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nicotinamide moiety has been replaced by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.