Three Arabidopsis UMP kinases have different roles in pyrimidine nucleotide biosynthesis and (deoxy)CMP salvage.

Pyrimidine nucleotide monophosphate biosynthesis ends in the cytosol with uridine monophosphate (UMP). UMP phosphorylation to uridine diphosphate (UDP) by UMP KINASEs (UMKs) is required for the generation of all pyrimidine (deoxy)nucleoside triphosphates as building blocks for nucleic acids and central metabolites like UDP-glucose. The Arabidopsis (Arabidopsis thaliana) genome encodes five UMKs and three belong to the AMP KINASE (AMK)-like UMKs, which were characterized to elucidate their contribution to pyrimidine metabolism. Mitochondrial UMK2 and cytosolic UMK3 are evolutionarily conserved, whereas cytosolic UMK1 is specific to the Brassicaceae. In vitro, all UMKs can phosphorylate UMP, cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP), but with different efficiencies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced null mutants were generated for UMK1 and UMK2, but not for UMK3, since frameshift alleles were lethal for germline cells. However, a mutant with diminished UMK3 activity showing reduced growth was obtained. Metabolome analyses of germinating seeds and adult plants of single- and higher-order mutants revealed that UMK3 plays an indispensable role in the biosynthesis of all pyrimidine (deoxy)nucleotides and UDP-sugars, while UMK2 is important for dCMP recycling that contributes to mitochondrial DNA stability. UMK1 is primarily involved in CMP recycling. We discuss the specific roles of these UMKs referring also to the regulation of pyrimidine nucleoside triphosphate synthesis.

Repair and DNA Polymerase Bypass of Clickable Pyrimidine Nucleotides.

Clickable nucleosides, most often 5-ethynyl-2'-deoxyuridine (EtU), are widely used in studies of DNA replication in living cells and in DNA functionalization for bionanotechology applications. Although clickable dNTPs are easily incorporated by DNA polymerases into the growing chain, afterwards they might become targets for DNA repair systems or interfere with faithful nucleotide insertion. Little is known about the possibility and mechanisms of these post-synthetic events. Here, we investigated the repair and (mis)coding properties of EtU and two bulkier clickable pyrimidine nucleosides, 5-(octa-1,7-diyn-1-yl)-U (C8-AlkU) and 5-(octa-1,7-diyn-1-yl)-C (C8-AlkC). In vitro, EtU and C8-AlkU, but not C8-AlkC, were excised by SMUG1 and MBD4, two DNA glycosylases from the base excision repair pathway. However, when placed into a plasmid encoding a fluorescent reporter inactivated by repair in human cells, EtU and C8-AlkU persisted for much longer than uracil or its poorly repairable phosphorothioate-flanked derivative. DNA polymerases from four different structural families preferentially bypassed EtU, C8-AlkU and C8-AlkC in an error-free manner, but a certain degree of misincorporation was also observed, especially evident for DNA polymerase ß. Overall, clickable pyrimidine nucleotides could undergo repair and be a source of mutations, but the frequency of such events in the cell is unlikely to be considerable.

Expanding the Available RNA Labeling Toolbox With CutA Nucleotidyltransferase for Efficient Transcript Labeling with Purine and Pyrimidine Nucleotide Analogs.

RNA labeling is an invaluable tool for investigation of the function and localization of nucleic acids. Labels are commonly incorporated into 3' end of RNA and the primary enzyme used for this purpose is RNA poly(A) polymerase (PAP), which belongs to the class of terminal nucleotidyltransferases (NTases). However, PAP preferentially adds ATP analogs, thus limiting the number of available substrates. Here, we report the use of another NTase, CutA from the fungus Thielavia terrestris. Using this enzyme, we were able to incorporate into the 3' end of RNA not only purine analogs, but also pyrimidine analogs. We engaged strain-promoted azide-alkyl cycloaddition (SPAAC) to obtain fluorescently labeled or biotinylated transcripts from RNAs extended with azide analogs by CutA. Importantly, modified transcripts retained their biological properties. Furthermore, fluorescently labeled mRNAs were suitable for visualization in cultured mammalian cells. Finally, we demonstrate that either affinity studies or molecular dynamic (MD) simulations allow for rapid screening of NTase substrates, what opens up new avenues in the search for the optimal substrates for this class of enzymes.

2',3'-Protected Nucleotides as Building Blocks for Enzymatic de novo RNA Synthesis.

Besides being a key player in numerous fundamental biological processes, RNA also represents a versatile platform for the creation of therapeutic agents and efficient vaccines. The production of RNA oligonucleotides, especially those decorated with chemical modifications, cannot meet the exponential demand. Due to the inherent limits of solid-phase synthesis and in vitro transcription, alternative, biocatalytic approaches are in dire need to facilitate the production of RNA oligonucleotides. Here, we present a first step towards the controlled enzymatic synthesis of RNA oligonucleotides. We have explored the possibility of a simple protection step of the vicinal cis-diol moiety to temporarily block ribonucleotides. We demonstrate that pyrimidine nucleotides protected with acetals, particularly 2',3'-O-isopropylidene, are well-tolerated by the template-independent RNA polymerase PUP (polyU polymerase) and highly efficient coupling reactions can be achieved within minutes - an important feature for the development of enzymatic de novo synthesis protocols. Even though purines are not equally well-tolerated, these findings clearly demonstrate the possibility of using cis-diol-protected ribonucleotides combined with template-independent polymerases for the stepwise construction of RNA oligonucleotides.

Reprogramming of pyrimidine nucleotide metabolism supports vigorous cell proliferation of normal and malignant T cells.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is triggered by infection with human T-cell lymphotropic virus-1 (HTLV-1). Here, we describe the reprogramming of pyrimidine biosynthesis in both normal T cells and ATL cells through regulation of uridine-cytidine kinase 2 (UCK2), which supports vigorous proliferation. UCK2 catalyzes the monophosphorylation of cytidine/uridine and their analogues during pyrimidine biosynthesis and drug metabolism. We found that UCK2 was overexpressed aberrantly in HTLV-1-infected T cells but not in normal T cells. T-cell activation via T-cell receptor (TCR) signaling induced expression of UCK2 in normal T cells. Somatic alterations and epigenetic modifications in ATL cells activate TCR signaling. Therefore, we believe that expression of UCK2 in HTLV-1-infected cells is induced by dysregulated TCR signaling. Recently, we established azacitidine-resistant (AZA-R) cells showing absent expression of UCK2. AZA-R cells proliferated normally in vitro, whereas UCK2 knockdown inhibited ATL cell growth. Although uridine and cytidine accumulated in AZA-R cells, possibly because of dysfunction of pyrimidine salvage biosynthesis induced by loss of UCK2 expression, the amount of UTP and CTP was almost the same as in parental cells. Furthermore, AZA-R cells were more susceptible to an inhibitor of dihydroorotic acid dehydrogenase, which performs the rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, and more resistant to dipyridamole, an inhibitor of pyrimidine salvage biosynthesis, suggesting that AZA-R cells adapt to UCK2 loss by increasing de novo pyrimidine nucleotide biosynthesis. Taken together, the data suggest that fine-tuning pyrimidine biosynthesis supports vigorous cell proliferation of both normal T cells and ATL cells.

Recent advances on patents of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors as antimalarial agents.

INTRODUCTION: Pyrimidine nucleotides are essential for the parasite's growth and replication. Parasites have only a de novo pathway for the biosynthesis of pyrimidine nucleotides. Dihydroorotate dehydrogenase (DHODH) enzyme is involved in the rate-limiting step of the pyrimidine biosynthesis pathway. DHODH is a biochemical target for the discovery of new antimalarial agents. AREA COVERED: This review discussed the development of patented PfDHODH inhibitors published between 2007 and 2023 along with their chemical structures and activities. EXPERT OPINION: PfDHODH enzyme is involved in the rate-limiting fourth step of the pyrimidine biosynthesis pathway. Thus, inhibition of PfDHODH using species-selective inhibitors has drawn much attention for treating malaria because they inhibit parasite growth without affecting normal human functions. Looking at the current scenario of antimalarial drug resistance with most of the available antimalarial drugs, there is a huge need for targeted newer agents. Newer agents with unique mechanisms of action may be devoid of drug toxicity, adverse effects, and the ability of parasites to quickly gain resistance, and PfDHODH inhibitors can be those newer agents. Many PfDHODH inhibitors were patented in the past, and the dependency of Plasmodium on de novo pyrimidine provided a new approach for the development of novel antimalarial agents.

The Warburg effect modulates DHODH role in ferroptosis: a review.

Ferroptosis is an iron-dependent regulated cell death that suppresses tumor growth. It is activated by extensive peroxidation of membrane phospholipids caused by oxidative stress. GPX4, an antioxidant enzyme, reduces these peroxidized membrane phospholipids thereby inhibiting ferroptosis. This enzyme has two distinct subcellular localization; the cytosol and mitochondria. Dihydroorotate dehydrogenase (DHODH) complements mitochondrial GPX4 in reducing peroxidized membrane phospholipids. It is the rate-limiting enzyme in de novo pyrimidine nucleotide biosynthesis. Its role in ferroptosis inhibition suggests that DHODH inhibitors could have two complementary mechanisms of action against tumors; inhibiting de novo pyrimidine nucleotide biosynthesis and enhancing ferroptosis. However, the link between mitochondrial function and ferroptosis, and the involvement of DHODH in the ETC suggests that its role in ferroptosis could be modulated by the Warburg effect. Therefore, we reviewed relevant literature to get an insight into the possible effect of this metabolic reprogramming on the role of DHODH in ferroptosis. Furthermore, an emerging link between DHODH and cellular GSH pool has also been highlighted. These insights could contribute to the rational design of ferroptosis-based anticancer drugs. Video Abstract.

ENT1 blockade by CNX-774 overcomes resistance to DHODH inhibition in pancreatic cancer.

Inhibitors of dihydroorotate dehydrogenase (DHODH), a key enzyme for de novo synthesis of pyrimidine nucleotides, have failed in clinical trials for various cancers despite robust efficacy in preclinical animal models. To probe for druggable mediators of DHODH inhibitor resistance, we performed a combination screen with a small molecule library against pancreatic cancer cell lines that are highly resistant to the DHODH inhibitor brequinar (BQ). The screen revealed that CNX-774, a preclinical Bruton tyrosine kinase (BTK) inhibitor, sensitizes resistant cell lines to BQ. Mechanistic studies showed that this effect is independent of BTK and instead results from inhibition of equilibrative nucleoside transporter 1 (ENT1) by CNX-774. We show that ENT1 mediates BQ resistance by taking up extracellular uridine, which is salvaged to generate pyrimidine nucleotides in a DHODH-independent manner. In BQ-resistant cell lines, BQ monotherapy slowed proliferation and caused modest pyrimidine nucleotide depletion, whereas combination treatment with BQ and CNX-774 led to profound cell viability loss and pyrimidine starvation. We also identify N-acetylneuraminic acid accumulation as a potential marker of the therapeutic efficacy of DHODH inhibitors. In an aggressive, immunocompetent pancreatic cancer mouse model, combined targeting of DHODH and ENT1 dramatically suppressed tumor growth and prolonged mouse survival. Overall, our study defines CNX-774 as a previously uncharacterized ENT1 inhibitor and provides strong proof of concept support for dual targeting of DHODH and ENT1 in pancreatic cancer.

Coordination Chemistry of Phosphate Groups in Systems Including Copper(II) Ions, Phosphoethanolamine and Pyrimidine Nucleotides.

The activity of phosphate groups of phosphoethanolamine and pyrimidine nucleotides (thymidine 5-monophosphate, cytidine 5-monophosphate and uridine 5'monophosphate) in the process of complexation metal ions in aqueous solution was studied. Using the potentiometric method with computer calculation of the data and spectroscopic methods such as UV-Vis, EPR, 13C and 31P NMR as well as FT-IR, the overall stability constants of the complexes as well as coordination modes were obtained. At lower pH, copper(II) ions are complexed only by phosphate groups, whereas the endocyclic nitrogen atom of nucleotides has been identified as a negative center interacting with the -NH3+ groups of phosphoethanolamine.

Azoles as Auxiliaries and Intermediates in Prebiotic Nucleoside Synthesis.

4,5-Dicyanoimidazole and 2-aminothiazole are azoles that have previously been implicated in prebiotic nucleotide synthesis. The former compound is a byproduct of adenine synthesis, and the latter compound has been shown to be capable of separating C2 and C3 sugars via crystallization as their aminals. We now report that the elusive intermediate cyanoacetylene can be captured by 4,5-dicyanoimidazole and accumulated as the crystalline compound N-cyanovinyl-4,5-dicyanoimidazole, thus providing a solution to the problem of concentration of atmospherically formed cyanoacetylene. Importantly, this intermediate is a competent cyanoacetylene surrogate, reacting with ribo-aminooxazoline in formamide to give ribo-anhydrocytidine ─ an intermediate in the divergent synthesis of purine and pyrimidine nucleotides. We also report a prebiotically plausible synthesis of 2-aminothiazole and examine the mechanism of its formation. The utilization of each of these azoles enhances the prebiotic synthesis of ribonucleotides, while their syntheses comport with the cyanosulfidic scenario we have previously described.

Structure-Based Prototyping of Allosteric Inhibitors of Human Uridine/Cytidine Kinase 2 (UCK2).

Pyrimidine nucleotide biosynthesis in humans is a promising chemotherapeutic target for infectious diseases caused by RNA viruses. Because mammalian cells derive pyrimidine ribonucleotides through a combination of de novo biosynthesis and salvage, combined inhibition of dihydroorotate dehydrogenase (DHODH; the first committed step in de novo pyrimidine nucleotide biosynthesis) and uridine/cytidine kinase 2 (UCK2; the first step in salvage of exogenous nucleosides) strongly attenuates viral replication in infected cells. However, while several pharmacologically promising inhibitors of human DHODH are known, to date there are no reports of medicinally viable leads against UCK2. Here, we use structure-based drug prototyping to identify two classes of promising leads that noncompetitively inhibit UCK2 activity. In the process, we have identified a hitherto unknown allosteric site at the intersubunit interface of this homotetrameric enzyme. By reducing the kcat of human UCK2 without altering its KM, these new inhibitors have the potential to enable systematic dialing of the fractional inhibition of pyrimidine salvage to achieve the desired antiviral effect with minimal host toxicity.

Ferrocene as a potential electrochemical reporting surrogate of abasic sites in DNA.

Methods for the real-time monitoring of the substrate acceptance of modified nucleotides by DNA polymerases are in high demand. In a step towards this aim, we have incorporated ferrocene-based abasic nucleotides into DNA templates and evaluated their compatibility with enzymatic synthesis of unmodified and modified DNA. All canonical nucleotides can be incorporated opposite ferrocene sites with a strong preference for purines. DNA polymerases with lesion-bypass capacity such as Dpo4 allow DNA synthesis to be resumed beyond the site of incorporation. Modified purine nucleotides can readily be incorporated opposite ferrocene basic site analogs, while pyrimidine nucleotides decorated with simple side-chains are also readily tolerated. These findings open up directions for the design of electrochemical sensing devices for the monitoring of enzymatic synthesis of natural or modified DNA.

Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-ß-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.

Although 2'-deoxy-2'-α-F-2'-ß-C-methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.

Epigenetic Pyrimidine Nucleotides in Competition with Natural dNTPs as Substrates for Diverse DNA Polymerases.

Five 2'-deoxyribonucleoside triphosphates (dNTPs) derived from epigenetic pyrimidines (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, 5-hydroxymethyluracil, and 5-formyluracil) were prepared and systematically studied as substrates for nine DNA polymerases in competition with natural dNTPs by primer extension experiments. The incorporation of these substrates was evaluated by a restriction endonucleases cleavage-based assay and by a kinetic study of single nucleotide extension. All of the modified pyrimidine dNTPs were good substrates for the studied DNA polymerases that incorporated a significant percentage of the modified nucleotides into DNA even in the presence of natural nucleotides. 5-Methylcytosine dNTP was an even better substrate for most polymerases than natural dCTP. On the other hand, 5-hydroxymethyl-2'-deoxyuridine triphosphate was not the best substrate for SPO1 DNA polymerase, which naturally synthesizes 5hmU-rich genomes of the SPO1 bacteriophage. The results shed light onto the possibility of gene silencing through recycling and random incorporation of epigenetic nucleotides and into the replication of modified bacteriophage genomes.

Control of a pyrimidine ribonucleotide salvage pathway in Pseudomonas oleovorans.

The control of a pyrimidine ribonucleotide salvage pathway in the bacterium Pseudomonas oleovorans ATCC 8062 was studied. This bacterium is important for its ability to synthesize polyesters as well as for its increasing clinical significance in humans. The pyrimidine salvage pathway enzymes pyrimidine nucleotide N-ribosidase and cytosine deaminase were investigated in P. oleovorans ATCC 8062 under selected culture conditions. Initially, the effect of carbon source on the two pyrimidine salvage enzymes in ATCC 8062 cells was examined and it was observed that cell growth on the carbon source succinate generally produced higher enzyme activities than did glucose or glycerol as a carbon source when ammonium sulfate served as the nitrogen source. Using succinate as a carbon source, growth on dihydrouracil as nitrogen source caused a 1.9-fold increase in the pyrimidine nucleotide N-ribosidase activity and a 4.8-fold increase in cytosine deaminase activity compared to the ammonium sulfate-grown cells. Growth of ATCC 8062 cells on cytosine or dihydrothymine as a nitrogen source elevated deaminase activity by more than double that observed for ammonium sulfate-grown cells. The findings indicated a relationship between this pyrimidine salvage pathway and the pyrimidine reductive catabolic pathway since growth on dihydrouracil appeared to increase the degradation of the pyrimidine ribonucleotide monophosphates to uracil. The uracil produced could be degraded by the pyrimidine base reductive catabolic pathway to ß-alanine as a source of nitrogen. This investigation could prove helpful to future work examining the metabolic relationship between pyrimidine salvage pathways and pyrimidine reductive catabolism in pseudomonads.

Total Synthesis and Stereochemistry Assignment of Nucleoside Antibiotic A-94964.

A collective total synthesis of eight diastereoisomers associated with NMR analysis leads to a full stereochemistry assignment of the structurally unique nucleoside antibiotic A-94964, which features an octuronic acid uridine core decorated with an α-D-mannopyranosyl residue and an α-D-N-acylglucosaminopyranosyl residue via a phosphodiester bridge.

Saliva Metabolomics in Dry Mouth Patients with Head and Neck Cancer or Sjögren's Syndrome.

The etiology of dry mouth conditions is multi-faceted. Patients radiated after head and neck cancer (HNC) and those with primary Sjögren's syndrome (pSS) share many of the same symptoms despite different causes. With the aim of better understanding the pathophysiology and biochemical processes behind dry mouth with different etiologies, we investigated the metabolic profile of 10 HNC patients, 9 pSS patients and 10 healthy controls using high-performance liquid chromatography-high resolution mass spectrometry (HPLC-MS) metabolomics. Principal component analysis (PCA) revealed different metabolic profiles when comparing all subjects included in the study. Both patient groups showed higher ratios of several pyrimidine nucleotides and nucleosides when compared to controls. This finding may indicate that purinergic signaling plays a role in dry mouth conditions. Moreover, significantly increased levels of DL-3-aminoisobutyric acid were found in HNC patients when compared to controls, and a similar tendency was observed in the pSS patients. Furthermore, a dysregulation in amino acid metabolism was observed in both patient groups. In conclusion, metabolomics analysis showed separate metabolic profiles for HNC and pSS patients as compared to controls that could be useful in diagnostics and for elucidating the different pathophysiologies. The demonstrated dysregulation of pyrimidine nucleotides and levels of metabolites derived from amino acids in the patient groups should be studied further.

Substrate binding modes of purine and pyrimidine nucleotides to human ecto-5'-nucleotidase (CD73) and inhibition by their bisphosphonic acid derivatives.

Human ecto-5-nucleotidase (CD73) is involved in purinergic signalling, which influences a diverse range of biological processes. CD73 hydrolyses AMP and is the major control point for the levels of extracellular adenosine. Inhibitors of CD73 thus block the immunosuppressive action of adenosine, a promising approach for cancer immunotherapy. Interestingly, ADP and ATP are competitive inhibitors of CD73, with the most potent small-molecule inhibitors to date being non-hydrolysable ADP analogues. While AMP is the major substrate of the enzyme, CD73 has been reported to hydrolyse other 5'-nucleoside monophosphates. Based on a fragment screening campaign at the BESSY II synchrotron, we present the binding modes of various deoxyribo- and ribonucleoside monophosphates and of four additional fragments binding to the nucleoside binding site of the open form of the enzyme. Kinetic analysis of monophosphate hydrolysis shows that ribonucleotide substrates are favoured over their deoxyribose equivalents with AMP being the best substrate. We characterised the initial step of AMP hydrolysis, the binding mode of AMP to the open conformation of CD73 and compared that to other monophosphate substrates. In addition, the inhibitory activity of various bisphosphonic acid derivatives of nucleoside diphosphates was determined. Although AMPCP remains the most potent inhibitor, replacement of the adenine base with other purines or with pyrimidines increases the Ki value only between twofold and sixfold. On the other hand, these nucleobases offer new opportunities to attach substituents for improved pharmacological properties.