tiazofurin and selenazofurin

Inosine 5 -monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme for the synthesis of GTP and dGTP. Two isoforms of IMPDH have been identified. IMPDH Type I is ubiquitous and predominantly present in normal cells, whereas IMPDH Type II is predominant in malignant cells. IMPDH plays an important role in the expression of cellular genes, such as p53, c-myc and Ki-ras. IMPDH activity is transformation and progression linked in cancer cells. IMPDH inhibitors, tiazofurin, selenazofurin, and benzamide riboside share similar mechanism of action and are metabolized to their respective NAD analogues to exert antitumor activity. Tiazofurin exhibits clinical responses in patients with acute myeloid leukemia and chronic myeloid leukemia in blast crisis. These responses relate to the level of the NAD analogue formed in the leukemic cells. Resistance to tiazofurin and related IMPDH inhibitors relate mainly to a decrease in NMN adenylyltransferase activity. IMPDH inhbitors induce apoptosis. IMPDH inhitors are valuable probes for examining biochemical functions of GTP as they selectively reduce guanylate concentration. Incomplete depletion of cellular GTP level seems to down-regulate G-protein function, thereby inhibit cell growth or induce apoptosis. Inosine 5'-monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) catalyzes the dehydrogenation of IMP to XMP utilizing NAD as the proton acceptor. Studies have demonstrated that IMPDH is a rate-limiting step in the de novo synthesis of guanylates, including GTP and dGTP. The importance of IMPDH is central because dGTP is required for the DNA synthesis and GTP plays a major role not only for the cellular activity but also for cellular regulation. Two isoforms of IMPDH have been demonstrated. IMPDH Type I is ubiquitous and predominately present in normal cells, whereas the IMPDH Type II enzyme is predominant in malignant cells. Although guanylates could be salvaged from guanine by the enzyme hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8), the level of circulating guanine is low in dividing cells and this route is probably insufficient to satisfy the needs of guanylates in the cells.

Topics: Antineoplastic Agents; Apoptosis; cdc25 Phosphatases; Clinical Trials as Topic; Clinical Trials, Phase I as Topic; Clinical Trials, Phase II as Topic; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Guanosine Triphosphate; HL-60 Cells; Humans; IMP Dehydrogenase; Leukemia, Myeloid; Neoplasms; Nucleosides; Organoselenium Compounds; Ovarian Neoplasms; Protein Tyrosine Phosphatases; Ribavirin; Ribonucleosides; RNA, Messenger; Time Factors; Tumor Cells, Cultured

IMP dehydrogenase (IMPDH) is an enzyme which catalyzes the NAD-dependent conversion of inosine 5 -monophosphate (IMP) to xanthosine 5 -monophosphate (XMP) at the metabolic branch point in the de novo purine nucleotide synthetic pathway. IMPDH was shown to be increased significantly in cancer cells and therefore considered to be a sensitive target for cancer chemotherapy. By blocking the conversion of IMP to XMP, IMPDH inhibitors lead to depletion of the guanylate (GMP, GDP, GTP and dGTP) pools. Two isoforms of human IMPDH, designed type I and type II, have been identified and sequenced. Type I is constitutively expressed and is the predominant isoform in normal cells, while type II is selectively up-regulated in neoplastic and replicating cells. Two types of IMPDH inhibitors, endowed with antineoplastic, antiviral and immunosoppressive activity, have been discovered so far: nucleoside inhibitors, such as ribavirin and tiazofurin, and non-nucleoside, such as mycophenolic acid. Ribavirin produces IMPDH inhibition via its anabolite 5 -monophosphate. Tiazofurin inhibits the enzyme after metabolic conversion into thiazole-4-carboxamide adenine dinucleotide (TAD), an analogue of the cofactor NAD. It was hypothesized that the inhibitory activity of tiazofurin is due to an attractive electrostatic interaction between the heterocyclic sulphur atom and the furanose oxygen 1 which constrain rotation about the C-glycosidic bond in tiazofurin and in its active anabolite TAD. To check this hypothesis, we studied several C-nucleosides related to tiazofurin and their NAD analogues. Non-nucleoside IMPDH inhibitors are also reviewed.

Topics: Antineoplastic Agents; Antiviral Agents; Humans; IMP Dehydrogenase; Inhibitory Concentration 50; K562 Cells; Mycophenolic Acid; Nucleosides; Organoselenium Compounds; Oxazoles; Ribavirin; Ribonucleosides; Ribose; Tumor Cells, Cultured

The broad spectrum of antiviral activity of ribavirin (RBV) lies in its ability to inhibit IMP dehydrogenase, which lowers cellular GTP. However, RBV can act as a potent mutagen for some RNA viruses. Previously we have shown a lack of correlation between antiviral activity and GTP repression for Hantaan virus (HTNV) and evidence for RBV's ability to promote error-prone replication. To further explore the mechanism of RBV, GTP levels, specific infectivity, and/or mutation frequency was measured in the presence of RBV, mycophenolic acid (MPA), selenazofurin, or tiazofurin. While all four drugs resulted in a decrease in the GTP levels and infectious virus, only RBV increased the mutation frequency of viral RNA (vRNA). MPA, however, could enhance RBV's mutagenic effect, which suggests distinct mechanisms of action for each. Therefore, a simple drop in GTP levels does not drive the observed error-prone replication. To further explore RBV's mechanism of action, we made a comprehensive analysis of the mutation frequency over several RBV concentrations. Of importance, we observed that the viral population reached a threshold after which mutation frequency did not correlate with a dose-dependent decrease in the level of vRNA, PFU, or [RTP]/[GTP] (where RTP is ribavirin-5'-triphosphate) over these same concentrations of RBV. Modeling of the relationship of mutation frequency and drug concentration showed an asymptotic relationship at this point. After this threshold, approximately 57% of the viral cDNA population was identical to the wild type. These studies revealed a lethal threshold, after which we did not observe a complete loss of the quasispecies structure of the wild-type genome, although we observed extinction of HTNV.

Topics: Animals; Antiviral Agents; Chlorocebus aethiops; Dose-Response Relationship, Drug; Gene Frequency; Genome, Viral; Guanosine Triphosphate; Hantaan virus; Mutation; Mycophenolic Acid; Organoselenium Compounds; Ribavirin; Ribonucleosides; RNA, Viral; Vero Cells

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that is emerging as a significant human pathogen in many regions of the world, including Africa, Asia, and Europe. In this report, we describe a simple screening method for discovering new antiviral compounds directed against CCHFV. Antiviral activity was determined by assaying infected SW-13 cells (human adrenal gland carcinoma) for protection from cytopathic effect (CPE). By using an in vitro neutral red uptake assay, we were able to quantitatively measure CPE induced by CCHFV. As a proof of concept, we used this method to evaluate the antiviral activity of ribavirin and a series of structural analogs (ribamidine, 6-azauridine, selenazofurin, and tiazofurin) against four geographically diverse strains of CCHFV. Ribavirin inhibited the replication of CCHFV as reported previously using plaque reduction assays. One drug, ribamidine, showed antiviral activity that was 4.5- to 8-fold less than that of ribavirin, and the other three drugs (6-azauridine, selenazofurin, and tiazofurin) did not show significant antiviral activity. There were no significant differences in drug sensitivities among the CCHFV strains. Development of this simple and reliable assay will potentially allow high-throughput screening for discovering additional antiviral drugs to combat this important public health threat.

Topics: Animals; Antiviral Agents; Azauridine; Cell Line; Chlorocebus aethiops; Coloring Agents; Cricetinae; Fibroblasts; Hemorrhagic Fever Virus, Crimean-Congo; Humans; Microbial Sensitivity Tests; Neutral Red; Organoselenium Compounds; Ribavirin; Ribonucleosides; Staining and Labeling; Vero Cells; Viral Plaque Assay; Virus Replication

Antineoplastic activity of tiazofurin (Tz) and selenazofurin (Se) depends on their conversion to substances which are analogs of NAD. NAD performs pleiotropic and essential cellular functions, both as a cofactor in oxidation-reduction reactions and as a substrate for poly- and mono-ADP-ribosylation reactions. The therapeutic potential of modulating intracellular NAD levels and activity of NAD-dependent enzymes by concomitant administration of conventional anticancer agents merits further research. Our aim was to investigate the cytotoxic effects of Tz and Se in hematopoietic cells and to test their ability to potentiate the effects of DNA strand-disrupting agents.. THP-1, a cell line, derived from human acute monoblastic leukemia, was used. CLL lymphocytes were obtained from 8 patients with CLL.. The WST-l test was used to detect the function of NAD(P)-dependent dehydrogenases after exposure of THP-1 cells to Tz or Se. Cytotoxicity of Tz, Se, MNNG and chlorambucil was assessed using the membrane permeability assay (PI test).. THP-1 cells were sensitive to cytotoxic effects of Tz and Se, with IC50 values of 2.5 x 10(-5) M for Tz and 2 x 10(-6) M for Se, as determined with the WST-1 test; 10 microM Se induced cell membrane disruption in more than 20% of THP-1 cells 48 hours after commencement of treatment, whereas the same concentration of Tz failed to increase membrane permeability. Pretreatment of THP-1 cells with 0.5 - 1.5 microM Se had no effect on the time course of cell death, induced by treatment with the DNA-damaging agent 1-methyl-3-nitro-1 - nitrosoguanidinium (MNNG) for 36 hours. However, when incubation of THP-1 cells with MNNG was prolonged (72 hours) without changing the incubation medium, pretreatment with Se had the following effects: the relative number of cells that died spontaneously decreased, and the cytotoxicity of MNNG was diminished. This effect was also demonstrated ex vivo in 6 of 8 cases of CLL, treated with MNNG and chlorambucil.. Contrary to other investigations, we here demonstrate that preincubation with Se may partially protect cells from cell death induced by the alkylating agents MNNG and chlorambucil in the THP-1 cell line and in CLL lymphocytes presumably by affecting spontaneous cell death.

Topics: Antineoplastic Agents; Cell Death; Cell Line; Cell Survival; Chlorambucil; Dose-Response Relationship, Drug; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Monocytic, Acute; Methylnitronitrosoguanidine; Organoselenium Compounds; Ribavirin; Ribonucleosides

Oncolytic C-nucleosides, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and benzamide riboside (3-beta-D-ribofuranosylbenzamide) are converted in cell into active metabolites thiazole-4-carboxamide- and benzamide adenine dinucleotide, TAD and BAD, respectively. TAD and BAD as NAD analogues were found to bind at the nicotinamide adenine dinucleotide (cofactor NAD) site of inosine monophosphate dehydrogenase (IMPDH), an important target in cancer treatment. The synthesis and evaluation of anticancer activity of a number of C-nucleosides related to tiazofurin and nicotinamide riboside then followed and are reviewed herein. Interestingly, pyridine C-nucleosides (such as C-nicotinamide riboside) are not metabolized into the corresponding NAD analogues in cell. Their conversion by chemical methods is described. As dinucleotides these compounds show inhibition of IMPDH in low micromolar level. Also, the synthesis of BAD in metabolically stable bis(phosphonate) form is discussed indicating the usefulness of such preformed inhibitors in drug development. Among tiazofurin analogues, Franchetti and Grifantini found, that the replacement of the sulfur by oxygen (as in oxazafurin) but not the removal of nitrogen (tiophenfurin) of the thiazole ring resulted in inactive compounds. The anti cancer activity of their synthetic dinucleotide analogues indicate that inactive compounds are not only poorly metabolized in cell but also are weak inhibitors of IMPDH as dinucleotides.

Topics: Antineoplastic Agents; Cell Survival; Humans; IMP Dehydrogenase; NAD; Niacinamide; Organoselenium Compounds; Pyridinium Compounds; Ribavirin; Ribonucleosides; Ribonucleotides; Tumor Cells, Cultured

Benzamide is a well known inhibitor of poly(ADP-ribose)polymerase, an enzyme involved in DNA repair. However, benzamide exhibited neuotoxicity in animals and hence, in the hope of overcoming this problem, benzamide riboside (BR) was synthesized. Our mechanism of action studies on BR suggested that the agent was being metabolized to its 5'-monophosphate and then to its NAD analogue (BAD, benzamide adenine dinucleotide) that inhibits Inosine 5'-monophosphate dehydrogenase (IMPDH). IMPDH is the rate-limiting enzyme of the branched purine nucleotide synthetic pathway that provides guanylates including GTP and dGTP. There are two isoforms of IMPDH, type I that is constitutively present in all cells, and type II that is inducible and is present in highly proliferating cells such as cancer. Ongoing studies with BR analogues suggest that they are more selective in inhibiting IMPDH type II. Our studies have characterized the metabolites of BR, especially its NAD analogue, BAD, by synthesizing this active metabolite by enzymatic means, and identifying its structure by NMR and mass spectrometry. We have partially purified IMPDH from tumor cells and have examined the kinetics of inhibition of IMPDH by BAD. We have also compared biochemical and cytotoxic activities of BR with tiazofurin and selenazofurin, that share similar mechanisms of action with BR. Our studies demonstrated that 2-3-fold more BAD is formed compared to TAD and SAD, the active metabolites of tiazofurin and selenazofurin, respectively. BR has demonstrated potent cytotoxic activity in a diverse group of human tumor cells, specifically more active in sarcomas and CNS neoplasms compared to tiazofurin or selenazofurin. Future in vivo animal studies should set a stage for determining its effectiveness in clinical Phase I studies.

Topics: Antineoplastic Agents; Cell Survival; Central Nervous System Neoplasms; Humans; IMP Dehydrogenase; Isoenzymes; Nucleosides; Organoselenium Compounds; Ribavirin; Ribonucleosides; Sarcoma; Tumor Cells, Cultured

The synthesis and biological activity of selenophenfurin (5-beta-D-ribofuranosylselenophene-3-carboxamide, 1), the selenophene analogue of selenazofurin, are described. Glycosylation of ethyl selenophene-3-carboxylate (6) under stannic chloride-catalyzed conditions gave 2- and 5-glycosylated regioisomers, as a mixture of alpha- and beta-anomers, and the beta-2,5-diglycosylated derivative. Deprotected ethyl 5-beta-D-ribofuranosylselenophene-3-carboxylate (12 beta) was converted into selenophenfurin by ammonolysis. The structure of 12 beta was determined by 1H- and 13C-NMR, crystallographic, and computational studies. Selenophenfurin proved to be antiproliferative against a number of leukemia, lymphoma, and solid tumor cell lines at concentrations similar to those of selenazofurin but was more potent than the thiophene and thiazole analogues thiophenfurin and tiazofurin. Incubation of K562 cells with selenophenfurin resulted in inhibition of IMP dehydrogenase (IMPDH) (76%) and an increase in IMP pools (14.5-fold) with a concurrent decrease in GTP levels (58%). The results obtained confirm the hypothesis that the presence of heteroatoms such as S or Se in the heterocycle in position 2 with respect to the glycosidic bond is essential for both cytotoxicity and IMP dehydrogenase inhibitory activity in this type of C-nucleosides.

Topics: Animals; Antineoplastic Agents; Cell Division; Computer Simulation; Crystallography, X-Ray; Enzyme Inhibitors; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Inosine Monophosphate; Leukemia; Lymphoma; Magnetic Resonance Spectroscopy; Mice; Models, Molecular; Molecular Structure; Neoplasms; Organoselenium Compounds; Ribavirin; Ribonucleosides; Tumor Cells, Cultured

The biochemical and cytotoxic activities of the IMP dehydrogenase (IMPDH) inhibitors benzamide riboside, tiazofurin, and selenazofurin were compared. These three C-nucleosides exert their cytotoxicity by forming an analogue of NAD, wherein nicotinamide is replaced by the C-nucleoside base. The antiproliferative activities of these three agents were compared in a panel of 60 human cancer cell lines. To examine the relationship of benzamide riboside and selenazofurin to tiazofurin, COMPARE computer analysis was performed, and correlation coefficients of 0.761 and 0.815 were obtained for benzamide riboside and selenazofurin, respectively. The biochemical activities of these agents were examined in human myelogenous leukemia K562 cells. Incubation of K562 cells for 4 hr with 10 microM each of benzamide riboside, selenazofurin and tiazofurin resulted in a 49, 71, and 26% decrease in IMPDH activity with a concurrent increase in intracellular IMP pools. As a consequence of IMPDH inhibition, GTP and dGTP concentrations were curtailed. These studies demonstrated that selenazofurin was the most potent of the three agents. To compare the cellular synthesis of NAD analogues of these agents, K562 cells were incubated with 10 microM each of benzamide riboside, tiazofurin and selenazofurin after prelabeling the cells with [2,8-3H]adenosine. The results demonstrated that benzamide riboside produced 2- and 3-fold more of NAD analogue (BAD) than tiazofurin and selenazofurin did. To elucidate the effects of the three compounds on other NAD-utilizing enzymes, the inhibitory activities of purified benzamide adenine dinucleotide (BAD), thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD) were studied in commercially available purified preparations of lactate dehydrogenase, glutamate dehydrogenase and malate dehydrogenase. TAD and SAD did not inhibit these three dehydrogenases. Although BAD did not influence lactate and glutamate dehydrogenases, it selectively inhibited 50% of malate dehydrogenase activity at a 3.2 microM concentration. These studies demonstrate similarities and differences in the biochemical actions of the three C-nucleosides, even though they share similar mechanisms of action.

Topics: Antineoplastic Agents; Cell Survival; Glutamate Dehydrogenase; Humans; IMP Dehydrogenase; L-Lactate Dehydrogenase; Malate Dehydrogenase; Nucleosides; Organoselenium Compounds; Ribavirin; Ribonucleosides; Ribonucleotides; Tumor Cells, Cultured

Unlike conventional enzymes, receptors that activate G proteins do not catalyze the direct formation or cleavage of covalent bonds but act instead as a catalyst for the exchange of GTP vs GDP, which results in major conformational changes in the alpha subunit of G proteins and dissociation and selective binding of the alpha subunit which provokes direct enzyme activation eventually resulting in stimulation of protein kinase A, B or C. Each of these kinases can phosphorylate specific DNA binding proteins which allow new portions of DNA to be read and expressed. Such a series of events can act as switches to control cellular genetic expression resulting in cellular proliferation, differentiation or hormonal secretion of growth factors (Scheme I). Examples of nucleosides and nucleotides which appear to exert their therapeutic effects via G protein control of cellular proliferation resulting in differentiation are tiazofurin, selenazofurin, and 8-chloro-cAMP which have been synthesized and studied in our laboratories. The clinical application of these nucleosides in cancer treatment is presently underway and offers a viable alternative to chemotherapy with highly cytotoxic agents. The use of these derivatives result in down-regulation of the G protein regulatory pathways responsible for rapid cell division. Alternatively, a series of guanosine analogs prepared in our laboratories, 8-bromoguanosine, 8-mercaptoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, all activate various aspects of the immune response by up-regulation of G protein regulatory pathways in various lymphocyte derived cells. Guanosine-like nucleosides which function in this manner could have major clinical application as antitumor, antiviral and antimetastatic agents providing the desired specificity can be achieved. Specific immune enhancement of the aged might be an attainable goal if suitable orally active guanosine derivatives with high specificity can be achieved. The G protein regulatory pathways for modulation of genetic expression in specific cell types provide a major modern approach to new chemotherapeutic agents.

Topics: Animals; Antineoplastic Agents; Antiviral Agents; Down-Regulation; Gene Expression Regulation, Neoplastic; GTP-Binding Proteins; Humans; Nucleosides; Nucleotides; Organoselenium Compounds; Phosphorylation; Ribavirin; Ribonucleosides; Selenium; Signal Transduction; Tumor Cells, Cultured

The synthetic nucleoside tiazofurin(2-beta-ribofuranosylthiazole-4-carboxyamide) and its selenium analog selenazofurin inhibited the growth of L1210 leukemia cell culture in a dose dependent manner with IC50 value of 2.0 and 0.2 Um respectively. The GTP/ATP ratio was diminished 4-6 fold as measured by HPLC, while IMP/ATP increased 6-8 fold. The decreased guanylate pools may explain the 30% reduction in cyclic GMP levels and GTPase activity measured after the treatment with the nucleosides. Inhibition of phospholipase C activity is suggested since diacylglycerol content, protein kinase C activity and phorbol ester binding of the membrane fraction were also reduced 20-40%. These results reveal a novel aspect in the action of these compounds which may play a role in their therapeutic action and selectivity.

Topics: Adenosine Triphosphate; Animals; Antineoplastic Agents; Caenorhabditis elegans Proteins; Carrier Proteins; Cyclic GMP; Diglycerides; Guanosine Triphosphate; Inosine Monophosphate; Leukemia L1210; Mice; Organoselenium Compounds; Phorbol 12,13-Dibutyrate; Phosphates; Phosphatidylinositols; Protein Kinase C; Receptors, Drug; Ribavirin; Ribonucleosides; Selenium

Tiazofurin, an anti-cancer drug, which induces remissions in human leukemia, and ribavirin, an anti-viral agent, bind at separate sites (NADH and IMP-XMP sites, respectively) on the target enzyme, IMP dehydrogenase. Now we show that the binding to IMP dehydrogenase of these drugs at two separate sites is translated into synergistic inhibition of de novo guanylate biosynthesis and synergistic toxicity in rat hepatoma 3924A cells. These results may be utilized in the chemotherapy of neoplastic diseases and in the treatment of hepatitis virus infection and hepatocellular carcinoma.

Topics: Animals; Cell Line; Cell Survival; Drug Synergism; Guanosine Monophosphate; IMP Dehydrogenase; Liver Neoplasms, Experimental; Organoselenium Compounds; Purines; Rats; Ribavirin; Ribonucleosides; Selenium

The synthetic "C" nucleoside, tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide), its selenium analogue selenazofurin, and the related inhibitor of inosine 5'-phosphate (IMP) dehydrogenase, mycophenolic acid, are effective inducers of the terminal differentiation of HL-60 promyelocytic leukemia cells. The inhibition of cellular replication and the induced maturation produced by these agents appears to be a consequence of the inhibition of IMP dehydrogenase, since growth inhibition is partially reversed and differentiation is completely prevented by the simultaneous exposure of cells treated with inhibitors of IMP dehydrogenase to exogenous guanosine, which serves to circumvent the effects of the blockage of IMP dehydrogenase. The exposure of HL-60 leukemia cells to inhibitors of IMP dehydrogenase caused a marked reduction in the incorporation of [3H]mannose into both cellular glycoproteins and their lipid-linked oligosaccharide precursors; these effects are presumably due to the pronounced decrease in intracellular levels of guanosine triphosphate produced by blockage of IMP dehydrogenase. Maximum effects on glycoprotein biosynthesis occurred within 8 h of exposure to the inhibitors of IMP dehydrogenase. The simultaneous incubation of cells with guanosine and these inducers of differentiation partially prevented the reduction in [3H]mannose incorporation into glycoproteins, supporting a relationship between glycoprotein biosynthesis and guanosine triphosphate formation in the induction of differentiation by inhibitors of IMP dehydrogenase.

Topics: Cell Cycle; Cell Differentiation; Cell Line; Dolichol Monophosphate Mannose; Glycoproteins; Guanosine; Guanosine Triphosphate; Hematopoiesis; Humans; IMP Dehydrogenase; Ketone Oxidoreductases; Leukemia, Myeloid, Acute; Mycophenolic Acid; Organoselenium Compounds; Phagocytosis; Ribavirin; Ribonucleosides; Selenium

A series of dinucleotides, analogous to nicotinamide adenine dinucleotide but containing the five-membered base nucleosides tiazofurin (1a), selenazofurin (1b), ribavirin (2), and AICAR (3) in place of nicotinamide ribonucleoside, were prepared in greater than 50% yield by reacting the corresponding nucleotide imidazolidates (6a-d) with adenosine 5'-monophosphate (AMP). The symmetric dinucleotides of tiazofurin (TTD, 8e) and selenazofurin (SSD, 8f) were also prepared by a similar methodology. These dinucleotides were characterized by 1H NMR and fast atom bombardment MS and were evaluated for their inhibitory potency against a partially purified preparation of tumoral inosine monophosphate dehydrogenase (IMPD) from P388 cells. The order of potency found was SAD (8b) greater than TAD (8a) much greater than SSD (8f) congruent to TTD (8e) congruent to RAD (8c) much much greater than ZAD (8d). On kinetic analysis none of the dinucleotides produced competitive inhibition of IMPD with NAD as a variable substrate. In addition to their superior IMPD inhibitory activity, SAD and TAD appear to be the only dinucleotides, besides NAD, that are formed naturally by the NAD pyrophosphorylase from P388 lymphoblasts.

Topics: Animals; IMP Dehydrogenase; Ketone Oxidoreductases; Kinetics; Leukemia P388; Mice; NAD; Organoselenium Compounds; Ribavirin; Ribonucleosides; Selenium

The mechanism of the cellular toxicity of four inosinate dehydrogenase (IMP-DH) inhibitors with different antitumor and antiviral pharmacological profiles was investigated in mouse lymphoma (S-49) cell culture. Drug effects on cell growth, nucleotide pools, and DNA and RNA synthesis were measured in the presence and absence of guanine salvage supplies. Both guanine and guanosine were capable of bypassing the IMP-DH block, while they also demonstrated some growth-inhibitory effects when added alone in high concentrations. All four drugs reduced cellular guanosine triphosphate levels and caused secondary changes of the uridine, cytidine, and adenosine triphosphate pools that were similar among the four drugs. However, several drug effects in addition to IMP-DH inhibition were observed except with mycophenolic acid which may represent a pure IMP-DH inhibitor. Both tiazofurin and selenazofurin interfered with the uptake and/or metabolism of uridine and thymidine tracers; however, this effect appeared not to contribute to their cellular toxicity in vitro. Moreover, selenazofurin and tiazofurin impaired the utilization of exogenous guanine salvage supplies for DNA and RNA synthesis, and guanine was particularly ineffective in reversing the toxic effects of tiazofurin on cell growth. This finding is important in view of the available guanine salvage supplies in vivo. Since tiazofurin, selenazofurin, and their known metabolites failed to inhibit hypoxanthine-guanine-phosphoribosyl transferase, guanosine monophosphate kinase, and guanosine diphosphate kinase in cell extracts or permeabilized cells, these drugs may interfere with salvage transport across cellular membranes. The toxic effects of mycophenolic acid and ribavirin were similarly reversed by salvage supplies of up to 200 microM guanine, which suggests that ribavirin primarily acts as an IMP-DH inhibitor under these conditions. This result could explain the rather low antitumor efficacy of both mycophenolic acid and ribavirin in vivo. However, increasing the guanine salvage supply in the medium above 200 microM further reversed the toxic effects of mycophenolic acid to maximum rescue, while it increased the toxicity of ribavirin (300 microM). This finding suggests the presence of a toxic mechanism of ribavirin at higher concentrations that is dependent upon the presence of guanine supplies sufficient to fully overcome the IMP-DH inhibition. This study documents that each antimetabolite displays a unique s

Topics: Adenosine Triphosphate; Animals; Cell Division; Cells, Cultured; DNA, Neoplasm; Guanine; Guanylate Kinases; Hypoxanthine Phosphoribosyltransferase; IMP Dehydrogenase; Ketone Oxidoreductases; Lymphoma; Mice; Mycophenolic Acid; Nucleoside-Diphosphate Kinase; Nucleoside-Phosphate Kinase; Organoselenium Compounds; Ribavirin; Ribonucleosides; RNA, Neoplasm; Selenium; Tritium; Uridine

Tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) and selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) are synthetic "C" nucleosides whose antineoplastic activity depends on their conversion to tiazofurin-adenine dinucleotide and selenazofurin-adenine dinucleotide which are analogs of NAD. The present study was conducted to determine whether these nucleoside analogs and their dinucleotide derivatives interfere with NAD metabolism and in particular with the NAD-dependent enzyme, poly(ADP-ribose) polymerase. Incubation of L1210 cells with 10 microM tiazofurin or selenazofurin resulted in inhibition of cell growth, reduction of cellular NAD content, and interference with NAD synthesis. Using [14C]nicotinamide to study the uptake of nicotinamide and its conversion to NAD, we showed that the analogs interfere with NAD synthesis, apparently by blocking formation of nicotinamide mononucleotide. The analogs also serve as weak inhibitors of poly(ADP-ribose) polymerase, which is an NAD-utilizing, chromatin-bound enzyme, whose function is required for normal DNA repair processes. Continuous incubation of L1210 cells in tiazofurin or selenazofurin resulted in progressive and synergistic potentiation of the cytotoxic effects of DNA-damaging agents, such as 1,3-bis(2-chloroethyl)-1-nitrosourea or N-methyl-N'-nitro-N-nitrosoguanidine. These studies provide a basis for designing chemotherapy combinations in which tiazofurin or selenazofurin are used to modulate NAD and poly(ADP-ribose) metabolism to synergistically potentiate the effects of DNA strand-disrupting agents.

Topics: Animals; Antineoplastic Agents; Cells, Cultured; Leukemia L1210; Mice; NAD; Nucleoside Diphosphate Sugars; Organoselenium Compounds; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Ribavirin; Ribonucleosides; Selenium

2-beta-D-Ribofuranosyl-4-selenazolecarboxamide (selenazofurin, CI-935), the selenium analog of tiazofurin (CI-909), was 3- to 10-fold more cytotoxic to murine or human tumor cells in vitro than tiazofurin and was also more active against P388 mouse leukemia in vivo. In vitro cytotoxicity could be reversed by guanosine or guanine but not by other purine nucleosides or bases. Three human tumor cell lines selected for selenazofurin or tiazofurin resistance showed cross resistance between selenazofurin and tiazofurin. Treatment with tiazofurin, selenazofurin, or mycophenolic acid decreased guanylate pools and caused an accumulation of IMP in WIL2 human lymphoma cells. The decrease in guanylate pools was accompanied by inhibition of RNA and DNA synthesis. The NAD analogs of tiazofurin and selenazofurin were inhibitors of L1210 IMP dehydrogenase (IMP:NAD oxidoreductase, EC 1.2.1.14), and both showed uncompetitive inhibition with respect to NAD having Kii values of 5.7 X 10(-8)M and 3.3 X 10(-8)M respectively.

Topics: Animals; Antineoplastic Agents; Cells, Cultured; Humans; Mice; Neoplasms; Nucleic Acids; Organoselenium Compounds; Ribavirin; Ribonucleosides; Ribonucleotides; Selenium

Binary combinations of the N-nucleoside ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) and the C-nucleoside analog selenazofurin (2-beta-D-ribofuranosylselenazole-4-carboxamide) or tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide) were tested in vitro for activity against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, Rift Valley fever, Korean hemorrhagic fever, and Pichinde viruses. The 50% effective dose for each compound alone or in a series of combinations was determined with a plaque reduction assay. Combinations of ribavirin and selenazofurin were synergistic against Venezuelan equine encephalomyelitis, Japanese encephalitis, yellow fever, and Pichinde viruses, with fractional inhibitory concentrations of 0.1, 0.2, 0.4, 0.4, respectively, but showed additive effects against Korean hemorrhagic fever and Rift Valley fever viruses. Combinations of ribavirin and tiazofurin were synergistic against yellow fever and Japanese encephalitis (fractional inhibitory concentrations, 0.41 and 0.48, respectively) but showed additive effects against Korean hemorrhagic fever virus. Combinations of selenazofurin and tiazofurin had additive effects against Japanese encephalitis, yellow fever, and Korean hemorrhagic fever viruses. The effect of combinations on cell toxicity was additive, both in monolayers of nondividing cells incubated under agar for the same period as the plaque assay and for rapidly dividing cells given short-term exposure (4 h), followed by determination of the proportion of surviving cells with a colony forming assay.

Topics: Antiviral Agents; Arenaviridae; Bunyaviridae; Drug Combinations; Drug Synergism; IMP Dehydrogenase; Organoselenium Compounds; Ribavirin; Ribonucleosides; Selenium; Togaviridae

Topics: Animals; Gas Chromatography-Mass Spectrometry; IMP Dehydrogenase; Ketone Oxidoreductases; Leukemia P388; Leukemia, Experimental; Mice; NAD; Organoselenium Compounds; Ribavirin; Ribonucleosides; Selenium

2-beta-D-Ribofuranosylselenazole-4-carboxamide, a selenazole analog of the antitumor agent Tiazofurin, is severalfold more cytotoxic to murine tumor cells in culture than Tiazofurin. Like Tiazofurin, the cytotoxicity of the selenazole analog is reversed by exogenous guanosine, and both nucleosides specifically inhibit IMP dehydrogenase activity in cultured P388 cells. The dose-dependency for this inhibition correlates with the relative cytotoxicities of both drugs, indicating that a more potent inhibition of IMP dehydrogenase by the selenazole analog is primarily responsible for its increased cytotoxicity. The specific inhibition of the isolated enzyme by potential metabolites of the selenazole analog is discussed.

Topics: Animals; Antineoplastic Agents; Cell Survival; IMP Dehydrogenase; Leukemia L1210; Leukemia P388; Leukemia, Experimental; Mice; Nucleoside-Diphosphate Kinase; Organoselenium Compounds; Ribavirin; Ribonucleosides; Selenium