Cytotoxicity and characterization of an active metabolite of benzamide riboside, a novel inhibitor of IMP dehydrogenase.
Benzamide riboside exhibits significant cytotoxicity against a variety of human tumor cells in culture. On the basis of metabolic studies, the primary target of this drug's action appears to be IMP dehydrogenase (IMPDH). Incubation of human myelogenous leukemia K562 cells with an IC50 concentration of benzamide riboside resulted in an expansion of IMP pools (5.9-fold), with a parallel reduction in the concentration of GMP (90%), GDP (63%), GTP (55%) and dGTP (40%). On kinetic grounds, it was deduced that benzamide riboside (whose Ki versus IMPDH is 6.4 mM, while that of its 5'-monophosphate is 3.9 mM) or its 5'-monophosphate were unlikely to be responsible for inhibition of this target enzyme, IMPDH, since only micromolar concentrations of benzamide riboside were needed to exert potent inhibition of tumor-cell growth. Studies on the metabolism of this C-nucleoside have revealed the presence of a new peak eluting in the nucleoside diphosphate area on HPLC. Treatment of this peak with venom phosphodiesterase degraded it and concurrently nullified its inhibitory activity versus IMPDH; alkaline phosphatase, on the other hand, totally failed to digest the anabolite. These results suggest that the metabolite in question is the phosphodiester, benzamide adenine dinucleotide (BAD). Evidence that the inhibitor was an analog of NAD, wherein the nicotinamide moiety has been replaced by benzamide, was provided by both NMR and mass spectrometric analysis and confirmed by enzymatic synthesis. Further insight into the nature of the active principle was obtained from kinetic studies, which established that BAD competitively inhibited NAD utilization by partially purified IMPDH from K562 cells with a Ki of 0.118 microM. In concert, these studies establish that benzamide riboside exhibits potent antiproliferative activity by inhibiting IMPDH through BAD.
Gharehbaghi
K
K
Laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis 46202-5200.
Paull
K D
KD
Kelley
J A
JA
Barchi
J J
JJ
Marquez
V E
VE
Cooney
D A
DA
Monks
A
A
Scudiero
D
D
Krohn
K
K
Jayaram
H N
HN
D002289:Carcinoma, Non-Small-Cell Lung / Q000188:drug therapy; D003110:Colonic Neoplasms / Q000188:drug therapy; D004354:Drug Screening Assays, Antitumor; D006151:Guanosine / Q000008:administration & dosage / Q000494:pharmacology; D006801:Humans; D007168:IMP Dehydrogenase / Q000037:antagonists & inhibitors*; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000378:metabolism; D008175:Lung Neoplasms / Q000188:drug therapy; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009705:Nucleosides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D009711:Nucleotides / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D014407:Tumor Cells, Cultured
D009705:Nucleosides; D009711:Nucleotides; D012265:Ribonucleotides; D006151:Guanosine; C076145:3-(1-deoxyribofuranosyl)benzamide; D007168:IMP Dehydrogenase