Dihydro-orotate

Lieberman 1, A Komberg (1953) Enzymatic synthesis and breakdown of a pyrimidine, orotic acid I. Dihydro-orotic dehydrogenase. Biochim Biophys Acta 12 223-234. 

HMR 1726, Terifluno-mide Dihydro-orotate dehydrogenase inhibitor Reduced number of active lesions Multiple Sclerosis... 

L- and DL-dihydro-orotic acid supported growth of Lactobacillus bulgaricus 09. The D-isomer alone is not only without activity, but is reported to reversibly inhibit the growth-promoting property exerted by ureidosuccinic or orotic acid [74, 91]. The dihydro-orotic acid prepared by fusion of maleic acid and urea [130] is inactive in bothi. bulgaricus (B [128] and an enzyme system [72, 131]. A comparison of the urea fusion product and that prepared by catalytic hydrogenation of orotic acid revealed that the former is actually fumarylurea [132]. 

The phenoxyquinolines are a recent addition to crop fungicides and only one compound has been described. LY214352, an experimental fungicide, inhibits a novel target, dihydro-orotate dehydrogenase (DHO-DH) in the pyrimidine biosynthesis pathway that catalyses the conversion of dihydro-orotate into orotic acid9 (Figure 4.13). 

In vivo, Leflunomide is rapidly converted into its pharmacologically active metabolite A77 1726 (Herrmann et al., 2000). Although the precise mode of action of Leflunomide in vivo remains elusive, A77 1726 has been shown in vitro to inhibit reversibly dihydro-orotate dehydrogenase (DHODH), which catalyzes a rate-limiting step in the de novo synthesis of pyrimidines (Cherwinski et al., 1995 Williamson et al., 1996). The inhibition of DHODH activity by A77 1726 might explain part of its mechanism of action in suppressing inflammation. 

Isoindoline li/-Isoindole, 2,3-dihydro- Orotic acid 4-Pyrimidinecarboxylicacid,l,2,3,6-tetrahydro-2,6-... 

Brequinar (DUP 785, NSC 368390) is a quinoline carboxylic acid derivative that inhibits pyrimidine synthesis by inhibiting dihydro-orotate dehydrogenase. It was originally developed as an anticancer drug, but has also been investigated for its immunosuppressant activity after transplantation. Some data suggest that that the immunosuppressant activity of brequinar may be partly due to inhibition of tyrosine phosphorylation in lymphocytes (1). 

Leflunomide Inhibits dihydro-orotic acid dehydrogenase (DHOD) —4 ump —4 ribonucleotides -> arrests lymphocytes in Gj Alopecia, rash, diarrhea, hepatotoxicity... 

B. leflunomide inhibition of dihydro-orotic acid dehydrogenase... 

Answer B. Leflunomide, used in rheumatoid arthritis, inhibits dihydro-orotic acid dehydrogenase >4 formation of UMP ->4- de novo synthesis of ribonucleotides — arrest of lymphocytes in the GI phase. Glucocorticoids do not decrease expression of lipoxygenase, but by preventing arachidonate formation they decrease activity of the pathway. Misoprostol, used in NSAID-induced GI ulcers, activates receptors colchicine decreases microtubular polymerization ketorolac is a potent NSAID but a nonselective inhibitor of cyclooxygenases. 

Dihydroorotase carbamoylaspartic acid amidohydrolase l-5, 6-dihydro-orotate 3.5.2.3... 

Ans B Leflunomide, used in rheumatoid arthritis, inhibits dihydro-orotic... 

Spirobarbiturates are of interest as inhibitors of dihydro-orotate dehydrogenase enzyme system (DHODase) <90JCS(P1)3137>. 

Leflunomide is an antirheumatic agent. It is an isoxazole immunomodulatory agent that inhibits dihydro-orotate dehydrogenase and has antiproliferative and antiinflammatory activity. It is indicated in the treatment of active rheumatoid arthritis (RA) to reduce signs and symptoms and to retard structural damage. 

Leflunomide (Arava) is a pyrimidine-synthesis inhibitor indicated for the treatment of adults with rheumatoid arthritis. This drug has found utility in the treatment of polyo-mavirus nephropathy seen in immunosuppressed renal transplant recipients and is increasingly being used for that purpose. There are no studies showing efficacy, however, compared with control patients treated with withdrawal or reduction of immune-suppression alone in BK virus nephropathy. The drug inhibits dihydro-orotate dehydrogenase in the de novo pathway of pyrimidine synthesis. It is hepa-totoxic and can cause fetal injury when administered to pregnant women. 

Orotate is an intermediate in de novo synthesis of pyrimidines. It is made by dehydrogenation of dihydroo rotate in a reaction catalyzed by dihydro orotate dehydrogenase (Figure 22.10). 

FIG. 6.12 Pyrimidine de novo synthesis pathway. Enzymes are as follows (1) carbamoyl-phosphate synthetase II (2) asparate carbamoyl-transferase (3) dihydro-orotase (4) dihydro-orotate oxidase (5) orotate phosphoribosyltransferase (6) orotidine-5 -phosphate decarboxylase (7) nucleoside monophosphate kinase (8) nucleotide diphospho kinase (9) CTP synthetase. 

The first three reactions are catalyzed by a trifunctional protein which contains carbamoyl-phosphate synthetase II, aspartate carbamoyltransferase and dihydro-orotase. This set of reactions begins with the synthesis of carbamoyl phosphate followed by its condensation with aspartic acid. The third step involves the closure of the ring through the removal of water by the action of dihydro-orotase to yield dihydro-orotate. The fourth enzyme, dihydro-orotate oxidase, oxidizes dihydro-orotate to orotate and is a mitochondrial flavoprotein enzyme located on the outer surface of the inner membrane and utilizes NAD" " as the electron acceptor. The synthesis of UMP from orotate is catalyzed by a bifunctional protein which comprises orotate PRTase and orotidine 5 -phosphate (OMP) decarboxylase. The former phosphoribosylates orotate to give OMP the latter decarboxylates OMP to UMP, the immediate precursor for the other pyrimidine nucleotides. It is interesting to note that whereas five molecules of ATP (including the ATP used in the synthesis of PRPP) are used in the de novo synthesis of IMP, no net ATP is used in the de novo synthesis of UMP. In de novo pyrimidine synthesis, two ATP molecules are used to synthesize carbamoyl phosphate and one ATP is needed to synthesize the PRPP used by orotate PRTase but 3 ATPs... 

In summary, pyrimidine metabolism in the kinetoplastids is functionally similar to that found in mammalian cells. In the de novo synthesis of UMP, these parasites differ in two respects dihydro-orotate oxidase is cytoplasmic and not mitochondrial and the last two enzymes of UMP synthesis are glycosomal instead of cytoplasmic. Pyrimidine salvage by these parasites is more diverse than that of mammalian cells but, unlike the purines, de novo synthesis plays the major role. 

The enzymes are functionally similar to their mammalian counterparts. The dihydro-orotate oxidase, unlike that of the kinetoplastids, is associated with the parasite mitochondrion (99). However, the last two enzymes, the cytoplasmic orotate PRTase and OMP decarboxylase, are separate enzymes instead of being arranged in a bifunctional complex as is found in mammalian cells (99,100). Metabolic studies indicate that CTP synthetase must be present. 

All of the enzyme activities involved in de novo UMP biosynthesis have been identified in the trematodes Schistosoma mansoni (81,106,107) and S. japonicum (108). Carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydro-orotase have been partially purified from the cytosol and appear to exist, as in mammalian cells, as a multienzyme protein (81,106). Dihydro-orotate oxidase is membrane bound (81), but its electron acceptor has not been identified. Orotate PRTase and OMP decarboxylase are also cytosolic. In mammalian cells these last two enzymes exist as part of a multienzyme protein that efficiently channels orotate to UMP since little free OMP is present in the cell. In S. mansoni they also exist as a multienzyme protein but apparently the channeling is less efiBcient since levels of free OMP are significantly higher than those of UMP (107). 

The conversion of carbamoyl aspartate to orotic acid became evident in work by Lieberman and Kornberg [97] who studied the degradation of orotic acid by an orotate-fermenting bacterium, Zymobacterium oroticum, and isolated two intermediates, dihydro-orotate and carbamoyl aspartate. The degradation reactions were found to be reversible and the cell-free extract of Z. oroticum converted carbamoyl aspartate back to orotic acid. 

The enzyme catalysing the reversible cyclization of carbamoyl aspartate to dihydro-orotate is called dihydro-orotase (L-4,5-dihydro-orotate amino-hydrolase, EC 3.5.2.3). Dihydro-orotase was found in various animal tissues and for the catalytic function requires Zn + ions [98]. Orotic acid was found to be a competitive inhibitor of dihydro-orotate synthesis though a variety of other pyrimidines had no effect on enzyme activity [99]. 

Dihydro-orotate-oxidizing activity in rat liver homogenates can be recovered completely in the mitochondrial fraction [103,104].With the exception of this system all the other enzymes of the orotate pathway appear to be present in the soluble cytosolic fraction. Dihydro-orotate dehydrogenase from rat liver was found to be located on the outer surface of the inner membrane of mitochondria [105]. Dihydro-orotate can diffuse freely from the cytosol into the mitochondria and orotate can diffuse freely from the mitochondria into the cytosol. Therefore no active transport of either dihydro-orotate or orotate is required in pyrimidine synthesis [105]. In addition to inhibiting dihydro-orotase, orotic acid strongly blocks [103] dihydro-orotate oxidation. 

B25 Blattmann, P. and Retey, J. Stereospecificity of the dihydro-orotate-dehydrogenase reaction. Eur. J. Biochem., 30,130-137 (1972)... 

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