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, t he primary target of this drug's action appears to be IMP dehydrogenas e (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 o f GMP (90%), GDP (63%), GTP (55%) and dGTP (40%). On kinetic grounds, it was deduced that benzamide riboside (whose K-i versus IMPDH is 6.4 mM, while that of its 5'-monophosphate is 3.9 mM) or its 5'-monophosph ate were unlikely to be responsible for inhibition of this target enzy me, IMPDH, since only micromolar concentrations of benzamide riboside were needed to exert potent inhibition of tumor-cell growth. Studies o n 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 concurrentl y nullified its inhibitory activity verses IMPDH; alkaline phosphatase , on the other hand, totally failed to digest the anabolite. These res ults suggest that the metabolite in question is the phosphodiester, be nzamide adenine dinucleotide (BAD). Evidence that the inhibitor was an analog of NAD, wherein the nicotinamide moiety has been replaced by b enzamide, was provided by both NMR and mass spectrometric analysis and confirmed by enzymatic synthesis.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 establis hed that BAD competitively inhibited NAD utilization by partially puri fied IMPDH from K562 cells with a K-i of 0.118 mu M. In concert, these studies establish that benzamide riboside exhibits potent antiprolife rative activity by inhibiting IMPDH through BAD. (C) 1994 Wiley-Liss, Inc.