Monika Löffler

Kinetics of inhibition of human and rat dihydroorotate dehydrogenase by atovaquone, lawsone derivatives, brequinar sodium and polyporic acid.

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 microM, respectively) and by the derivatives dichloroally-lawsone (67 and 10 nM), lapachol (618 and 61 nM) and atovaquone (15 microM and 698 nM). With respect to the quinone co-substrate of the dihydroorotate dehydrogenase, atovaquone (Kic = 2.7 microM) and dichloroally-lawsone (Kic = 9.8 nM) were shown to be competitive inhibitors of human dihydroorotate dehydrogenase. Atovaquone (Kic = 60 nM) was also acompetitive inhibitor of the rat enzyme. Dichloroally]-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 microM) and a non-competitive inhibitor of the rat enzyme (Kiu = 905/ Kic = 1,012 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%.

Inhibitor binding in a class 2 dihydroorotate dehydrogenase causes variations in the membrane-associated N-terminal domain.

The flavin enzyme dihydroorotate dehydrogenase (DHOD; EC 1.3.99.11) catalyzes the oxidation of dihydroorotate to orotate, the fourth step in the de novo pyrimidine biosynthesis of UMP. The enzyme is a promising target for drug design in different biological and clinical applications for cancer and arthritis. The first crystal structure of the class 2 dihydroorotate dehydrogenase from rat has been determined in complex with its two inhibitors brequinar and atovaquone. These inhibitors have shown promising results as anti‐proliferative, immunosuppressive, and antiparasitic agents. A unique feature of the class 2 DHODs is their N‐terminal extension, which folds into a separate domain comprising two α‐helices. This domain serves as the binding site for the two inhibitors and the respiratory quinones acting as the second substrate for the class 2 DHODs. The orientation of the first N‐terminal helix is very different in the two complexes of rat DHOD (DHODR). Binding of atovaquone causes a 12 Å movement of the first residue in the first α‐helix. Based on the information from the two structures of DHODR, a model for binding of the quinone and the residues important for the interactions could be defined. His 56 and Arg 136, which are fully conserved in all class 2 DHODs, seem to play a key role in the interaction with the electron acceptor. The differences between the membrane‐bound rat DHOD and membrane‐associated class 2 DHODs exemplified by the Escherichia coli DHOD has been investigated by GRID computations of the hydrophobic probes predicted to interact with the membrane.

Catalytic enzyme histochemistry and biochemical analysis of dihydroorotate dehydrogenase/oxidase and succinate dehydrogenase in mammalian tissues, cells and mitochondria

Dihydroorotate dehydrogenase (EC 1.3.3.1 or EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. In eukaryotes it is located in the inner mitochondrial membrane, with ubiquinone as the proximal and cytochrome oxidase as the ultimate electron transfer system, whereas the rest of pyrimidine biosynthesis takes place in the cytosol. Here, the distribution of dihydroorotate dehydrogenase activity in cryostat sections of various rat tissues, and tissue samples of human skin and kidney, was visualized by light microscopy using the nitroblue tetrazolium technique. In addition, a hydrogen peroxide-producing oxidase side-reactivity of dihydroorotate dehydrogenase could be visualized by trapping the peroxide with cerium-diaminobenzidine. The pattern of activity was similar to that of succinate dehydrogenase, but revealed a less intensive staining. High activities of dihydroorotate dehydrogenase were found in tissues with known proliferative, regenerative, absorptive or excretory activities, e.g., mucosal cells of the ileum and colon crypts in the gastro-intestinal tract, cultured Ehrlich ascites tumor cells, and proximal tubules of the kidney cortex, whilst lower activities were present in the periportal area of the liver, testis and spermatozoa, prostate and other glands, and skeletal muscle. Dihydroorotate dehydrogenase and succinate dehydrogenase activity in Ehrlich ascites tumor cells grown in suspension culture were quantified by application of nitroblue tetrazolium or cyanotolyl tetrazolium and subsequent extraction of the insoluble formazans with organic solvents. The ratio of dihydroorotate dehydrogenase to succinate dehydrogenase activity was 1∶4. This was in accordance with that of 1∶5 obtained from oxygen consumption measurement of isolated mitochondria on addition of dihydroorotate or succinate. The ratio determined with mitochondria from animal tissues was up to 1∶15 (rat liver, bovine heart). The application of the enzyme inhibitors brequinar sodium and toltrazuril verified the specificity of the histochemical and biochemical methods applied.

Redoxal as a new leadstructure for dihydroorotate dehydrogenase inhibitors: a kinetic study of the inhibition mechanism

Mitochondrial dihydroorotate dehydrogenase (DHOdehase; EC 1.3.99.11) is a target of anti‐proliferative, immunosuppressive and anti‐parasitic agents. Here, redoxal, (2,2′‐[3,3′‐dimethoxy[1,1′‐biphenyl]‐4,4′‐diyl)diimino]bis‐benzoic acid, was studied with isolated mitochondria and the purified recombinant human and rat enzyme to find out the mode of kinetic interaction with this target. Its pattern of enzyme inhibition was different from that of cinchoninic, isoxazol and naphthoquinone derivatives and was of a non‐competitive type for the human (K ic=402 nM; K iu=506 nM) and the rat enzyme (K ic=116 nM; K iu=208 nM). The characteristic species‐related inhibition of DHOdehase found with other compounds was less expressed with redoxal. In human and rat mitochondria, redoxal did not inhibit NADH‐induced respiration, its effect on succinate‐induced respiration was marginal. This was in contrast to the sound effect of atovaquone and dichloroallyl‐lawsone, studied here for comparison. In human mitochondria, the IC50 value for the inhibition of succinate‐induced respiration by atovaquone was 6.1 μM and 27.4 μM for the DHO‐induced respiration; for dichlorallyl‐lawsone, the IC50 values were 14.1 μM and 0.23 μM.

Restimulation of cell cycle progression by hypoxic tumour cells with deoxynucleosides requires ppm oxygen tension

Continuous exposure of Ehrlich ascites tumour cells to argon-CO2 under in vitro conditions caused rapid cessation of cell proliferation. On fixing the O2 level at 10 ppm in the protective atmosphere (0.001% in comparison with about 20% in normoxic atmosphere), G1 and early S cells remained stationary while G2 cells continued to pass from G2 into mitosis, to remain in a non-growing state in G1 of the subsequent cycle. Re-aeration of cells following 12 h hypoxia induced up to 25% of the population to continue DNA synthesis without a preceding cell division, as revealed by flow-cytometric analysis. Supplementation of cells cultured under hypoxia with a combination of deoxynucleosides (100 microM deoxycytidine, 10 microM deoxyadenosine, 10 microM deoxyguanosine) was found to stimulate reprogression through the cycle, provided the residual oxygen tension in the protective atmosphere exceeded 40 ppm. The increase in the number of cells with a DNA content of more than 4 C and in the number of binucleate cells observed after re-aeration of hypoxic cells was not prevented by deoxynucleosides or by uridine, which were present in the medium either during hypoxia of from the beginning of reoxygenation. These results indicate that the development of polyploidy as a result of oxygen deficiency cannot be influenced by improvement of RNA and DNA synthetic precursors.

Localization of dihydroorotate oxidase in myocardium and kidney cortex of the rat. An electron microscopic study using the cerium technique.

Biochemical studies have demonstrated that dihydroorotate dehydrogenase (DHOdehase; EC 1.3.3.1 or 1.3.99.11) is the sole enzyme of de novo pyrimidine synthesis in mitochondria, whereas the rest of the pathway takes place in the cytosol. The dehydrogenation of dihydroorotate to orotate is linked to the respiratory chain via ubiquinone. In this study, we show for the first time the ultrastructural localization of DHOdehase. Since the purified enzyme was found to act both as dehydrogenase and as oxidase, the cerium capture technique for detecting enzymatically generated hydrogen peroxide could be applied to pin-point the in situ activity of DHOdehase oxidase in mitochondria of rat heart and kidney cortex. Cerium perhydroxide as the final reaction product was detected predominantly in the matrix with some focal condensation along the inner membrane, but not in the intermembrane space. From this pattern of localization, it is concluded that the active site of the membrane-bound enzyme could face the mitochondrial matrix similar to succinate dehydrogenase. The reliability of the applied method for the demonstration of DHOdehase oxidase was demonstrated by the addition of Brequinar sodium to the incubation medium. This quinoline-carboxylic acid derivative is a potent inhibitor of DHOdehase and has proven anti-proliferative activity. The present observations do not ascertain whether the oxidase is permanently active as a constant portion of the enzyme in vivo, similar to xanthine oxidase/dehydrogenase. However, DHOdehase should be considered as a source of radical oxygen species under pathophysiological conditions.

Deoxycytidylate shortage is a cause of G1 arrest of ascites tumor cells under oxygen deficiency

not received Cell cycle Anaerobiosis Deoxyribonucleotide pool Ribonucleotide reduction

Plant dihydroorotate dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes.

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.

Towards a further understanding of the growth-inhibiting action of oxygen deficiency. Evaluation of the effect of antimycin on proliferating Ehrlich ascites tumour cells.

The effect of 1 microM antimycin on the proliferative properties, metabolism and basic cell composition of Ehrlich ascites tumour cells cultured in the second in vitro passage was studied. Continuous drug exposure of asynchronous cells caused rapid cessation of cell growth, characterized by the cell number and DNA, RNA and protein content of cultures. Cells cease to consume oxygen and enhance their glycolytic activity. Uptake of labelled thymidine into acid-insoluble material was far below that of the controls, whereas incorporation of labelled uridine exceeded that of controls, as was also observed with other inhibitors of the respiratory chain (sodium cyanide, 2-thenoyltrifluoroacetone, or anaerobiosis). The influence of antimycin on cells at different stages of the cell cycle was tested using cells enriched in either G1, S or G2 phase by centrifugal elutriation. DNA histograms (flow cytometry) and pulse-labelling index curves gave detailed insight into cell-cycle progression of antimycin-treated cells: G1 and early S cells remained stationary; G2 cells still passed from G2 into mitosis to remain subsequently in a non-growing state in G1; S cells were either slowed or halted. Supplementation of antimycin-containing cultures with exogenous pyrimidine nucleosides stimulated reprogression of G1 cells without changing their ATP content. The results of the current experiments are interpreted as supporting the concept that growth cessation of G1 cells under respiratory insufficiency is not predominantly caused by impairment of respiratory phosphorylation but may be the consequence of a lack of precursors for DNA and RNA synthesis.

Biochemical characterization of recombinant dihydroorotate dehydrogenase from the opportunistic pathogenic yeast Candida albicans

Candida albicans is the most prevalent yeast pathogen in humans, and recently it has become increasingly resistant to the current antifungal agents. In this study we investigated C. albicans dihydroorotate dehydrogenase (DHODH, EC 1.3.99.11), which catalyzes the fourth step of de novo pyrimidine synthesis, as a new target for controlling infection. We propose that the enzyme is a member of the DHODH family 2, which comprises mitochondrially bound enzymes, with quinone as the direct electron acceptor and oxygen as the final electron acceptor. Full‐length DHODH and N‐terminally truncated DHODH, which lacks the targeting sequence and the transmembrane domain, were subcloned from C. albicans, recombinantly expressed in Escherichia coli, purified, and characterized for their kinetics and substrate specificity. An inhibitor screening with 28 selected compounds was performed. Only the dianisidine derivative, redoxal, and the biphenyl quinoline‐carboxylic acid derivative, brequinar sodium, which are known to be potent inhibitors of mammalian DHODH, markedly reduced C. albicans DHODH activity. This study provides a background for the development of antipyrimidines with high efficacy for decreasing in situ pyrimidine nucleotide pools in C. albicans.