Inhibition of pyrimidine de novo synthesis by DUP-785 (NSC 368390)

Abstract

The mechanism of action of NSC 368390 (DUP-785, 6-fluoro-2-(2'-fluoro-1, 1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt) was studied using three different approaches. First, we studied growth inhibition by DUP-785 in L1210 leukemia cells and M5 melanoma cells. The concentrations causing 50% growth inhibition after 48 hr of culture were 5.8 and 0.6 microM, respectively. DUP-785 had to be present continuously throughout culture. Growth inhibition by 25 microM DUP-785 could be prevented by addition of 1 mM uridine or orotic acid to cultures of these cell lines; in M5 cells cytidine was also able to prevent growth inhibition. Dihydro-orotic acid (DHO) and carbamyl-aspartate were not able to prevent growth inhibition by DUP-785. Second, we studied accumulation of orotic acid and of orotidine induced by incubation with 1 microM pyrazofurin, an inhibitor of the orotate phosphoribosyl-transferase-orotidine-monophosphate decarboxylase complex. Addition of DUP-785 to the culture medium prevented the orotic acid accumulation. Furthermore, DUP-785 prevented accumulation of H14CO3- into orotic acid of pyrazofurin-treated L1210 cells. Third, we measured the effect of DUP-785 on DHO-dehydrogenase (DHO-DH), since the results indicated that this enzyme was affected by DUP-785. DHO-DH was assayed in isolated rat liver mitochondria. The Km for L-DHO was about 12 microM. DUP-785 appeared to be a potent inhibitor of DHO-DH with an apparent Ki of about 0.1 microM and an apparent Ki' of about 0.8 microM. The mode of inhibition appeared to be linear mixed type. After exposure of L1210 cells to 25 microM DUP-785 for 2 hr DHO-DH was almost completely inhibited. After suspension in fresh medium without drug, DHO-DH activity was recovered to about 60% after 24 hr. In conclusion, DUP-785 is a potent inhibitor of pyrimidine de novo biosynthesis, by inhibition of the mitochondrial enzyme DHO-DH.

In vitro and in vivo studies on the combination of Brequinar sodium (DUP-785; NSC 368390) with 5-fluorouracil; effects of uridine

“…Biochemical effects of Brequinar at the dose range from 600-2,250 mg m-2 (Peters et al, 1990a) were related to the toxicity of the drug and consisted of the inhibition of DHO-DH and a depletion of pyrimidine nucleotides in lymphocytes of the patients; in plasma uridine levels decreased followed by a rebound. Biochemical effects observed in patients were comparable to those in mice and in vitro (Peters et al, 1987a;1990b;Schwartsmann et al, 1988). Drug exposure resulted in accumulation of cells in the S phase (Schwartsmann et al, 1988).…”

“…Theoretically Brequinar would be an ideal compound to modulate the effects of 5FU, as the drug is a very potent inhibitor of the pyrimidine de novo nucleotide synthesis (Chen et al, 1986;Peters et al, 1987a). A fundamental requirement for in vivo application of biochemical modulation is a clear-cut demonstration of the biochemical effects, which has been fulfilled in the case of Brequinar (Peters et al, 1987a(Peters et al, , 1990bSchwartsmann et al, 1988). However, physiological concentrations of uridine can already prevent growth-inhibition by Brequinar, but only at concentrations lower than 30 ILM Brequinar.…”

“…Drug exposure resulted in accumulation of cells in the S phase (Schwartsmann et al, 1988). Growth-inhibitory effects of Brequinar could be prevented and reversed by addition of uridine or cytidine (Peters et al, 1987a;Schwartsmann et al, 1988), but not by thymidine or deoxycytidine. Depletion of pyrimidine deoxyribonucleotides was proportional to that of the ribonucleotides (Schwartsmann et al, 1988), while both could be reversed by uridine.…”

“…Brequinar Sodium (Brequinar; DUP-785; NSC 368390) is a potent inhibitor of dihydroorotic acid dehydrogenase (DHO-DH), the fourth enzyme in the de novo pyrimidine nucleotide synthesis (Figure 1) which is located on the outer site of the inner membrane of the mitochondrion (Chen et al, 1986;Peters et al, 1987a (Peters et al, 1987a;1990b;Schwartsmann et al, 1988). Drug exposure resulted in accumulation of cells in the S phase (Schwartsmann et al, 1988).…”

“…plasma (Darnowski et al, 1986;Peters et al, 1987b); this might affect the antitumour activity of Brequinar. In vitro this is reflected by the higher sensitivity of cell lines cultured in dialysed serum compared to culturing in non-dialysed serum (Peters et al, 1987a), which contains a considerable amount of uridine. Since Brequinar can decrease pyrimidine nucleotides both in vitro and in vivo (Chen et al, 1986;Peters et al, 1990b;Schwartsmann et al, 1988;Anderson et al, 1989), the compound theoretically can enhance the activity of 5FU.…”