SARS-CoV-2 couples evasion of inflammatory response to activated nucleotide synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolves rapidly under the pressure of host immunity, as evidenced by waves of emerging variants despite effective vaccinations, highlighting the need for complementing antivirals. We report that targeting a pyrimidine synthesis enzyme restores inflammatory response and depletes the nucleotide pool to impede SARS-CoV-2 infection. SARS-CoV-2 deploys Nsp9 to activate carbamoyl-phosphate synthetase, aspartate transcarbamoylase, and dihydroorotase (CAD) that catalyzes the rate-limiting steps of the de novo pyrimidine synthesis. Activated CAD not only fuels de novo nucleotide synthesis but also deamidates RelA. While RelA deamidation shuts down NF-κB activation and subsequent inflammatory response, it up-regulates key glycolytic enzymes to promote aerobic glycolysis that provides metabolites for de novo nucleotide synthesis. A newly synthesized small-molecule inhibitor of CAD restores antiviral inflammatory response and depletes the pyrimidine pool, thus effectively impeding SARS-CoV-2 replication. Targeting an essential cellular metabolic enzyme thus offers an antiviral strategy that would be more refractory to SARS-CoV-2 genetic changes.

Identification of Small Molecule Inhibitors against Staphylococcus aureus Dihydroorotase via HTS.

Drug-resistant Staphylococcus aureus is an imminent threat to public health, increasing the importance of drug discovery utilizing unexplored bacterial pathways and enzyme targets. De novo pyrimidine biosynthesis is a specialized, highly conserved pathway implicated in both the survival and virulence of several clinically relevant pathogens. Class I dihydroorotase (DHOase) is a separate and distinct enzyme present in gram positive bacteria (i.e., S. aureus, B. anthracis) that converts carbamoyl-aspartate (Ca-asp) to dihydroorotate (DHO)-an integral step in the de novo pyrimidine biosynthesis pathway. This study sets forth a high-throughput screening (HTS) of 3000 fragment compounds by a colorimetry-based enzymatic assay as a primary screen, identifying small molecule inhibitors of S. aureus DHOase (SaDHOase), followed by hit validation with a direct binding analysis using surface plasmon resonance (SPR). Competition SPR studies of six hit compounds and eight additional analogs with the substrate Ca-asp determined the best compound to be a competitive inhibitor with a KD value of 11 µM, which is 10-fold tighter than Ca-asp. Preliminary structure-activity relationship (SAR) provides the foundation for further structure-based antimicrobial inhibitor design against S. aureus.

Plumbagin, a Natural Product with Potent Anticancer Activities, Binds to and Inhibits Dihydroorotase, a Key Enzyme in Pyrimidine Biosynthesis.

Dihydroorotase (DHOase) is the third enzyme in the de novo biosynthesis pathway for pyrimidine nucleotides, and an attractive target for potential anticancer chemotherapy. By screening plant extracts and performing GC-MS analysis, we identified and characterized that the potent anticancer drug plumbagin (PLU), isolated from the carnivorous plant Nepenthes miranda, was a competitive inhibitor of DHOase. We also solved the complexed crystal structure of yeast DHOase with PLU (PDB entry 7CA1), to determine the binding interactions and investigate the binding modes. Mutational and structural analyses indicated the binding of PLU to DHOase through loop-in mode, and this dynamic loop may serve as a drug target. PLU exhibited cytotoxicity on the survival, migration, and proliferation of 4T1 cells and induced apoptosis. These results provide structural insights that may facilitate the development of new inhibitors targeting DHOase, for further clinical anticancer chemotherapies.

Combined small molecule and loss-of-function screen uncovers estrogen receptor alpha and CAD as host factors for HDV infection and antiviral targets.

OBJECTIVE: Hepatitis D virus (HDV) is a circular RNA virus coinfecting hepatocytes with hepatitis B virus. Chronic hepatitis D results in severe liver disease and an increased risk of liver cancer. Efficient therapeutic approaches against HDV are absent. DESIGN: Here, we combined an RNAi loss-of-function and small molecule screen to uncover host-dependency factors for HDV infection. RESULTS: Functional screening unravelled the hypoxia-inducible factor (HIF)-signalling and insulin-resistance pathways, RNA polymerase II, glycosaminoglycan biosynthesis and the pyrimidine metabolism as virus-hepatocyte dependency networks. Validation studies in primary human hepatocytes identified the carbamoyl-phosphatesynthetase 2, aspartate transcarbamylase and dihydroorotase (CAD) enzyme and estrogen receptor alpha (encoded by ESR1) as key host factors for HDV life cycle. Mechanistic studies revealed that the two host factors are required for viral replication. Inhibition studies using N-(phosphonoacetyl)-L-aspartic acid and fulvestrant, specific CAD and ESR1 inhibitors, respectively, uncovered their impact as antiviral targets. CONCLUSION: The discovery of HDV host-dependency factors elucidates the pathogenesis of viral disease biology and opens therapeutic strategies for HDV cure.

Targeting Pyrimidine Pathway of Plasmodium knowlesi: New Strategies Towards Identification of Novel Antimalarial Chemotherapeutic Agents.

AIM AND OBJECTIVE: Plasmodium knowlesi has been recently recognized as a human malarial parasite, particularly in the region of south-east Asia. Unlike human host, P. knowlesi cannot salvage pyrimidine bases and relies solely on nucleotides synthesized from de novo pyrimidine pathway. The enzymes involved in this are also unique in terms of their structure and function to its human counterpart. Thus, targeting Dihydroorotase, an enzyme involved in the pyrimidine biosynthesis, provides a promising route for novel drug development. MATERIALS AND METHODS: The 3D structure of P. knowlesi Dihydroorotase was predicted, refined and validated. Multiple docking was performed and the resultant complex was used for 3D structurebased pharmacophore modelling. A combinatorial library of 2,664,779 molecules was generated and used for structure based virtual screening. The stability of resultant compounds was checked using simulation studies. RESULTS: The modelled 3D structure of P. knowlesi Dihydroorotase enzyme is relaxed by running an MD simulation of 20 ns, and structure is validated by using Ramachandran plot and G-factor analysis. A five point based pharmacophore model was created and used as a query for screening in house database. The stability of two negatively charged compounds was studied, and ZINC22066495-DHOase complex was more stable throughout the simulation. CONCLUSION: The present study shows that ZINC22066495 compound has a high potential for disrupting P. knowlesi DHOase enzyme and may be used as a potential lead molecule for effective pyrimidine biosynthesis inhibition in P. knowlesi.

Biochemical characterization of dihydroorotase of Leishmania donovani: Understanding pyrimidine metabolism through its inhibition.

De novo pyrimidine biosynthesis pathway is well developed and functional in protozoan parasite Leishmania donovani. The dihydroorotase (LdDHOase) is third enzyme of the pathway. The enzyme was cloned, expressed in E. coli BL21 (DE3), purified to homogeneity and biochemically characterized. The estimated kcat for the forward reaction and reverse reactions were 2.1 ± 0.1 s-1 and 1.1 ± 0.15 s-1, respectively. Homology modeling and docking studies were done to find out potential inhibitors for LdDHOase. Biotin sulfone and Kaempferol were found to be potential inhibitors of LdDHOase based on docking studies. These inhibitors were verified using recombinant LdDHOase and their anti-leishmanial effect was evaluated. Moreover, alterations in expressions of de novo as well as salvage pathways enzymes, after treatment of L. donovani with dihydroorotase inhibitor(s) were evaluated and discussed as survival mechanism of the pathogen. Further, effect of inhibition of cytidine deaminase, a key enzyme of salvage pathway of pyrimidine biosynthesis, was also evaluated on parasitic survival and alteration in gene expression of enzymes of both pathways. Further, effect of both pathways inhibition was also evaluated. The data suggests that the inhibition of single pathway can be overcome by increased expression of enzyme(s) of alternate pathway and both pathways seem to be equally important in the pathogen. When both pathways are simultaneously inhibited, parasite shows significant DNA damage and parasitic death.

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 (KD) 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.

Allantoinase and dihydroorotase binding and inhibition by flavonols and the substrates of cyclic amidohydrolases.

Allantoinase and dihydroorotase are members of the cyclic amidohydrolases family. Allantoinase and dihydroorotase possess very similar binuclear metal centers in the active site and may use a similar mechanism for catalysis. However, whether the substrate specificities of allantoinase and dihydroorotase overlap and whether the substrates of other cyclic amidohydrolases inhibit allantoinase and dihydroorotase remain unknown. In this study, the binding and inhibition of allantoinase (Salmonella enterica serovar Typhimurium LT2) and dihydroorotase (Klebsiella pneumoniae) by flavonols and the substrates of other cyclic amidohydrolases were investigated. Hydantoin and phthalimide, substrates of hydantoinase and imidase, were not hydrolyzed by allantoinase and dihydroorotase. Hydantoin and dihydroorotate competitively inhibited allantoinase, whereas hydantoin and allantoin bind to dihydroorotase, but do not affect its activity. We further investigated the effects of the flavonols myricetin, quercetin, kaempferol, and galangin, on the inhibition of allantoinase and dihydroorotase. Allantoinase and dihydroorotase were both significantly inhibited by kaempferol, with IC50 values of 35 ± 3 µM and 31 ± 2 µM, respectively. Myricetin strongly inhibited dihydroorotase, with an IC50 of 40 ± 1 µM. The double reciprocal of the Lineweaver-Burk plot indicated that kaempferol was a competitive inhibitor for allantoinase but an uncompetitive inhibitor for dihydroorotase. A structural study using PatchDock showed that kaempferol was docked in the active site pocket of allantoinase but outside the active site pocket of dihydroorotase. These results constituted a first study that naturally occurring product flavonols inhibit the cyclic amidohydrolases, allantoinase, and dihydroorotase, even more than the substrate analogs (>3 orders of magnitude). Thus, flavonols may serve as drug leads for designing compounds that target several cyclic amidohydrolases.

The nucleotide synthesis enzyme CAD inhibits NOD2 antibacterial function in human intestinal epithelial cells.

BACKGROUND & AIMS: Polymorphisms that reduce the function of nucleotide-binding oligomerization domain (NOD)2, a bacterial sensor, have been associated with Crohn's disease (CD). No proteins that regulate NOD2 activity have been identified as selective pharmacologic targets. We sought to discover regulators of NOD2 that might be pharmacologic targets for CD therapies. METHODS: Carbamoyl phosphate synthetase/aspartate transcarbamylase/dihydroorotase (CAD) is an enzyme required for de novo pyrimidine nucleotide synthesis; it was identified as a NOD2-interacting protein by immunoprecipitation-coupled mass spectrometry. CAD expression was assessed in colon tissues from individuals with and without inflammatory bowel disease by immunohistochemistry. The interaction between CAD and NOD2 was assessed in human HCT116 intestinal epithelial cells by immunoprecipitation, immunoblot, reporter gene, and gentamicin protection assays. We also analyzed human cell lines that express variants of NOD2 and the effects of RNA interference, overexpression and CAD inhibitors. RESULTS: CAD was identified as a NOD2-interacting protein expressed at increased levels in the intestinal epithelium of patients with CD compared with controls. Overexpression of CAD inhibited NOD2-dependent activation of nuclear factor κB and p38 mitogen-activated protein kinase, as well as intracellular killing of Salmonella. Reduction of CAD expression or administration of CAD inhibitors increased NOD2-dependent signaling and antibacterial functions of NOD2 variants that are and are not associated with CD. CONCLUSIONS: The nucleotide synthesis enzyme CAD is a negative regulator of NOD2. The antibacterial function of NOD2 variants that have been associated with CD increased in response to pharmacologic inhibition of CAD. CAD is a potential therapeutic target for CD.

The dihydroorotase inhibitor 5-aminoorotic acid inhibits the metabolism in the rat of the cardioprotective drug dexrazoxane and its one-ring open metabolites.

Dexrazoxane (ICRF-187) is clinically used as a doxorubicin cardioprotective agent and to prevent anthracycline extravasation injury. It may act by preventing iron-based oxygen free radical damage through the iron-chelating ability of its metabolite N,N'-[(1S)-1-methyl-1,2-ethanediyl]bis[(N-(2-amino-2-oxoethyl)]glycine (ADR-925). Dexrazoxane undergoes an initial metabolism to its two one-ring open intermediates [N-(2-amino-2-oxoethyl)-N-[(1S)-2-(3,5-dioxo-1-piperazinyl)-1-methylethyl]glycine (B) and N-(2-amino-2-oxoethyl)-N-[(2S)-2-(3,5-dioxo-1-piperazinyl)propyl]glycine (C)] and is then further metabolized to its presumably active metal-chelating form ADR-925. We previously showed that the first ring opening reaction is catalyzed by dihydropyrimidinase and the second by dihydroorotase (DHOase), but not vice versa. To determine whether DHOase was important in the metabolism of dexrazoxane, its metabolism and that of B and C to ADR-925 were measured in rats that were pretreated with the DHOase inhibitor 5-aminoorotic acid. In rats pretreated with 5-aminoorotic acid the area-under-the-curve concentration of ADR-925 was reduced 5.3-fold. In rats treated with a mixture of B and C, the maximum concentration of ADR-925 in the plasma was significantly decreased in rats pretreated with 5-aminoorotic acid, which indicates that DHOase directly metabolized B and C. Both heart and liver tissue levels of ADR-925 in rats were also greatly reduced by pretreatment with 5-aminoorotic acid. Together these results indicate that the metabolism of dexrazoxane and of B and C is mediated by DHOase. These results provide a mechanistic basis for the antioxidant cardioprotective activity of dexrazoxane.

Structures of ligand-free and inhibitor complexes of dihydroorotase from Escherichia coli: implications for loop movement in inhibitor design.

Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-L-aspartate (CA-asp) to L-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. DHOase is a potential anti-malarial drug target as malarial parasites can only synthesize pyrimidines via the de novo pathway and do not possess a salvage pathway. Here we report the structures of Escherichia coli DHOase crystallized without ligand (1.7 A resolution) and in the presence of the inhibitors 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate (HDDP; 2.0 A) and 5-fluoroorotate (FOA, 2.2 A). These are the first crystal structures of DHOase-inhibitor complexes, providing structural information on the mode of inhibitor binding. HDDP possesses features of both the substrate and product, and ligates the Zn atoms in the active site. In addition, HDDP forms hydrogen bonds to the flexible loop (residues 105-115) stabilizing the "loop-in" conformation of the flexible loop normally associated with the presence of CA-asp in the active site. By contrast, FOA, a product-like inhibitor, binds to the active site in a similar fashion to DHO but does not ligate the Zn atoms directly nor stabilize the loop-in conformation. These structures define the necessary features for the future design of improved inhibitors of DHOase.

Structure of the T109S mutant of Escherichia coli dihydroorotase complexed with the inhibitor 5-fluoroorotate: catalytic activity is reflected by the crystal form.

Crystals of a single-point mutant (T109S) of Escherichia coli dihydroorotase (DHOase) with diminished activity grown in the presence of L-dihydroorotate (L-DHO) are tetragonal, with a monomer in the asymmetric unit. These crystals are extremely unstable and disintegrate shortly after formation, which is followed by the growth of orthorhombic crystals from the remnants of the tetragonal crystals or at new nucleation sites. Orthorhombic crystals, for which a structure has previously been reported [Thoden et al. (2001), Biochemistry, 40, 6989-6997; Lee et al. (2005), J. Mol. Biol. 348, 523-533], contain a dimer of DHOase in the asymmetric unit; the active site of one monomer contains the substrate N-carbamyl-L-aspartate (L-CA-asp) and the active site of the other monomer contains the product of the reaction, L-DHO. In the subunit with L-DHO in the active site, a surface loop (residues 105-115) is 'open'. In the other subunit, with L-CA-asp in the active site, the loop folds inwards, forming specific hydrogen bonds from the loop to the L-CA-asp. The tetragonal crystal form can be stabilized by crystallization in the presence of the inhibitor 5-fluoroorotate (FOA), a product (L-DHO) mimic. Crystals of the complex of T109S DHOase with FOA are tetragonal, space group P4(1)2(1)2, with unit-cell parameters a = b = 72.6, c = 176.1 A. The structure has been refined to R and R(free) values of 0.218 and 0.257, despite severe anisotropy of the diffraction. In this structure, the flexible loops are both in the 'open' conformation, which is consistent with FOA, like L-DHO, binding at both sites. The behaviour of the T109S mutant crystals of DHOase in the presence of L-DHO is explained by initial binding of L-DHO to both subunits, followed by slow conversion to L-CA-asp, with consequent movement of the flexible loop and dissolution of the crystals. Orthorhombic crystals are then able to grow in the presence of L-DHO and L-CA-asp.

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction.

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.

Inhibitors designed for the active site of dihydroorotase.

Four new compounds have been synthesized as potential inhibitors of dihydroorotase from Escherichia coli. NMR spectroscopy was used to show that 4,6-dioxo-piperidine-2(S)-carboxylic acid (3), exists in solution as a mixture of the hydrate (7), enol (8), and enolate (9) tautomeric forms. This compound was found to be a competitive inhibitor versus dihydroorotate and thio-dihydroorotate at pH values of 7-9. The K(i) of 76 microM was lowest at pH7.0 where the ketone and hydrate forms of the inhibitor 3 predominate in solution. Compound 3 was reduced to the two diastereomeric 4-hydroxy derivatives (4 and 5) and then dehydrated to yield the alkene derivative, 1,2,3,6-tetrahydro-6-oxopyridine-2(S)-carboxylic acid (6). Compounds 4-6 were competitive inhibitors versus thio-dihydroorotate at pH 8.0 with K(i) values of 3.0, 1.6, and 2.3 mM. Dihydroorotase was unable to dehydrate the 4-hydroxy derivative 4 or 5 to the alkene 6 or catalyze the reverse reaction.

Experimental charge density of a potential DHO synthetase inhibitor: dimethyl-trans-2-oxohexahydro-pyrimidine-4,6-dicarboxylate.

The experimental charge density distribution of dimethyl-trans-2-oxohexahydro-pyrimidine-4,6-dicarboxylate 1 has been determined using single-crystal X-ray diffraction data measured at 100 K, in terms of the rigid-pseudoatom formalism. Multipole refinement converged at R(F) = 0.034 for 7283 reflections with I > 3 sigma (I) and sin theta/lambda < or = 1.13 A(-1). Covalent and hydrogen bonding interactions are analyzed using a topological analysis of the Laplacian of the charge density. The experimentally derived electrostatic potential mapped onto the reactive surface of the molecule reveals the potential binding sites of 1.

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.

Regulation of carbamoyl phosphate synthetase by MAP kinase.

The de novo synthesis of pyrimidine nucleotides is required for mammalian cells to proliferate. The rate-limiting step in this pathway is catalysed by carbamoyl phosphate synthetase (CPS II), part of the multifunctional enzyme CAD. Here we describe the regulation of CAD by the mitogen-activated protein (MAP) kinase cascade. When phosphorylated by MAP kinase in vitro or activated by epidermal growth factor in vivo, CAD lost its feedback inhibition (which is dependent on uridine triphosphate) and became more sensitive to activation (which depends upon phosphoribosyl pyrophosphate). Both these allosteric regulatory changes favour biosynthesis of pyrimidines for growth. They were accompanied by increased epidermal growth factor-dependent phosphorylation of CAD in vivo and were prevented by inhibition of MAP kinase. Mutation of a consensus MAP kinase phosphorylation site abolished the changes in CAD allosteric regulation that were stimulated by growth factors. Finally, consistent with an effect of MAP kinase signalling on CPS II activity, epidermal growth factor increased cellular uridine triphosphate and this increase was reversed by inhibition of MAP kinase. Hence these studies may indicate a direct link between activation of the MAP kinase cascade and de novo biosynthesis of pyrimidine nucleotides.

Synthesis and enzymic evaluation of 4-mercapto-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide as an inhibitor of mammalian dihydroorotase.

The design, synthesis, and enzymic evaluation of cis- and trans-4-mercapto-6-oxo-1,4-azaphosphinane-2-carboxylic acid 4-oxide 5 against mammalian dihydroorotase is presented. The design strategy for 5 was based on the strong affinity of phosphinothioic acids for zinc and that 5 also resembles the postulated tetrahedral transition state for the enzyme-catalyzed reaction. The synthesis of 5 utilized a novel protection/deprotection sequence upon 4-hydroxy-6-oxo-1, 4-azaphosphinane-2-carboxylic acid 4-oxide 4, followed by incorporation of alpha-phenyl benzenemethanethiol and exhaustive deprotection to afford 5 in 40% overall yield from 4. The activities of both isomers of 5 as inhibitors of mammalian dihydroorotase were marginally greater than that of the parent phosphinic acid 4, indicating a weak binding enhancement due to the phosphinothioic acid moiety.

dCTP levels are maintained in Plasmodium falciparum subjected to pyrimidine deficiency or excess.

The pyrimidine antagonists, 6-L-thiodihydroorotate (TDHO) and atovaquone, are known to induce inhibition of de-novo pyrimidine biosynthesis in Plasmodium falciparum growing in erythrocytic culture, at reactions catalysed by dihydroorotase and dihydroorotate dehydrogenase, respectively. In the present study, TDHO and atovaquone induced decreases in the levels of UTP, CTP and dTTP but not dCTP in P. falciparum. Addition of orotate with either antagonist increased UTP, CTP and dTTP but depressed GTP, ATP, dATP and dCTP, suggesting that these drugs indirectly modulate the activity of ribonucleotide reductase. The changes induced in the levels of dNTP by these pyrimidine antagonists are similar to those previously described for the antifolates, cycloguanil and WR99210.