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Monika Loffler - One of the best experts on this subject based on the ideXlab platform.
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Dihydroorotate Dehydrogenase mrna and protein expression analysis in normal and drug resistant cells
Nucleosides Nucleotides & Nucleic Acids, 2004Co-Authors: Monika Loffler, Wolfgang Knecht, A Klein, M Hayekouassini, L KonradAbstract:To follow the expression of the fourth enzyme of pyrimidine de novo synthesis Dihydroorotate Dehydrogenase (DHODH) in cells and tissues, we studied the DHODH mRNA expression by means of RT-PCR in rat tissues. Rabbit polyclonal anti-DHODH immunoglobulins were applied for immunochemical quantification of the enzyme protein by Western blotting. In mouse B-lymphocytes, which were adapted to tolerate up to a 50-fold concentration of the DHODH inhibitor leflunomide, a 20 fold protein overexpression was measured. Southern blotting indicated DHODH gene amplification.
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Synthesis of β-hydroxy-propenamide derivatives and the inhibition of human Dihydroorotate Dehydrogenase
Archives of Pharmacal Research, 2003Co-Authors: Taek Hyeon Kim, Monika LofflerAbstract:Novel β-hydroxy propenamides as analogues of the active metabolite of leflunomide (A 771726) were synthesized and evaluated for their inhibitory activity on Dihydroorotate Dehydrogenase (DHODH) in an investigation into their immunosuppressive activity. Compounds 2a, 3a , and 3h were approximately 4–40 times more potent than leflunomide in their activity while they wereless active than A 771726.
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Plant Dihydroorotate Dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes.
FEBS letters, 2002Co-Authors: Alexandra Ullrich, Wolfgang Knecht, Jure Piskur, Monika LofflerAbstract:The mitochondrial membrane bound Dihydroorotate Dehydrogenase (DHODH; EC 1.3.99.11) catalyzes the fourth step of pyrimidine biosynthesis. By the present correction of a known cDNA sequence for Arabidopsis thaliana DHODH we revealed the importance of the very C-terminal part for its catalytic activity and the reason why--in contrast to mammalian and insect species--the recombinant plant flavoenzyme was unaccessible to date for in vitro characterization. Structure-activity relationship studies explained that potent inhibitors of animal DHODH do not significantly affect the plant enzyme. These difference could be exploited for a novel approach to herb or pest growth control by limitation of pyrimidine nucleotide pools.
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Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme Dihydroorotate Dehydrogenase. A versatile tool to rate inhibitor effects?
European journal of biochemistry, 2001Co-Authors: Alexandra Ullrich, Wolfgang Knecht, Markus Fries, Monika LofflerAbstract:Mammalian Dihydroorotate Dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse Dihydroorotate Dehydrogenase. Based on our recent functional expression of the full-length rat and human Dihydroorotate Dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K(m) = 11 microM Dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM Dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K(m) = 26 microM Dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of Dihydroorotate Dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.
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Indirect inhibition of mitochondrial Dihydroorotate Dehydrogenase activity by nitric oxide.
Free radical biology & medicine, 2000Co-Authors: Claire Beuneu, Monika Loffler, Rodolphe Auger, Annie Guissani, Geneviève Lemaire, Michel LepoivreAbstract:Abstract Dihydroorotate Dehydrogenase (DHODH) catalyzes the oxidation of Dihydroorotate to orotate in the pyrimidine biosynthesis pathway. It is functionally connected to the respiratory chain, delivering electrons to ubiquinone. We report here that inhibition of cytochrome c oxidase by nitric oxide (NO) indirectly inhibits DHODH activity. In digitonin-permeabilized cells, DEA/NO, a chemical NO donor, induced a dramatic decrease in DHO-dependent O 2 consumption. The inhibition was reversible and more pronounced at low O 2 concentration; it was correlated with a decrease in orotate synthesis. Since orotate is the precursor of all pyrimidine nucleotides, indirect inhibition of DHODH by NO may significantly contribute to NO-dependent cytotoxicity.
Jon Clardy - One of the best experts on this subject based on the ideXlab platform.
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brequinar derivatives and species specific drug design for Dihydroorotate Dehydrogenase
Bioorganic & Medicinal Chemistry Letters, 2006Co-Authors: Darrell E Hurt, Amanda E Sutton, Jon ClardyAbstract:Therapeutic agents brequinar sodium and leflunomide (Arava™) work by binding in a hydrophobic tunnel formed by a highly variable N-terminus of family 2 Dihydroorotate Dehydrogenase (DHODH). The X-ray crystallographic structure of an analog of brequinar bound to human DHODH was determined. In silico screening of a library of compounds suggested another subset of brequinar analogs that do not inhibit human DHODH as potentially effective inhibitors of Plasmodium falciparum DHODH.
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structure of plasmodium falciparum Dihydroorotate Dehydrogenase with a bound inhibitor
Acta Crystallographica Section D-biological Crystallography, 2006Co-Authors: Darrell E Hurt, Joanne Widom, Jon ClardyAbstract:Membrane-associated Dihydroorotate Dehydrogenase (DHODH) is an antimalarial therapeutic target without an effective inhibitor. Studies on human DHODH (HsDHODH) led to a structural mechanistic model in which respiratory quinones bind in a tunnel formed by the highly variable N-terminus that leads to the flavin mononucleotide-binding site. The therapeutic agents leflunomide (Arava) and brequinar sodium inhibit HsDHODH by binding in this tunnel. Plasmodium falciparum DHODH (PfDHODH) and HsDHODH have markedly different sensitivities to the two drugs. To understand the structural basis of this differential sensitivity and begin a structure-based drug-design cycle for PfDHODH inhibitors, the three-dimensional structure (2.4 A, R = 20.1%) of PfDHODH bound to the active metabolite of leflunomide was determined by X-ray crystallography. Comparison of the structures of HsDHODH and PfDHODH reveals a completely different binding mode for the same inhibitor in these two catalytically identical enzymes and explains the previously observed species-specific preferential binding. Because no effective inhibitors have been described for PfDHODH, this structure provides critical insight for the design of potential antimalarials.
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The Synthesis of Potentially Selective Inhibitors of Dihydroorotate Dehydrogenase. The Utilization of Chemoselective Suzuki Cross‐Coupling Reactions in a Parallel Synthesis.
ChemInform, 2001Co-Authors: Amanda E Sutton, Jon ClardyAbstract:Abstract A series of potentially selective inhibitors of Dihydroorotate Dehydrogenase (DHODH) were synthesized via iterative, chemoselective Suzuki cross-couplings utilizing biaryl chlorides as key intermediates.
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the synthesis of potentially selective inhibitors of Dihydroorotate Dehydrogenase the utilization of chemoselective suzuki cross coupling reactions in a parallel synthesis
Tetrahedron Letters, 2001Co-Authors: Amanda E Sutton, Jon ClardyAbstract:Abstract A series of potentially selective inhibitors of Dihydroorotate Dehydrogenase (DHODH) were synthesized via iterative, chemoselective Suzuki cross-couplings utilizing biaryl chlorides as key intermediates.
Zhenjiang Zhao - One of the best experts on this subject based on the ideXlab platform.
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synthesis design and structure activity relationship of the pyrimidone derivatives as novel selective inhibitors of plasmodium falciparum Dihydroorotate Dehydrogenase
Molecules, 2018Co-Authors: Le Xu, Yanyan Diao, Hongchang Zhou, Wenjie Li, Honglin Li, Zhenjiang ZhaoAbstract:The inhibition of Plasmodium falciparum Dihydroorotate Dehydrogenase (PfDHODH) potentially represents a new treatment option for malaria, as P. falciparum relies entirely on a de novo pyrimidine biosynthetic pathway for survival. Herein, we report a series of pyrimidone derivatives as novel inhibitors of PfDHODH. The most potent compound, 26, showed high inhibition activity against PfDHODH (IC50 = 23 nM), with >400-fold species selectivity over human Dihydroorotate Dehydrogenase (hDHODH). The brand-new inhibitor scaffold targeting PfDHODH reported in this work may lead to the discovery of new antimalarial agents.
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Synthesis, Design, and Structure⁻Activity Relationship of the Pyrimidone Derivatives as Novel Selective Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase.
Molecules, 2018Co-Authors: Le Xu, Yanyan Diao, Hongchang Zhou, Wenjie Li, Honglin Li, Zhenjiang ZhaoAbstract:The inhibition of Plasmodium falciparum Dihydroorotate Dehydrogenase (PfDHODH) potentially represents a new treatment option for malaria, as P. falciparum relies entirely on a de novo pyrimidine biosynthetic pathway for survival. Herein, we report a series of pyrimidone derivatives as novel inhibitors of PfDHODH. The most potent compound, 26, showed high inhibition activity against PfDHODH (IC50 = 23 nM), with >400-fold species selectivity over human Dihydroorotate Dehydrogenase (hDHODH). The brand-new inhibitor scaffold targeting PfDHODH reported in this work may lead to the discovery of new antimalarial agents.
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structure based design of potent human Dihydroorotate Dehydrogenase inhibitors as anticancer agents
MedChemComm, 2016Co-Authors: Wenlin Song, Zhenjiang Zhao, Lili Zhu, Yi Tong, Jiawei Wang, Lina Quan, Zhuo Chen, Xuhong QianAbstract:It has been proven that inhibiting human Dihydroorotate Dehydrogenase (hDHODH) restricts the growth of rapidly proliferating cells, thus hDHODH can be developed as a promising target for the treatment of immunological disease and cancer. Here, a succession of substituted hydrazino-thiazole derivatives were designed, synthesized, and biologically evaluated through structure-based optimization, of which compound 22 was the most potent inhibitor of hDHODH with an IC50 value of 1.8 nM. Furthermore, 22 exhibited much better antiproliferative activity than brequinar, both in HCT-116 and BxPC-3 cancer cell lines. Flow cytometry analysis revealed that 22 induced S phase cell cycle arrest and promoted induction of apoptosis. All results established a proof that blocking the pyrimidine de novo synthesis pathway by inhibiting the rate-limiting enzyme hDHODH is an attractive therapy for cancer.
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discovery of diverse human Dihydroorotate Dehydrogenase inhibitors as immunosuppressive agents by structure based virtual screening
Journal of Medicinal Chemistry, 2012Co-Authors: Yanyan Diao, Zhenjiang Zhao, Huangtao Jin, Junsheng Zhu, Le Han, Rui Gao, Xu Shen, Xiaofeng Liu, Jin HuangAbstract:This study applied an efficient virtual screening strategy integrating molecular docking with MM-GBSA rescoring to identify diverse human Dihydroorotate Dehydrogenase (hDHODH) inhibitors. Eighteen compounds with IC50 values ranging from 0.11 to 18.8 μM were identified as novel hDHODH inhibitors that exhibited overall species-selectivity over Plasmodium falciparum Dihydroorotate Dehydrogenase (pfDHODH). Compound 8, the most potent one, showed low micromolar inhibitory activity against hDHODH with an IC50 value of 0.11 μM. Moreover, lipopolysaccharide-induced B-cell assay and mixed lymphocyte reaction assay revealed that most of the hits showed potent antiproliferative activity against B and T cells, which demonstrates their potential application as immunosuppressive agents. In particular, compound 18 exhibited potent B-cell inhibitory activity (IC50 = 1.78 μM) and presents a B-cell-specific profile with 17- and 26-fold selectivities toward T and Jurkat cells, respectively.
Martin D Brand - One of the best experts on this subject based on the ideXlab platform.
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production of superoxide h2o2 by Dihydroorotate Dehydrogenase in rat skeletal muscle mitochondria
Free Radical Biology and Medicine, 2014Co-Authors: Martin Heymogensen, Renata L S Goncalves, Adam L Orr, Martin D BrandAbstract:Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate Dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate Dehydrogenase oxidizes Dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that Dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min(-1)·mg protein(-1) when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of Dihydroorotate Dehydrogenase. Eighty percent of this rate is indirect, originating from site IIF of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF), myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), Dihydroorotate Dehydrogenase generates superoxide/H2O2, at a small but significant rate (23pmol H2O2·min(-1)·mg protein(-1)), from the ubiquinone-binding site. We conclude that Dihydroorotate Dehydrogenase can generate superoxide and/or H2O2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.
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Production of superoxide/H2O2 by Dihydroorotate Dehydrogenase in rat skeletal muscle mitochondria.
Free radical biology & medicine, 2014Co-Authors: Martin Hey-mogensen, Renata L S Goncalves, Adam L Orr, Martin D BrandAbstract:Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate Dehydrogenase, are known to be direct generators of superoxide and/or H2O2. Dihydroorotate Dehydrogenase oxidizes Dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H2O2 directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that Dihydroorotate oxidation leads to superoxide/H2O2 production at a fairly high rate of about 300pmol H2O2·min(-1)·mg protein(-1) when oxidation of ubiquinol is prevented and complex II is uninhibited. This H2O2 production is abolished by brequinar or leflunomide, known inhibitors of Dihydroorotate Dehydrogenase. Eighty percent of this rate is indirect, originating from site IIF of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H2O2 production (rotenone to inhibit sites in complex I (site IQ and, indirectly, site IF), myxothiazol to inhibit site IIIQo in complex III, and malonate plus atpenin A5 to inhibit site IIF in complex II), Dihydroorotate Dehydrogenase generates superoxide/H2O2, at a small but significant rate (23pmol H2O2·min(-1)·mg protein(-1)), from the ubiquinone-binding site. We conclude that Dihydroorotate Dehydrogenase can generate superoxide and/or H2O2 directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.
Wolfgang Knecht - One of the best experts on this subject based on the ideXlab platform.
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Dihydroorotate Dehydrogenase mrna and protein expression analysis in normal and drug resistant cells
Nucleosides Nucleotides & Nucleic Acids, 2004Co-Authors: Monika Loffler, Wolfgang Knecht, A Klein, M Hayekouassini, L KonradAbstract:To follow the expression of the fourth enzyme of pyrimidine de novo synthesis Dihydroorotate Dehydrogenase (DHODH) in cells and tissues, we studied the DHODH mRNA expression by means of RT-PCR in rat tissues. Rabbit polyclonal anti-DHODH immunoglobulins were applied for immunochemical quantification of the enzyme protein by Western blotting. In mouse B-lymphocytes, which were adapted to tolerate up to a 50-fold concentration of the DHODH inhibitor leflunomide, a 20 fold protein overexpression was measured. Southern blotting indicated DHODH gene amplification.
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Plant Dihydroorotate Dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes.
FEBS letters, 2002Co-Authors: Alexandra Ullrich, Wolfgang Knecht, Jure Piskur, Monika LofflerAbstract:The mitochondrial membrane bound Dihydroorotate Dehydrogenase (DHODH; EC 1.3.99.11) catalyzes the fourth step of pyrimidine biosynthesis. By the present correction of a known cDNA sequence for Arabidopsis thaliana DHODH we revealed the importance of the very C-terminal part for its catalytic activity and the reason why--in contrast to mammalian and insect species--the recombinant plant flavoenzyme was unaccessible to date for in vitro characterization. Structure-activity relationship studies explained that potent inhibitors of animal DHODH do not significantly affect the plant enzyme. These difference could be exploited for a novel approach to herb or pest growth control by limitation of pyrimidine nucleotide pools.
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Recombinant expression of N-terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme Dihydroorotate Dehydrogenase. A versatile tool to rate inhibitor effects?
European journal of biochemistry, 2001Co-Authors: Alexandra Ullrich, Wolfgang Knecht, Markus Fries, Monika LofflerAbstract:Mammalian Dihydroorotate Dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse Dihydroorotate Dehydrogenase. Based on our recent functional expression of the full-length rat and human Dihydroorotate Dehydrogenase, here we expressed N-terminal-truncated C-terminal-histidine-tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N-terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal-chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 micromol x min(-1) x mg(-1) at pH optimum of 8.0--8.1. Flavin analyses by UV-visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6--1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (K(m) = 11 microM Dihydroorotate; K(m) = 7 microM ubiquinone) and human enzyme (K(m) = 10 microM Dihydroorotate; K(m) = 14 microM ubiquinone) were very close to those recently reported for the full-size enzymes. The constants for the mouse enzyme, K(m) = 26 microM Dihydroorotate and K(m) = 62 microM ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N-terminus of Dihydroorotate Dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77-1726, MNA715 and MNA279 with the rat and human enzyme, the N-termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N-terminal part of the human enzyme seemed to be of crucial importance for the 'slow-binding' features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti-proliferative and immunosuppressive action.
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kinetics of inhibition of human and rat Dihydroorotate Dehydrogenase by atovaquone lawsone derivatives brequinar sodium and polyporic acid
Chemico-Biological Interactions, 2000Co-Authors: Wolfgang Knecht, Johannes Henseling, Monika LofflerAbstract:Abstract Mitochondrially-bound Dihydroorotate Dehydrogenase (EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. The enzyme has been identified as or surmised to be the pharmacological target for isoxazol, triazine, cinchoninic acid and (naphtho)quinone derivatives, which exerted antiproliferative, immunosuppressive, and antiparasitic effects. Despite this broad spectrum of biological and clinical relevance, there have been no comparative studies on drug-Dihydroorotate Dehydrogenase interactions. Here, we describe a study of the inhibition of the purified recombinant human and rat Dihydroorotate Dehydrogenase by ten compounds. 1,4-Naphthoquinone, 5,8-hydroxy-naphthoquinone and the natural compounds juglon, plumbagin and polyporic acid (quinone derivative) were found to function as alternative electron acceptors with 10–30% of control enzyme activity. The human and rat enzyme activity was decreased by 50% by the natural compound lawsone (>500 and 49 μM, respectively) and by the derivatives dichloroally-lawsone (67 and 10 nM), lapachol (618 and 61 nM) and atovaquone (15 μM and 698 nM). With respect to the quinone co-substrate of the Dihydroorotate Dehydrogenase, atovaquone (Kic=2.7 μM) and dichloroally-lawsone (Kic=9.8 nM) were shown to be competitive inhibitors of human Dihydroorotate Dehydrogenase. Atovaquone (Kic=60 nM) was also a competitive inhibitor of the rat enzyme. Dichloroallyl-lawsone was found to be a time-dependent inhibitor of the rat enzyme, with the lowest inhibition constant (Ki*=0.77 nM) determined so far for mammalian Dihydroorotate Dehydrogenases. Another inhibitor, brequinar was previously reported to be a slow-binding inhibitor of the human Dihydroorotate Dehydrogenase [W. Knecht, M. Loffler, Species-related inhibition of human and rat dihyroorotate Dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives, Biochem. Pharmacol. 56 (1998) 1259–1264]. The slow binding features of this potent inhibitor (Ki*=1.8 nM) with the human enzyme, were verified and seen to be one of the reasons for the narrow therapeutic window (efficacy versus toxicity) reported from clinical trials on its antiproliferative and immunosuppressive action. With respect to the substrate Dihydroorotate, atovaquone was an uncompetitive inhibitor of human Dihydroorotate Dehydrogenase (Kiu=11.6 μM) and a non-competitive inhibitor of the rat enzyme (Kiu=905/Kic=1012 nM). 1.5 mM polyporic acid, a natural quinone from fungi, influenced the activity of the human enzyme only slightly; the activity of the rat enzyme was decreased by 30%.
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Species-related inhibition of human and rat Dihydroorotate Dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives
Biochemical pharmacology, 1998Co-Authors: Wolfgang KnechtAbstract: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-trifluoro methylphenyl-carbamoyl)-2-cyclopropyl-2oxo-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 (IC50 = 6.3 microM) and human (IC50 = 98 microM) Dihydroorotate Dehydrogenase, the influence of A77-1726, MNA 279, MNA715 and HR325 was of comparable efficacy for either the rat (range of IC50, 19-53 nM) or the human enzyme (range of IC50, 0.5-2.3 microM). From the IC50 values, it was deduced that brequinar was a more potent inhibitor of the human Dihydroorotate Dehydrogenase activity (IC50 = 10 nM) than of the rat enzyme (IC50 = 367 nM). The rat enzyme was influenced by all isoxazol derivatives to a greater extent (IC50 = 19 nM A77-1726) than the human enzyme (IC50 = 1.1 microM 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.