Malarial dihydroorotate dehydrogenase. Substrate and inhibitor specificity

The malarial parasite relies on de novo pyrimidine biosynthesis to maintain its pyrimidine pools, and unlike the human host cell it is unable to scavenge preformed pyrimidines. Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate (DHO) to produce orotate, a key step in pyrimidine biosynthesis. The enzyme is located in the outer membrane of the mitochondria of the malarial parasite. To characterize the biochemical properties of the malarial enzyme, an N-terminally truncated version of P. falciparum DHODH has been expressed as a soluble, active enzyme in E. coli. The recombinant enzyme binds 0.9 molar equivalents of the cofactor FMN and it has a pH maximum of 8.0 (k(cat) 8 s(-1), K(m)(app) DHO (40-80 microm)). The substrate specificity of the ubiquinone cofactor (CoQ(n)) that is required for the oxidation of FMN in the second step of the reaction was also determined. The isoprenoid (n) length of CoQ(n) was a determinant of reaction efficiency; CoQ(4), CoQ(6) and decylubiquinone (CoQ(D)) were efficiently utilized in the reaction, however cofactors lacking an isoprenoid tail (CoQ(0) and vitamin K(3)) showed decreased catalytic efficiency resulting from a 4 to 7-fold increase in K(m)(app). Five potent inhibitors of mammalian DHODH, Redoxal, dichloroallyl lawsone (DCL), and three analogs of A77 1726 were tested as inhibitors of the malarial enzyme. All five compounds were poor inhibitors of the malarial enzyme, with IC(50)'s ranging from 0.1-1.0 mm. The IC(50) values for inhibition of the malarial enzyme are 10(2)-10(4)-fold higher than the values reported for the mammalian enzyme, demonstrating that inhibitor binding to DHODH is species specific. These studies provide direct evidence that the malarial DHODH active site is different from the host enzyme, and that it is an attractive target for the development of new anti-malarial agents.     

Discovery of small molecule inhibitors of human uridine-cytidine kinase 2 by high-throughput screening

Clinically relevant inhibitors of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in mammalian de novo pyrimidine synthesis, have strong antiviral and anticancer activity in vitro. However, they are ineffective in vivo due to efficient uridine salvage by infected or rapidly dividing cells. The pyrimidine salvage enzyme uridine-cytidine kinase 2 (UCK2), a ∼29 kDa protein that forms a tetramer in its active state, is necessary for uridine salvage. Notwithstanding the pharmacological potential of this target, no medicinally tractable inhibitors of the human enzyme have been reported to date. We therefore established and miniaturized an in vitro assay for UCK2 activity and undertook a high-throughput screen against a ∼40,000-compound library to generate drug-like leads. The structures, activities, and modes of inhibition of the most promising hits are described. Notably, our screen yielded non-competitive UCK2 inhibitors which were able to suppress nucleoside salvage in cells both in the presence and absence of DHODH inhibitors.     

Type II NADH dehydrogenase of the respiratory chain of Plasmodium falciparum and its inhibitors

Plasmodium falciparum NDH2 (pfNDH2) is a non-proton pumping, rotenone-insensitive alternative enzyme to the multi-subunit NADH:ubiquinone oxidoreductases (Complex I) of many other eukaryotes. Recombinantly expressed pfNDH2 prefers coenzyme CoQ₀ as an acceptor substrate, and can also use the artificial electron acceptors, menadione and dichlorophenol–indophenol (DCIP). Previously characterized NDH2 inhibitors, dibenziodolium chloride (DPI), diphenyliodonium chloride (IDP), and 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) do not inhibit pfNDH2 activity. Here, we provide evidence that HDQ likely targets another P. falciparum mitochondrial enzyme, dihydroorotate dehydrogenase (pfDHOD), which is essential for de novo pyrimidine biosynthesis.     

Electron transport systems of Candida utilis. Purification and properties of the respiratory chain-linked external NADH dehydrogenase+

The respiratory chain-linked external NADH dehydrogenase has been isolated from Candida utilis in highly purified form. The enzyme is soluble and has a molecular weight of approx. 1.5 · 10⁶. The enzyme contains two moles of FMN per mole of enzyme and is composed of two large subunits of mol. wt. 270 000 and eight smaller subunits of mol. wt. 135 000. Iron and copper are present in the preparations, but appear to be contaminants. The enzyme catalyzes the oxidation of NADH and NADPH at nearly equal rates and reacts readily with 2,6-dichlorophenolindophenol, CoQ₆ and CoQ₁ derivatives as acceptors. Rotenone (10^?5 M) and seconal (10^?3 M) do not inhibit enzymatic activity.     

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.     

Crystal structure of Trypanosoma cruzi dihydroorotate dehydrogenase from Y strain

Trypanosoma cruzi is the etiological agent of Chagas’ disease, a pathogenesis that affects millions of people in Latin America. Here, we report the crystal structure of dihydroorotate dehydrogenase (DHODH) from T. cruzi strain Y solved at 2.2 Å resolution. DHODH is a flavin mononucleotide containing enzyme, which catalyses the oxidation of l-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. Genetic studies have shown that DHODH is essential for T. cruzi survival, validating the idea that this enzyme can be considered an attractive target for the development of antichagasic drugs. In our work, a detailed analysis of T. cruzi DHODH crystal structure has allowed us to suggest potential sites to be further exploited for the design of highly specific inhibitors through the technology of structure-based drug design.     

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.     

Expression and characterization of E. coli-produced soluble, functional human dihydroorotate dehydrogenase: a potential target for immunosuppression

Human dihydroorotate dehydrogenase (huDHODH) is essential for de novo biosynthesis of pyrimidines and the target of two immunosuppressive drugs, brequinar and the leflunomide metabolite A77-1726 (Chen et al., 1992; Davis et al., 1996). Using a T7 RNA polymerase expression system, we produced huDHODH as a fusion protein containing an amino-terminal decahistidine tag. Escherichia coli growth and expression conditions were optimized to enhance huDHODH solubility and to permit purification of the enzyme in the absence of detergent. Soluble huDHODH, purified by a simple two-step procedure, was catalytically active, monomeric, and contained a flavin mononucleotide (FMN) cofactor in a 1:1 FMN/protein molar ratio. Kinetic analysis showed that huDHODH uses a two site ping-pong mechanism, where DHO is oxidized at one site and the second substrate, ubiquinone, is reduced at the other. This result is consistent with the mechanism proposed for bovine liver DHODH (Hines and Johnston, 1989).     

Mammalian dihydroorotate dehydrogenase: physical and catalytic properties of the primary enzyme.

A number of physical and catalytic properties of purified dihydroorotate dehydrogenase from rat liver mitochondria are described. The only potentially reducible cofactor detected was iron. The enzyme was also found to contain zinc. The primary enzyme does not contain FAD, FMN, covalently bound flavin, ubiquinone, or labile sulfide. Certain metal chelators were shown to behave as noncompetitive inhibitors of dihydroorotate oxidation and as competitive inhibitors of the reduction of phenazine methosulfate. The purified preparation can use oxygen as sole electron acceptor, although the reaction rate is relatively slow. The activity of the purified enzyme differs from that of the membrane bound form in a number of ways: the pH maximum is apparently shifted, the effect of thenoyltrifluoroacetone and its K_i are markedly changed and the mode of electron transfer to dichlorophenolindophenol is altered. Therefore, only tentative extrapolations to the membrane system regarding activities such as Superoxide production can be made.     

The structures of human dihydroorotate dehydrogenase with and without inhibitor reveal conformational flexibility in the inhibitor and substrate binding sites

Inhibitors of dihydroorotate dehydrogenase (DHODH) have been suggested for the treatment of rheumatoid arthritis, psoriasis, autoimmune diseases, Plasmodium, and bacterial and fungal infections. Here we present the structures of N-terminally truncated (residues Met30-Arg396) DHODH in complex with two inhibitors: a brequinar analogue (6) and a novel inhibitor (a fenamic acid derivative) (7), as well as the first structure of the enzyme to be characterized without any bound inhibitor. It is shown that 7 uses the "standard" brequinar binding mode and, in addition, interacts with Tyr356, a residue conserved in most class 2 DHODH proteins. Compared to the inhibitor-free structure, some of the amino acid side chains in the tunnel in which brequinar binds and which was suggested to be the binding site of ubiquinone undergo changes in conformation upon inhibitor binding. Using our data, the loop regions of residues Leu68-Arg72 and Asn212-Leu224, which were disordered in previously studied human DHODH structures, could be built into the electron density. The first of these loops, which is located at the entrance to the inhibitor-binding pocket, shows different conformations in the three structures, suggesting that it may interfere with inhibitor/cofactor binding. The second loop has been suggested to control the access of dihydroorotate to the active site of the enzyme and may be an important player in the enzymatic reaction. These observations provide new insights into the dynamic features of the DHODH reaction and suggest new approaches to the design of inhibitors against DHODH.     

Functional expression of human dihydroorotate dehydrogenase (DHODH) in pyr4 mutants of ustilago maydis allows target validation of DHODH inhibitors in vivo

Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.     

Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (K_D) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.     

Tetrahydro-1H,5H-pyrazolo[1,2-a]pyrazole-1-carboxylates as inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase

The reactions between 5-substituted pyrazolidine-3-ones, aldehydes, and methyl methacrylate provided tetrahydropyrazolo[1,2-a]pyrazole-1-carboxylates as mixtures of syn- and anti-diastereomers. Testing for inhibition of dihydroorotate dehydrogenase of Plasmodium falciparum (PfDHODH) revealed high activity of some anti-isomers of the methyl esters, while the corresponding carboxylic acids and carboxamides were not active. The most active representative, methyl (1S*,3S*,5R*)-1,5-dimethyl-7-oxo-3-phenyltetrahydro-1H,5H-pyrazolo[1,2-a]pyrazole-1-carboxylate (IC₅₀ = 2.9 ± 0.3 μM), also exhibited very high selectivity of the parasite enzyme vs. the human enzyme, PfDHODH/HsDHODH > 350. According to the molecular docking score, this high activity is explainable by synergic interactions of the methyl, phenyl and the CO₂Me substituent with the hydrophobic pockets in the active site of the enzyme. The carboxylic acid and carboxamides derived from this compound did not inhibit PfDHODH.     

Structures of human dihydroorotate dehydrogenase in complex with antiproliferative agents

BACKGROUND: Dihydroorotate dehydrogenase (DHODH) catalyzes the fourth committed step in the de novo biosynthesis of pyrimidines. As rapidly proliferating human T cells have an exceptional requirement for de novo pyrimidine biosynthesis, small molecule DHODH inhibitors constitute an attractive therapeutic approach to autoimmune diseases, immunosuppression, and cancer. Neither the structure of human DHODH nor any member of its family was known. RESULTS: The high-resolution crystal structures of human DHODH in complex with two different inhibitors have been solved. The initial set of phases was obtained using multiwavelength anomalous diffraction phasing with selenomethionine-containing DHODH. The structures have been refined to crystallographic R factors of 16.8% and 16.2% at resolutions of 1. 6 A and 1.8 A for inhibitors related to brequinar and leflunomide, respectively. CONCLUSIONS: Human DHODH has two domains: an alpha/beta-barrel domain containing the active site and an alpha-helical domain that forms the opening of a tunnel leading to the active site. Both inhibitors share a common binding site in this tunnel, and differences in the binding region govern drug sensitivity or resistance. The active site of human DHODH is generally similar to that of the previously reported bacterial active site. The greatest differences are that the catalytic base removing the proton from dihydroorotate is a serine rather than a cysteine, and that packing of the flavin mononucleotide in its binding site is tighter.     

Development of a scintillation proximity binding assay for high-throughput screening of hematopoietic prostaglandin D2 synthase

Prostaglandin D₂ synthase (PGDS) catalyzes the isomerization of prostaglandin H₂ (PGH₂) to prostaglandin D₂ (PGD₂). PGD₂ produced by hematopoietic prostaglandin D₂ synthase (H-PGDS) in mast cells and Th2 cells is proposed to be a mediator of allergic and inflammatory responses. Consequently, inhibitors of H-PGDS represent potential therapeutic agents for the treatment of inflammatory diseases such as asthma. Due to the instability of the PGDS substrate PGH₂, an in-vitro enzymatic assay is not feasible for large-scale screening of H-PGDS inhibitors. Herein, we report the development of a competition binding assay amenable to high-throughput screening (HTS) in a scintillation proximity assay (SPA) format. This assay was used to screen an in-house compound library of approximately 280,000 compounds for novel H-PGDS inhibitors. The hit rate of the H-PGDS primary screen was found to be 4%. This high hit rate suggests that the active site of H-PGDS can accommodate a large diversity of chemical scaffolds. For hit prioritization, these initial hits were rescreened at a lower concentration in SPA and tested in the LAD2 cell assay. 116 compounds were active in both assays with IC₅₀s ranging from 6 to 807 nM in SPA and 82 nM to 10 μM in the LAD2 cell assay.     

Disruption of uridine homeostasis links liver pyrimidine metabolism to lipid accumulation

We report in this study an intrinsic link between pyrimidine metabolism and liver lipid accumulation utilizing a uridine phosphorylase 1 transgenic mouse model UPase1-TG. Hepatic microvesicular steatosis is induced by disruption of uridine homeostasis through transgenic overexpression of UPase1, an enzyme of the pyrimidine catabolism and salvage pathway. Microvesicular steatosis is also induced by the inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme of the de novo pyrimidine biosynthesis pathway. Interestingly, uridine supplementation completely suppresses microvesicular steatosis in both scenarios. The effective concentration (EC₅₀) for uridine to suppress microvesicular steatosis is approximately 20 µM in primary hepatocytes of UPase1-TG mice. We find that uridine does not have any effect on in vitro DHODH enzymatic activity. On the other hand, uridine supplementation alters the liver NAD⁺/NADH and NADP⁺/NADPH ratios and the acetylation profile of metabolic, oxidation-reduction, and antioxidation enzymes. Protein acetylation is emerging as a key regulatory mechanism for cellular metabolism. Therefore, we propose that uridine suppresses fatty liver by modulating the liver protein acetylation profile. Our findings reveal a novel link between uridine homeostasis, pyrimidine metabolism, and liver lipid metabolism.     

Mechanism of leflunomide-induced proliferation of mitochondria in mammalian cells

Leflunomide (LFM) is an inhibitor of mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) that catalyzes the conversion of dihydroorotate to orotate coupled with the generation of reactive oxygen species (ROS) from mitochondria. We demonstrate here that LFM causes an unrestrained proliferation of mitochondria both in human osteosarcoma cell line 143B cells and rat liver derived RL-34 cells. Increases in the total mass of mitochondria per cell in LFM-treated cells were evidenced by the application of Green FM or 10-n-nonyl acridine orange to flow cytometry, an enhanced replication of mtDNA and electron microscopy. Externally added uridine improved the disturbance in cell cycle progression in LFM-treated cells, but failed to suppress such unrestrained mitochondrial proliferation. On the contrary, lapacol and 5-fluoroorotate, inhibitors of DHODH besides LFM, suppressed the biogenesis of mitochondria during the cell cycle progression. LFM, but not lapacol or 5-fluoroorotate, caused increases of the intracellular level of acetylated alpha-tubulin. These data suggest that the inhibition of DHODH may not be at least primarily related to the LFM-induced abnormal proliferation of mitochondria, and support our recent published observation that changes in the physicochemical properties of microtubules may be in someway concerned with the biogenesis of mitochondria.     

Defects in vesicle core induced by escherichia coli dihydroorotate dehydrogenase

Dihydroorotate dehydrogenase (DHODH) catalyzes the oxidation of dihydroorotate to orotate during the fourth step of the de novo pyrimidine synthesis pathway. In rapidly proliferating mammalian cells, pyrimidine salvage pathway is insufficient to overcome deficiencies in that pathway for nucleotide synthesis. Moreover, as certain parasites lack salvage enzymes, relying solely on the de novo pathway, DHODH inhibition has turned out as an efficient way to block pyrimidine biosynthesis. Escherichia coli DHODH (EcDHODH) is a class 2 DHODH, found associated to cytosolic membranes through an N-terminal extension. We used electronic spin resonance (ESR) to study the interaction of EcDHODH with vesicles of 1,2-dioleoyl-sn-glycero-phosphatidylcholine/detergent. Changes in vesicle dynamic structure induced by the enzyme were monitored via spin labels located at different positions of phospholipid derivatives. Two-component ESR spectra are obtained for labels 5- and 10-phosphatidylcholine in presence of EcDHODH, whereas other probes show a single-component spectrum. The appearance of an additional spectral component with features related to fast-motion regime of the probe is attributed to the formation of a defect-like structure in the membrane hydrophobic region. This is probably the mechanism used by the protein to capture quinones used as electron acceptors during catalysis. The use of specific spectral simulation routines allows us to characterize the ESR spectra in terms of changes in polarity and mobility around the spin-labeled phospholipids. We believe this is the first report of direct evidences concerning the binding of class 2 DHODH to membrane systems.     

Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies

Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T. brucei did not inhibit growth in normal medium, but profoundly retarded growth in pyrimidine-depleted media or in the presence of the known pyrimidine uptake antagonist 5-fluorouracil (5-FU). These results have significant implications for the development of therapeutics to combat T. brucei infection. Specifically, a combination therapy including a T. brucei -specific DHODH inhibitor plus 5-FU may prove to be an effective therapeutic strategy. We also show that this trypanosomal enzyme is inhibited by known inhibitors of bacterial Class 1A DHODH, in distinction to the sensitivity of DHODH from human and other higher eukaryotes. This selectivity is supported by the crystal structure of the T. brucei enzyme, which is reported here at a resolution of 1.95 Å. Additional research, guided by the crystal structure described herein, is needed to identify potent inhibitors of T. brucei DHODH.