3-(1-deoxyribofuranosyl)benzamide and tiazofurin

Benzamide riboside, a recently discovered inhibitor of IMP dehydrogenase (IMPDH) exhibits oncolytic activity. IMPDH is the key enzyme of de novo guanylate biosynthesis and was shown to be linked with proliferation. Therefore, IMPDH is a very good target for antitumor therapy. In order to be active, benzamide riboside has to be converted to BAD, an NAD analogue that binds to the NAD site on IMPDH. Inhibition of the enzyme by benzamide riboside selectively inhibits tumor cell growth and induces apoptosis in various human tumor cell lines. In this manuscript we describe the induction of the CD71 transferrin receptor in human promyelocytic leukemia HL-60 cells following treatment with benzamide riboside. The results indicate a possible involvement of the iron metabolism in the action of this new compound. Benzamide riboside might be clinically used in the treatment of leukemia and solid tumors, alone or as part of combination therapy. Since transferrin receptors are overexpressed in certain cancers, such as glioma and colon cancer, a combination therapy that includes benzamide riboside in transferrin-coupled liposomes will not only target cancer cells but also leads to suicidal action because benzamide riboside will upregulate transferrin receptors on cancer cells thereby make it accessible to dose-intensive chemotherapy. We therefore believe that benzamide riboside itself or derivatives of benzamide riboside might become an important addition for the treatment to diseases that are otherwise fatal.

Topics: Antigens, CD; Antigens, Differentiation, B-Lymphocyte; Apoptosis; Cell Division; Drug Screening Assays, Antitumor; HL-60 Cells; Humans; IMP Dehydrogenase; Iron; Leukemia, Myeloid; Nucleosides; Receptors, Transferrin; Ribavirin; Tumor Cells, Cultured

Benzamide riboside (BR) is a novel anticancer agent exhibiting pronounced activity against several human tumor cell lines via the inhibition of inosine 5'-monophosphate dehydrogenase (IMPDH) that catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. Phosphorylation of BR to its 5'-monophosphate derivative appears to be ubiquitous in most cells catalyzed by the enzymes, adenosine kinase, nicotinamide nucleoside kinase and 5' nucleotidase. BR 5'-monophosphate is then converted to the active metabolite benzamide adenine dinucleotide (BAD) by NMN adenylyltransferase, the rate-limiting enzyme in the biosynthesis of NAD. As BAD is more potent in the inhibition of IMPDH than BR and BR 5'-monophosphate, cytotoxicity of BR is closely connected with intercellular metabolism to BAD. However, intracellular BAD level is also affected by BADase activity, a phosphodiesterase which hydrolyzes BAD to BR-5'-monophosphate and AMP. A recent study demonstrates enzymatic deamination of BR to non-cytotoxic benzene carboxylic acid (BR-COOH) as the main hepatic BR biotransformation product in rat liver. As the IMPDH inhibitors tiazofurin and ribavirin exhibit predominant accumulation and biotransformation in liver, hepatic metabolism may be an important factor also for BR activation and inactivation and should be considered in human liver during cancer therapy when BR is used as a single drug or in combination with other anticancer agents.

Topics: Animals; Antineoplastic Agents; Drug Resistance, Neoplasm; Enzyme Inhibitors; Forecasting; Humans; IMP Dehydrogenase; Isoenzymes; K562 Cells; Liver; Nucleosides; Rats; Ribavirin

Benzamide riboside (BR), a synthetic C-nucleoside, acts as a strong growth inhibitor of cancer cells in vitro and in vivo. BR, like TR and related nucleoside prodrugs, act by anabolism to NAD analogs. These analogs selectively inhibit IMPDH, leading to depletion of cellular GTP, growth cessation, and cell differentiation. To date only preclinical studies have been carried out. However, in tiazofurin (TR), a related drug, phase I/II clinical trials have been conducted in patients with acute leukemia and shown to be a very promising agent with a response rate of 85% in 26 patients in one of the trials. Tiazofurin is now undergoing phase III clinical trials as a result. Dose limiting toxicity of tiazofurin was headache, somnolence and nausea with no myelosuppression noted. By contrast, BR showed skeletal muscle toxicity, hepatotoxicity and myelosuppression in preclinical data. Skeletal muscle toxicity was noted in the paraspinal muscles and may represent dose-limiting toxicity. Since BR does exhibit myelosuppression, the most common chemotherapy-related side effect in humans, careful judgment is warranted should BR be included in multidrug regimens, although BR's potent cytotoxicity to tumor cells in preclinical models still makes it a promising drug.

Topics: Animals; Antineoplastic Agents; Cell Survival; Clinical Trials as Topic; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; IMP Dehydrogenase; Leukemia L1210; Mice; Mice, Nude; Nucleosides; Ribavirin; Tumor Cells, Cultured

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

Leishmania amazonensis undergoes apoptosis-like programmed cell death (PCD) under heat shock conditions. We identified a potential role for inosine 5' monophosphate dehydrogenase (IMPDH) in L. amazonensis PCD. Trypanosomatids do not have a "de novo" purine synthesis pathway, relying on the salvage pathway for survival. IMPDH, a key enzyme in the purine nucleotide pathway, is related to cell growth and apoptosis. Since guanine nucleotide depletion triggers cell cycle arrest and apoptosis in several organisms we analyzed the correlation between IMPDH and apoptosis-like death in L. amazonensis. The L. amazonensis IMPDH inhibition effect on PCD was evaluated through gene expression analysis, mitochondrial depolarization and detection of Annexin-V labeled parasites. We demonstrated a down-regulation of impdh expression under heat shock treatment, which mimics the natural mammalian host infection. Also, IMPDH inhibitors ribavirin and mycophenolic acid (MPA) prevented cell growth and generated an apoptosis-like phenotype in sub-populations of L. amazonensis promastigotes. Our results are in accordance with previous results showing that a subpopulation of parasites undergoes apoptosis-like cell death in the nutrient poor environment of the vector gut. Here, we suggest the involvement of purine metabolism in previously observed apoptosis-like cell death during Leishmania infection.

Topics: Animals; Apoptosis; Down-Regulation; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Heat-Shock Response; IMP Dehydrogenase; Leishmania mexicana; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mycophenolic Acid; Nucleosides; Purine Nucleotides; Ribavirin; RNA, Protozoan

Benzamide riboside (BR) and tiazofurin (TR) are converted to analogs of NAD that inhibit IMP dehydrogenase (IMPDH), resulting in cellular depletion of GTP and dGTP and inhibition of proliferation. The current work was undertaken to identify the human nucleoside transporters involved in cellular uptake of BR and TR and to evaluate their role in cytotoxicity. Transportability was examined in Xenopus laevis oocytes and Saccharomyces cerevisiae that produced individual recombinant human concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) types (hENT1, hENT2, hCNT1, hCNT2, or hCNT3). TR was a better permeant than BR with a rank order of transportability in oocytes of hCNT3 >> hENT1 > hENT2 > hCNT2 >> hCNT1. The concentration dependence of inhibition of [(3)H]uridine transport in S. cerevisiae by TR exhibited lower K(i) values than BR: hCNT3 (5.4 versus 226 microM), hENT2 (16 versus 271 microM), hENT1 (57 versus 168 microM), and hCNT1 (221 versus 220 microM). In cytotoxicity experiments, BR was more cytotoxic than TR to cells that were either nucleoside transport-defective or -competent, and transport-competent cells were more sensitive to both drugs. Exposure to nitrobenzylmercaptopurine ribonucleoside conferred resistance to BR and TR cytotoxicity to hENT1-containing CEM cells, thereby demonstrating the importance of transport capacity for manifestation of cytoxicity. A breast cancer cell line with mutant p53 exhibited 9-fold higher sensitivity to BR than the otherwise similar cell line with wild-type p53, suggesting that cells with mutant p53 may be potential targets for IMPDH inhibitors. Further studies are warranted to determine whether this finding can be generalized to other cell types.

Topics: Animals; Biological Transport; Cell Survival; Female; Humans; IMP Dehydrogenase; Membrane Transport Proteins; Nucleosides; Oocytes; Protein Isoforms; Recombinant Proteins; Ribavirin; Saccharomyces cerevisiae; Uridine; Xenopus laevis

NAD+ is essential for life in all organisms, both as a coenzyme for oxidoreductases and as a source of ADPribosyl groups used in various reactions, including those that retard aging in experimental systems. Nicotinic acid and nicotinamide were defined as the vitamin precursors of NAD+ in Elvehjem's classic discoveries of the 1930s. The accepted view of eukaryotic NAD+ biosynthesis, that all anabolism flows through nicotinic acid mononucleotide, was challenged experimentally and revealed that nicotinamide riboside is an unanticipated NAD+ precursor in yeast. Nicotinamide riboside kinases from yeast and humans essential for this pathway were identified and found to be highly specific for phosphorylation of nicotinamide riboside and the cancer drug tiazofurin. Nicotinamide riboside was discovered as a nutrient in milk, suggesting that nicotinamide riboside is a useful compound for elevation of NAD+ levels in humans.

Topics: Chromosomes, Human, Pair 9; Energy Metabolism; Evolution, Molecular; Fungi; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Humans; Intracellular Signaling Peptides and Proteins; Molecular Sequence Data; NAD; Niacinamide; Nucleosides; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Pyridinium Compounds; Ribavirin; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid

Ribavirin and mycophenolic acid (MPA) are known inhibitors of the IMPDH enzyme (E.C. 1.1.1.205). This enzyme catalyzes the conversion of inosine monophosphate to xanthine monophosphate, leading eventually to a decrease in the intracellular level of GTP and dGTP. The antiviral effect against bovine viral diarrhoea virus (BVDV) of 15 analogues related to MPA was determined. MDBK cells were infected with the cytopathic strain of BVDV in presence or absence of test compounds. Viral RNA was extracted from the cell supernatant fluids and quantified by RT-PCR. Ribavirin showed a potent antiviral effect against BVDV with 90% effective concentration (EC90) of 4 microM. MPA along with several analogues, including both its corresponding aldehyde and alcohol, and modifications in the length of the side chain (C2- and C4-derivatives) were tested. We have identified previously unreported IMPDH inhibitors that have potent anti-BVDV activity, namely: C6-MPAlc (5), C6-MPA-Me (7), C4-MPAlc (8), C4-MPA (10) and C2-MAD (20). Most of these compounds inhibited the IMPDH enzyme in the nanomolar range (4-800 nM) in cell-free assays. Some compounds, such as mizoribine, which is a potent inhibitor of IMPDH in vitro (enzyme 50% inhibitory concentration IC50=4 nM), had no detectable anti-BVDV activity up to 100 microM. The compounds were essentially non-toxic to a confluent monolayer of MDBK cells. However, in exponentially growing cells, they showed minimal toxicity at 100 microM over a 24 h period, but the toxicity was more pronounced after 3 days [50% cytotoxic concentration (CC50) value ranged from 5 to 30 microM].

Topics: Animals; Cattle; Cell Line; Computer Systems; Culture Media, Conditioned; Diarrhea Viruses, Bovine Viral; Dose-Response Relationship, Drug; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; Guanosine Triphosphate; IMP Dehydrogenase; Kidney; Molecular Structure; Mycophenolic Acid; NAD; Nucleosides; Reverse Transcriptase Polymerase Chain Reaction; Ribavirin; Ribonucleosides; Viral Plaque Assay; Virus Replication

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

Benzamide riboside (3-(1-deoxy-beta-D-ribofuranosyl)benzamide, BR) a new analog of nicotinamide riboside, is toxic to Chinese hamster ovary cells and inhibits guanine nucleotide synthesis in a manner comparable to that of tiazofurin (2-beta-D-ribofuranosylthiazole-4-carboxamide). Adenosine kinase deficient cells demonstrate slight resistance but retain the ability to form the NAD analog, benzamide adenine dinucleotide (BAD). HPLC analysis of BAD containing cells is described. A BR resistant cell line was isolated that demonstrates cross-resistance to both tiazofurin and 6-aminonicotinamide, suggesting a common metabolic step; enzymatic analysis indicates reduced levels of NAD pyrophosphorylase in these cells. BR toxicity was only partially reversed or prevented by the presence of guanosine, suggesting either that BR inhibits guanine salvage to some extent or, more probably, that BR can, at high concentration, inhibit cell growth by another mechanism in addition to inhibition of guanine nucleotide synthesis. Cells incubated with BR for several hours retain the ability to salvage exogenously provided guanosine. The demonstration that BAD can be phosphorylated by NAD kinase, presumably to form BADP, suggests that this metabolite may be formed in cells and may have inhibitory activity at high concentrations of BR.

Topics: Animals; Antineoplastic Agents; CHO Cells; Cricetinae; Drug Resistance; Enzyme Inhibitors; NAD; Nucleosides; Phosphorylation; Ribavirin

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

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.

Topics: Antineoplastic Agents; Cell Line; Cell Survival; Dose-Response Relationship, Drug; Humans; IMP Dehydrogenase; Kinetics; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Molecular Structure; Nucleosides; Ribavirin; Ribonucleotides