SITE OF ACTION OF 2 NOVEL PYRIMIDINE BIOSYNTHESIS INHIBITORS ACCURATELY PREDICTED BY THE COMPARE PROGRAM

The computer algorithm COMPARE provides information regarding the biological mechanism of action of a compound. In this study, excellent correlations were obtained for 2,2'-[3,3'-dimethoxy[1,1'-biphenyl]-4,4'-diyl)diimino]bis-benzoic acid (redoxal) and 1-(p-bromophenyl)-2-methyl-1H-naphth[2,3-d]imidazole-4,9-dione (BNID) and two well-studied dihydroorotate dehydrogenase (DHOD) inhibitors, dichloroallyl lawsone and brequinar, in terms of antiproliferative activity against tumor cell lines in vitro. When redoxal and BNID were incubated with MOLT-4 cells for 72 hr, 50% growth inhibition was achieved at 0.7 and 3.5 mu M, respectively. After 24 hr of incubation, pyrimidine triphosphate pools were shown to be decreased by 50% by redoxal (1 mu M) and BNID (0.25 mu M) Addition of either uridine (50 mu M) or cytidine (100 mu M) antagonized the cellular cytotoxicity caused by either drug; uridine corrected the UTP and CTP deficit, whereas cytidine corrected only the CTP deficit. Exposure of MOLT-4 cells to a 1 mu M concentration of either drug for 18 hr followed by a 1-hr exposure to [C-14]bicarbonate showed a 97% decrease of incorporation of [C-14] into pyrimidine triphosphates accompanied by a 91- and 82-fold increase in radioactive incorporation into L-dihydroorotate and N-carbamyl-L-aspartate, respectively. By direct exposure of DHOD prepared from MOLT-4 cell mitochondria to a range of concentrations of the two drugs, apparent K-i values of 0.33 mu M (redoxal) and 0.53 mu M (BNID) were determined. These data provide direct evidence for inhibition of DHOD by redoxal and BNID in MOLT-4 lymphoblasts.