Species-related inhibition of human and rat dihydroorotate dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives

Abstract

The isoxazol leflunomide (N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide) and its active metabolite A77-1726 (N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-crotonic acidamide) are promising disease-modifying antirheumatic drugs now in clinical trials. The malononitrilamides MNA279 (2-cyano-3-cyclopropyl-3-oxo-(4-cyanophenyl)propionamide) MNA715(N-(4-trifluoromethyl)-phenyl-2-cyano-3-hydroxy-hept-2-en-6-in-carboxylic acidamide) and HR325 (1(3-methyl-4-trifluoromethylphenyl-carbamoyl)-2-cyclopropyl-2-oxo-propionitrile) were shown to block rejection after allograft and xenograft transplantation in animals. Brequinar and other cinchoninic acid derivatives have also been evaluated as immuno-suppressive agents. A77-1726, HR325 and brequinar have been shown to have strong inhibitory effects on mitochondrial dihydroorotate dehydrogenase [EC 1.3.99.11], the fourth enzyme of pyrimidine de novo synthesis, with concomitant reduction of pyrimidine nucleotide pools. Pyrimidine nucleotides are essential for normal immune cell functions. Because most investigations had been carried out with cells, cell homogenates or mitochondrial fractions, it was the rationale of the present study to differentiate, under standardized conditions, the effect of leflunomide, A77-1726, MNA279, MNA715, HR 325 and brequinar on the recombinant rat and human enzymes, which were purified in our laboratory. Whereas leflunomide was a relatively weak inhibitor of the rat ( ic 50 = 6.3 μM) and human ( ic 50 = 98 μM) dihydroorotate dehydrogenase, the influence of A77-1726, MNA 279, MNA715 and HR325 was of comparable efficacy for either the rat (range of ic 50, 19–53 nM) or the human enzyme (range of ic 50, 0.5–2.3 μM). From the ic 50 values, it was deduced that brequinar was a more potent inhibitor of the human dihydroorotate dehydrogenase activity ( ic 50 = 10 nM) than of the rat enzyme ( ic 50 = 367 nM). The rat enzyme was influenced by all isoxazol derivatives to a greater extent ( ic 50 = 19 nM A77-1726) than the human enzyme ( ic 50 = 1.1 μM A77-1726). These results may provide a plausible explanation for the findings of other laboratories with cultured cell lines and lymphocytes: in comparison to cells derived from human tissues, rat and other rodent cells were more susceptible to the isoxazol derivatives and less susceptible to brequinar. Our detailed kinetic investigations of the bisubstrate reaction catalyzed by rat dihydroorotate dehydrogenase revealed a noncompetitive type of inhibition by A77-1726 with respect to the substrate dihydroorotate and the cosubstrates ubiquinone or decylubiquinone. For brequinar, the inhibition was noncompetitive with respect to the substrate dihydroorotate, whereas with the quinone it was found to follow the “mixed typed” inhibition. In addition, brequinar acted as a “slow-binding” inhibitor of the human dihydroorotate dehydrogenase, a feature that might be of consequence for the reversibility of the reaction with the target.