Targets and markers of selective action of tiazofurin.

Abstract 1. 1. The molecular correlation concept proposed that IMP dehydrogenase activity should be a sensitive target of chemotherapy. This hypothesis received support from an array of evidence. IMP dehydrogenase has the lowest activity in purine biosynthesis; it is the rate-limiting enzyme in GTP production; the enzymic activity is transformation- and progression-linked; it is elevated in all examined animal and human neoplastic cells. The activity of GMP synthetase and the concentrations of GMP and dGTP were increased in cancer cells. Whereas guanine salvage has a high potential activity, the low guanine content may well curtail actual salvage capacity. Ribonucleotide reductase activity was two orders of magnitude lower than that of IMP dehydrogenae. 2. 2. Tiazofurin, a C-nucleoside, had marked cytotoxicity on hepatoma cells in vitro and was the first drug that as a single agent profoundly inhibited the proliferation of the subcutaneously inoculated solid hepatoma 3924A in the rat. 3. 3. The impact of tiazofurin administration in hepatoma cells was revealed in a cascade of biochemical alterations involving primary, secondary and tertiary targets and markers of this drug action. The primary target was IMP dehydrogenase where the active metabolite of tiazofurin, TAD, was thought to be absorbed to the NADH site of the enzyme. As a consequence, the enzymic activity declined rapidly to about 30–40% and returned to normal range by 36 to 48 hr after injection. The secondary targets and markers are the profoundly decreased pools of guanylates (GMP, GDP, GTP). Concurrently, the concentrations of IMP and PRPP were increased 8- to 15-fold. The elevated IMP pools were attributed to the de-inhibition of the AMP deaminase activity subsequent to the decline in GTP concentration. The rise in PRPP pools was attributed to the selective inhibition of GPRT and HPRT activities by the high IMP pool which did not affect APRT activity. This interpretation is supported by the 6- to 8-fold increase in the concentrations of guanine and hypoxanthine and the lack of change in the adenine pools in the hepatomas after tiazofurin administration. The marked drop in NAD concentration which was drug dose- and time-dependent is attributed to the competition for NAD pyrophosphorylase activity by the precursors of NAD and tiazofurin monophosphate. The tertiary targets were dominated by the profound alterations in the concentrations of the dNTPs. This was characterized by a rapid and persistent drop (for 3 days) of the dGTP pool. The concentrations of dATP and dCTP also declined, but these alterations were less pronounced and the pools returned to normal after 2 days. In contrast, dTTP concentrations were continuously increased. The decline in the dNTP pool is attributed to desaturation by the decreased GDP concentration of ribonucleotide reductase. The increased dTTP concentration is attributed to the thymidine kinase activity which is highly elevated in the hepatoma. This interpretation is supported by the presence of markedly enlarged pools of thymidine and thymine in the hepatomas of tiazofurin-treated rats. 4. 4. The determinants of selectivity of tiazofurin action in hepatoma and liver entail the high concentrations of TAD observed in the hepatoma after tiazofurin injection. This is attributed to the high ratios of the activities of NAD pyrophosphorylase/phosphodiesterase in the tumors. Moreover, the hepatomas had low concentrations of NAD which decreased further after tiazofurin injection; there was no change in the liver. The IMP concentration in the hepatoma was markedly lower than that of the liver. The biochemical response to tiazofurin was much more pronounced in the hepatoma, whereas in the liver there was no change in the concentrations of guanylates and only a minor increase in IMP concentration. 5. 5. The results emphasize the differences in the biochemical phenotypes and responsiveness to tiazofurin of sensitive and non-sensitive tissues. The identification to tiazofurin of sensitve and non-sensitive tissues. The identification of targets and markers of tiazofurin suggest that for rapid screening of tiazofurin sensitivity the most meaningful indicators should be the drug-induced elevations in the concentrations of TAD and in the ratios of IMP/GTP or IMP/dGTP. Further indicators include a markedly depressed NAD concentration and a strongly elevated PRPP pool in the sensitive tissues after tiazofurin treatment.