Cytotoxicity and characterization of an active metabolite of benzamide riboside, a novel inhibitor of IMP dehydrogenase

Abstract

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 Evidence that the inhibitor was an analog of NAD, wherein the nicotinamide moiety has been replaced by benzamide, was provided by both NMR and mass spectrometric analysis and confirmed by enzymatic synthesis.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.

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside

“…13,14,40 The oncolytic activity of BR 3 was assumed to be due to its IMPDH-inhibitory and, therefore, dGTP-limiting action. 2,12 This hypothesis was strongly encouraged by the observation, that guanosine, a precursor of dGTP, prevented the oncolytic activity of BR. 12,41 Our findings show, that 5 mM BR induced apoptosis, whereas 20 mM BR provoked necrosis, although both concentrations of BR inhibited dGTP synthesis to a similar degree.…”

“…9 ± 11 BR, exhibited stronger antiproliferative activity in the K562 cells than TR 12 and was shown to induce apoptosis in HL-60 13 and N.1 ovarian carcinoma cells. 13,14 Higher BR-concentrations however, provoked necrosis, which is a common phenomenon of pro-apoptotic drugs 15 ± 17 and limits chemotherapy because of non-specific drug toxicity.…”

Maintenance of ATP favours apoptosis over necrosis triggered by benzamide riboside

“…7 TR is an inhibitor of IMPDH 8 and Phase I/II clinical trials conducted with this compound in acute myelogenous leukaemia patients, indicated a significant reduction in leukaemic cell burden. 9 ± 11 BR, exhibited stronger antiproliferative activity in the K562 cells than TR 12 and was shown to induce apoptosis in HL-60 13 and N.1 ovarian carcinoma cells. 13,14 Higher BR-concentrations however, provoked necrosis, which is a common phenomenon of pro-apoptotic drugs 15 ± 17 and limits chemotherapy because of non-specific drug toxicity.…”

“…17 ± 19 It was postulated that BR exerts its anti-tumour effects due to IMPDH inhibition. 2,12 Therefore dGTP and other dNTP levels were analyzed and correlated with cell death modes. The necrotic trigger of high BR-concentrations was identified as DNA-clastogenic activity, which subsequently led to ATP depletion.…”

Benzamide riboside induces apoptosis independent of Cdc25A expression in human ovarian carcinoma N.1 cells

“…10,11 BR exhibited stronger antiproliferative activity in the K562 cells than TR. 12 The two nucleoside antimetabolites, TR and BR are converted in cancer cells to their active dinucleotide metabolites, TAD (thiazole-4-carboxamide adenine dinucleotide) and BAD (benzamide adenine dinucleotide), which are analogues of NAD, wherein the nicotinamide moiety is replaced by their respective bases. 11,13 BAD potently inhibits NAD utilization by IMPDH, resulting in the depletion of intracellular guanylate concentrations including GTP and dGTP.…”