2-cyano-3-cyclopropyl-3-hydroxy-n-(3--methyl-4--(trifluoromethyl)phenyl)propenamide and brequinar

The objective was to demonstrate that the immunosuppressive agent HR325 (an inhibitor of dihydroorotate dehydrogenase, DHODH) inhibits immunoglobulin (Ig) secretion both in vitro and in vivo and that this effect can be reversed with exogenous uridine. In vitro, Ig secretion from mouse splenocytes was induced by lipopolysaccharide (LPS) for 5 days. HR325 inhibited the secretion of IgM and IgG with IC50 values of 2.5 and 2 microm, respectively. Adding uridine (50 microm) increased these values to 70 and 60 microm, respectively. Similarly, the IC50 values of another DHODH inhibitor, brequinar sodium, were also attenuated by uridine from 0.04 to 1 microm for IgM, and 0.012 to 10 microm for IgG. HR325 (and a structural analogue A771726) inhibited LPS-induced kappa light-chain cell surface expression on 70Z/3 cells, a property also reversed by uridine. In vivo, the secondary anti-sheep red blood cell (SRBC) antibody response (unaffected by uridine alone) was inhibited by HR325 and brequinar with respective ID50 values of 38 and 0.6 mg/kg per oral (p.o.). Immunosuppression with HR325 (50 mg/kg) and brequinar (1 mg/kg) was abrogated by uridine. Uridine had no effect on cyclophosphamide-induced (10 mg/kg p.o.) immunosuppression. These data are consistent with the immunosuppressive mechanism of HR325 being the result of pyrimidine depletion in vitro and in vivo.

Topics: Aniline Compounds; Animals; Biphenyl Compounds; Cell Membrane; Cells, Cultured; Crotonates; Dihydroorotate Dehydrogenase; Drug Antagonism; Enzyme Inhibitors; Erythrocytes; Hydroxybutyrates; Immunoglobulin G; Immunoglobulin kappa-Chains; Immunoglobulin M; Immunoglobulins; Immunosuppressive Agents; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Nitriles; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Pyrimidines; Sheep; Spleen; Toluidines; Uridine

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 microM 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 microM), with the exception of HR325, 10 microM 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

Topics: Aniline Compounds; Animals; Biphenyl Compounds; Crotonates; Dihydroorotate Dehydrogenase; Electron Transport; Hydroxybutyrates; Immunosuppressive Agents; Male; Mitochondria, Liver; Models, Molecular; NAD; Nitriles; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Oxygen Consumption; Rats; Rats, Wistar; Structure-Activity Relationship; Succinic Acid; Toluidines