Differential susceptibility of dihydroorotate dehydrogenase/oxidase to Brequinar Sodium (NSC 368 390) in vitro.
To verify the assumption of a specific and potent drug action on de novo pyrimidine biosynthesis, isolated dihydroorotate dehydrogenase (DHO-DH) (EC 1.3.3.1) was exposed to Brequinar Sodium (6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinoline carboxylic acid sodium salt, NSC 368 390) (Brequinar). The membrane-bound DHO-DH was purified to apparent homogeneity (25,000-fold) from rat liver mitochondria in six steps via detergent extraction and subsequent chromatography using the dye ligand Matrex Gel Orange A. Using molecular mechanistic studies (MM2) this ligand was found to mimic closely the stereochemical conformation of Brequinar. SDS-PAGE revealed two protein bands for the purified enzyme with apparent molecular masses of 58 (major) and 68 kDa (minor). In vitro, two modes of action of the DHO-DH are possible: (i) acting as a dehydrogenase in the presence of ubiquinone as proximal electron acceptor and (ii) direct reaction with oxygen as oxidase. A novel assay for the measurement of the oxidase activity was adapted using leuco-dichlorofluorescein-diacetate. Inhibition experiments revealed a striking difference in the susceptibility of DHO-dehydrogenase/oxidase to Brequinar: apparent Ki = 6.09 +/- 0.05 (SD) nM (DHO; ubiquinone n = 10), but Ki = 3.10 +/- 0.09 (SD) mM (DHO; O2). Analyses of initial velocity experiments showed non-competitive inhibition of Brequinar with respect to the substrate dihydroorotic acid in both assays (dehydrogenase and oxidase). The inhibitory effect of the latter was compared to that of the competitive inhibitor 5-aza-dihydroorotate (apparent Ki = 15 +/- 0.25 (SD) microM). The present kinetic data on the action of the purified rodent DHO-DH with Brequinar and computer-aided analyses provide a better insight into the drug-enzyme interaction.
Lakaschus
G
G
Department of Physiological Chemistry, School of Medicine, Philipps-University of Marburg, Federal Republic of Germany.
Löffler
M
M
D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology*; D004081:Dihydroorotate Oxidase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology; D008956:Models, Chemical; D008958:Models, Molecular; D051381:Rats; D013329:Structure-Activity Relationship; D011798:Quinaldines
D001713:Biphenyl Compounds; D004081:Dihydroorotate Oxidase; C046943:brequinar; D011798:Quinaldines