-methyladenine), 0.6% of the total product was S6-methyl-6-thioguanine. On the basis of thioguanine content, the formation of S6-methyl-6-thioguanine occurs 70-fold more efficiently than O6-methylguanine, which indicates that 6-thioguanine incorporated into DNA is very susceptible to chemical modification by alkylating agents. The ultraviolet and mass spectra of two of the major products of the reaction between 6-TdGuo and 2-chloroethyl methanesulfonate suggest that the structures are (1,S6-ethano)-6-thiodeoxyguanosine and (S6,7-ethano)-6-thiodeoxyguanosine, which are presumably formed by an internal cyclization reaction that proceeds through a sulfonium ion intermediate. In cells, this intermediate could react with DNA nucleophiles to form both DNA intra- and interstrand cross-links.

Topics: Alkylation; Antineoplastic Agents; Carmustine; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Deoxyribonucleotides; DNA Damage; DNA, Neoplasm; Guanosine; Methylnitrosourea; Spectrophotometry, Ultraviolet; Sulfuric Acid Esters; Thioguanine; Thionucleosides

Topics: Animals; Body Fluids; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Goats; Guanosine; Humans; Injections, Intravenous; Inosine; Mercaptopurine; Thioguanine; Thioinosine; Thionucleosides