3-(1-deoxyribofuranosyl)benzamide and Neoplasms

Benzamide riboside (BR) is a nucleoside prodrug that is phosphorylated to its 5'-monophosphate (BRMP) and then converted to its active metabolite, BAD (benzamide adenine dinucleotide), an analogue of NAD by the action of NMN adenylyltransferase (NMNAT). BAD is a potent, reversible, and noncompetitive inhibitor of inosine 5'-monophosphate dehydrogenase (IMPDH) resulting in depletion of guanylates (GTP and dGTP). IMPDH inhibitors such as BR induce differentiation and apoptosis as a consequence of GTP depletion. Tiazofurin (TR) and selenazofurin (SR) require similar metabolism by NMNAT. NMNAT is the rate-limiting step in the synthesis of NAD and NAD analogues. BR- and TR-sensitive leukemic cells contain high NMNAT activity, whereas resistant clones have greatly downregulated NMNAT activity (<0.1% of wild type). Perhaps the applicability of BR and analogues could be enhanced if combined with NMNAT gene expression in BR-resistant leukemic blasts. NAD has important regulatory role in repair of DNA damage and cell growth since it is a substrate for poly(ADP-ribose) polymerase (PARP). PARP appears to direct short-patch base excision repair and induce p53 upregulation leading to apoptosis. BR inhibits PARP at high concentrations when assayed in permeabilized leukemic cells. Several other IMPDH inhibitors (TR, mycophenolic acid, and ribavirin) exhibit similar PARP inhibitory activity. Although this inhibition was reversible, it was not prevented by the addition of guanosine, GTP, or its nonhydrolyzable analog gamma-S-GTP. Therefore, it can be concluded that IMPDH inhibitors directly inhibit PARP. Presumably, the shared IMP-NAD active site of IMPDH has a similar architecture to the NAD-binding pocket of PARP.

Topics: Antineoplastic Agents; Apoptosis; Cell Differentiation; Cell Survival; Enzyme Inhibitors; Guanosine Triphosphate; Humans; IMP Dehydrogenase; Neoplasms; Nicotinamide-Nucleotide Adenylyltransferase; Nucleosides; Tumor Cells, Cultured

Benzamide riboside (BR) after anabolism to an analogue of NAD, was shown to inhibit the activity of NAD-dependent enzymes such as inosine 5'-monophosphate dehydrogenase (IMPDH), the rate limiting enzyme in de novo guanylate biosynthesis, and malate dehydrogenase which is involved in the citric cycle and respiratory chain. BR exhibits strong anti-carcinogenic effects due to growth retardation and due to induction of apoptosis and necrosis. Apoptosis is ascribed to the inhibition of IMPDH because cell death can be blocked by restoring intracellular guanylate metabolism by the addition of guanosine. It is shown here, however, that also survival-relevant genes such as cdc25A, akt, bcl-2 and transferrin receptor become repressed by BR, whereas the expression level of the apoptosis enforcing gene c-myc persists. Even though BR-mediated growth retardation still allows BR to induce apoptosis, rapamycin-mediated cell cycle block and cell contact inhibition prevent cell death, it strongly suggests that BR induces a type of c-Myc-dependent apoptosis. At high concentrations BR induces DNA double strand breaks by yet to be determined mechanisms that occur hours before necrosis can be detected. This is accompanied by a dramatic decrease of intracellular ATP. The artificial restoration of ATP by addition of adenosine or sufficient provision of an energy source such as glucose prevents BR-promoted necrosis and favors apoptosis. This observation may be of clinical relevance.

Topics: Apoptosis; Cell Division; DNA Damage; Enzyme Inhibitors; Female; Gene Expression; Glucose; Guanosine Triphosphate; HL-60 Cells; Humans; IMP Dehydrogenase; Necrosis; Neoplasms; Nucleosides; 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