SPECIES-RELATED INHIBITION OF HUMAN AND RAT DIHYDROOROTATE DEHYDROGENASE BY IMMUNOSUPPRESSIVE ISOXAZOL AND CINCHONINIC ACID-DERIVATIVES

The isoxazol leflunomide luoromethylphenyl)-5-methylisoxazol-4-carboxa mide) and its active metabolite A77-1726 (N-(4-trifluoromethyl)-phenyl -2-cyano-3 -hydroxy-crotonic acidamide) are promising disease-modifyin g antirheumatic drugs now in clinical trials. The malononitrilamides M NA279 o-3-cyclopropyl-3-oxo-(4-cyanophenyl)propionamide) phenyl-2-cyan o-3-hydroxy-hept-2-en-6-in-carboxylic acidamide) and HR325 enyl-carbam oyl)-2-cyclopropyl-2-oxo-propionitrile) were shown to block rejection after allograft and xenograft transplantation in animals. Brequinar an d other cinchoninic acid derivatives have also been evaluated as immun e-suppressive agents. A77-1726, HR325 and brequinar have been shown to have strong inhibitory effects on mitochondrial dihydroorotate dehydr ogenase [EC 1.3.99.11], the fourth enzyme of pyrimidine de novo synthe sis, with concomitant reduction of pyrimidine nucleotide pools. Pyrimi dine nucleotides are essential for normal immune cell functions. Becau se most investigations had been carried out with cells, cell homogenat es or mitochondrial fractions, it was the rationale of the present stu dy to differentiate, under standardized conditions, the effect of lefl unomide, A77-1726, MNA279, MNA715, HR 325 and brequinar on the recombi nant rat and human enzymes, which were purified in our laboratory. Whe reas leflunomide was a relatively weak inhibitor of the rat (IC50 = 6. 3 mu M) and human (IC50 = 98 mu M) dihydroorotate dehydrogenase, the i nfluence of A77-1726, MNA 279, MNA715 and HR325 was of comparable effi cacy for either the rat (range of IC50, 19-53 nM) or the human enzyme (range of IC50, 0.5-2.3 mu M) From the IC50 values, it was deduced tha t brequinar was a more potent inhibitor of the human dihydroorotate de hydrogenase activity (IC50 = 10 nM) than of the rat enzyme (IC50 = 367 nM). The rat enzyme was influenced by all isoxazol derivatives to a g reater extent (IC50 = 19 nM A77-1726) than the human enzyme (IC50 = 1. 1 mu M A77-1726). These results may provide a plausible explanation fo r the findings of other laboratories with cultured cell lines and lymp hocytes: in comparison to cells derived from human tissues, rat and ot her 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 dehydrogenas e revealed a noncompetitive type of inhibition by A77-1726 with respec t to the substrate dihydroorotate and the cosubstrates ubiquinone or d ecylubiquinone. For brequinar, the inhibition was noncompetitive with respect to the substrate dihydroorotate, whereas with the quinone it w as found to follow the ''mixed typed'' inhibition. In addition, brequi nar acted as a ''slow-binding'' inhibitor of the human dihydroorotate dehydrogenase, a feature that might be of consequence for the reversib ility of the reaction with the target. (C) 1998 Elsevier Science Inc.