Structural and functional comparison of agents interfering with dihydroorotate, succinate and NADH oxidation of rat liver mitochondria

Abstract Mitochondrially bound dihydroorotate dehydrogenase (EC 1.3.99.11) catalyses the fourth sequential step in the de novo synthesis of uridine monophosphate; this enzyme uses ubiquinone as the proximal and cytochrome oxidase as is the ultimate electron transfer system. Here, seven compounds with proven antiproliferative activity and in vitro antipyrimidine effects were investigated with isolated functional mitochondria of rat tissues in order to differentiate their anti-dihydroorotate dehydrogenase potency versus putative effects on the respiratory chain enzymes. Ten μM of brequinar sodium, the leflunomide derivatives A77–1726, [2-cyano-3-cyclopropyl-3-hydroxy-enoic acid (4-trifluoromethylphenyl)-amide], MNA 279, (2-cyano- N -(4-cyanophenyl)-3-cyclopropyl-3-oxo-propanamide), MNA715 (2-cyano-3-hydroxy- N -(4-(trifluoromethyl)-phenyl-6-heptanamide), HR325 (2-cyano-3-cyclopropyl-3-hydroxy- N -[3′-methyl-4′-(trifluoromethyl)phenyl]-propenamide), and the diazine toltrazuril completely inhibited the dihydroorotate-induced oxygen consumption of liver mitochondria. Succinate and NADH oxidation were found to be influenced only at elevated drug concentration (100 μM), with the exception of HR325, 10 μM of which caused a 70% inhibition of NADH and 50% inhibition of succinate oxidation. This was comparable to the effects of toltrazuril, which caused an approximate 75% inhibition of NADH oxidation. Ciprofloxacin was shown here to have only marginal effects on the redox activities of the inner mitochondrial membrane. This differentiation of drug effects on mitochondrial functions will contribute to a better understanding of the in vivo pharmacological activity of these drugs, which are presently in clinical trials because of their immunosuppressive, cytostatic or anti-parasitic activity. A comparison of the influence of A77-1726, HR325, brequinar and 2,4-dinitrophenol on energetically coupled rat liver mitochondria revealed only a weak uncoupling potential of A77-1726 and brequinar. In addition, a modeling study was raised to search for common spatial arrangements of functional groups essential for binding of inhibitors to dihydroorotate dehydrogenase. From the structural comparison of different metabolites and inhibitors of pyrimidine metabolism, a 6-point model was obtained by conformational analysis for the drugs tested on mitochondrial functions, pharmacophoric perception and mapping. We propose our model in combination with kinetic data for a rational design of highly specific inhibitors of dihydroorotate dehydrogenase.