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.