Pyrimidine Research Articles

Brequinar and dipyridamole in combination exhibits synergistic antiviral activity against SARS-CoV-2 in vitro: Rationale for a host-acting antiviral treatment strategy for COVID-19

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19) and the associated global pandemic resulting in >400 million infections worldwide and several million deaths. The continued evolution of SARS-CoV-2 to potentially evade vaccines and monoclonal antibody (mAb)-based therapies and the limited number of authorized small-molecule antivirals necessitates the need for development of new drug treatments. There remains an unmet medical need for effective and convenient treatment options for SARS-CoV-2 infection. SARS-CoV-2 is an RNA virus that depends on host intracellular ribonucleotide pools for its replication. Dihydroorotate dehydrogenase (DHODH) is a ubiquitous host enzyme that is required for de novo pyrimidine synthesis. The inhibition of DHODH leads to a depletion of intracellular pyrimidines, thereby impacting viral replication in vitro. Brequinar (BRQ) is an orally available, selective, and potent low nanomolar inhibitor of human DHODH that has been shown to exhibit broad spectrum inhibition of RNA virus replication. However, host cell nucleotide salvage pathways can maintain intracellular pyrimidine levels and compensate for BRQ-mediated DHODH inhibition. In this report, we show that the combination of BRQ and the salvage pathway inhibitor dipyridamole (DPY) exhibits strong synergistic antiviral activity in vitro against SARS-CoV-2 by enhanced depletion of the cellular pyrimidine nucleotide pool. The combination of BRQ and DPY showed antiviral activity against the prototype SARS-CoV-2 as well as the Beta (B.1.351) and Delta (B.1.617.2) variants. These data support the continued evaluation of the combination of BRQ and DPY as a broad-spectrum, host-acting antiviral strategy to treat SARS-CoV-2 and potentially other RNA virus infections.

De novo pyrimidine synthesis is a targetable vulnerability in IDH mutant glioma.

SUMMARYMutations affecting isocitrate dehydrogenase (IDH) enzymes are prevalent in glioma, leukemia, and other cancers. Although mutant IDH inhibitors are effective against leukemia, they seem to be less active in aggressive glioma, underscoring the need for alternative treatment strategies. Through a chemical synthetic lethality screen, we discovered that IDH1-mutant glioma cells are hypersensitive to drugs targeting enzymes in the de novo pyrimidine nucleotide synthesis pathway, including dihydroorotate dehydrogenase (DHODH). We developed a genetically engineered mouse model of mutant IDH1-driven astrocytoma and used it and multiple patient-derived models to show that the brain-penetrant DHODH inhibitor BAY 2402234 displays monotherapy efficacy against IDH-mutant gliomas. Mechanistically, this reflects an obligate dependence of glioma cells on the de novo pyrimidine synthesis pathway and mutant IDH’s ability to sensitize to DNA damage upon nucleotide pool imbalance. Our work outlines a tumor-selective, biomarker-guided therapeutic strategy that is poised for clinical translation.

Inhibitors of Nucleotide Biosynthesis as Candidates for a Wide Spectrum of Antiviral Chemotherapy.

Emerging and re-emerging viruses have been a challenge in public health in recent decades. Host-targeted antivirals (HTA) directed at cellular molecules or pathways involved in virus multiplication represent an interesting strategy to combat viruses presently lacking effective chemotherapy. HTA could provide a wide range of agents with inhibitory activity against current and future viruses that share similar host requirements and reduce the possible selection of antiviral-resistant variants. Nucleotide metabolism is one of the more exploited host metabolic pathways as a potential antiviral target for several human viruses. This review focuses on the antiviral properties of the inhibitors of pyrimidine and purine nucleotide biosynthesis, with an emphasis on the rate-limiting enzymes dihydroorotate dehydrogenase (DHODH) and inosine monophosphate dehydrogenase (IMPDH) for which there are old and new drugs active against a broad spectrum of pathogenic viruses.

Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-β-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′-β-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.

ATP enhances the error-prone ribonucleotide incorporation by the SARS-CoV-2 RNA polymerase

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2 or COVID-19) has caused a global pandemic. The SARS-CoV-2 RNA genome is replicated by a conserved “core” replication-transcription complex (RTC) containing an error-prone RNA-dependent RNA polymerase holoenzyme (holo-RdRp, nsp12-nsp7-nsp8) and a RNA proofreading nuclease (nsp14-nsp10). Although structures and functions of SARS-CoV-2 holo-RdRp have been extensively studied and ribonucleotide-analog inhibitors, such as Remdesivir, have been treated for COVID-19 patients, the substrate and nucleotide specificity of SARS-CoV-2 holo-RdRp remain unknown. Here, our biochemical analysis of SARS-CoV-2 holo-RdRp reveals that it has a robust DNA-dependent RNA polymerase activity, in addition to its intrinsic RNA-dependent RNA polymerase activity. Strikingly, SARS-CoV-2 holo-RdRp fully extends RNAs with a low-fidelity even when only ATP and pyrimidine nucleotides, in particular CTP, are provided. This ATP-dependent error-prone ribonucleotide incorporation by SARS-CoV-2 holo-RdRp resists excision by the RNA proofreading nuclease in vitro. Our collective results suggest that a physiological concentration of ATP likely contributes to promoting the error-prone incorporation of ribonucleotides and ribonucleotide-analogs by SARS-CoV-2 holo-RdRp and provide a useful foundation to develop ribonucleotide analogs as an effective therapeutic strategy to combat coronavirus-mediated outbreak.

Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase.

Terminal deoxynucleotidyltransferase (TdT) is a member of the DNA polymerase X family that is responsible for random addition of nucleotides to single-stranded DNA. We present investigation into the role of metal ions and specific interactions of dNTP with active-site amino acid residues in the mechanisms underlying the recognition of nucleoside triphosphates by human TdT under pre-steady-state conditions. In the elongation mode, the ratios of translocation and dissociation rate constants, as well as the catalytic rate constant were dependent on the nature of the nucleobase. Preferences of TdT in dNTP incorporation were researched by molecular dynamics simulations of complexes of TdT with a primer and dNTP or with the elongated primer. Purine nucleotides lost the "summarised" H-bonding network after the attachment of the nucleotide to the primer, whereas pyrimidine nucleotides increased the number and relative lifetime of H-bonds in the post-catalytic complex. The effect of divalent metal ions on the primer elongation revealed that Me<sup>2+</sup> cofactor can significantly change parameters of the primer elongation by strongly affecting the rate of nucleotide attachment and the polymerisation mode.

5′-CMP and 5′-UMP promote myogenic differentiation and mitochondrial biogenesis by activating myogenin and PGC-1α in a mouse myoblast C2C12 cell line

Ribonucleotides are basic monomeric building blocks for RNA considered as conditionally essential nutrients. They are normally produced in sufficient quantity, but can become insufficient upon stressful challenges. The administration of pyrimidine nucleotides, such as cytidine-5′-monophosphate (5′-CMP) and uridine-5′-monophosphate (5′-UMP), enables rats to endure prolonged exercise. However, the underlying mechanisms have remained elusive. To investigate these mechanisms, we studied the effect of 5′-CMP and 5′-UMP on muscular differentiation and mitochondrial biogenesis in myoblast C2C12 cells. 5′-CMP and 5′-UMP were found to increase the mRNA levels of myogenin, which is a myogenic regulatory protein expressed during the final differentiation step and fusion of myoblasts into myotubes. 5′-CMP and 5′-UMP also promoted myoblast differentiation into myotube cells. 5′-CMP and 5′-UMP further increased the mRNA levels of PGC-1α which regulates mitochondrial biogenesis and skeletal muscle fiber type. In addition, 5′-CMP and 5′-UMP increased mitochondrial DNA copy number and enhanced mRNA levels of slow-muscle myosin heavy chains. Moreover, cytidine and uridine, nucleosides corresponding to 5′-CMP and 5′-UMP, markedly promoted myotube formation in C2C12 cells. Considering the metabolism and absorption of nucleotides, the active bodies underlying the effects observed with 5′-CMP and 5′-UMP could be cytidine and uridine. In conclusion, our results indicate that 5′-CMP and 5′-UMP can promote myogenic differentiation and mitochondrial biogenesis, as well as increase slow-twitch fiber via the activation of myogenin and PGC-1α. In addition, 5′-CMP and 5′-UMP may be considered as safe and effective agents to enhance muscle growth and improve the endurance in skeletal muscles.

PRS genes, a paradigm for gene duplication and its potential for yeast research?

TheS. cerevisiaegenome contains five highly homologous PRS genes, each capable of encoding PRPP (phosphoribosyl-pyrophosphate) synthetase, a key enzyme essential for the synthesis of purine and pyrimidine nucleotides. PRS1andPRS5may have arisen from the prototype Prs-encoding genes, PRS2, PRS3andPRS4by duplication followed by acquisition of non-homologous regions (NHRs), shown to link primary metabolism with intracellular signalling. ThePRSgenes are highly conserved from yeast to human. We have created genocopies in PRS1 associated with the human neuropathies, Arts syndrome and CMTX5 which caused increased caffeine sensitivity and elevated Rlm1 expression in the mutated strains. The insertions in Prs1 and Prs5 serve as a physical link to the cell wall integrity (CWI) pathway and explain why cell viability requires specific heterodimers for the provision of PRPP and the maintenance of CWI integrity. The interaction between Prs1 and Mpk1/Slt2, a component of the CWI pathway was demonstrated by immunoprecipitation. Prs3 contains a NLS (nuclear localisation site), deletion of which causes caffeine sensitivity, reduced Rlm1 expression and loss of the Prs1/Prs3 heterodimer. The contribution of Prs5 to CWI is shown by increased phosphorylation of Mpk1/Slt2 following mutation of the three phosphosites located within NHR5-2. We now intend to investigate the signalling pathway connecting the conserved master regulator TOR (target of rapamycin) with PRPP synthetase, thus adding a new dimension to the application of yeast research in the discovery of novel therapeutic targets for the treatment of human neuropathies, CMTX5 and Arts syndrome.

Enabling Role of Ligand-Driven Conformational Changes in Enzyme Evolution.

Many enzymes that show a large specificity in binding the enzymatic transition state with a higher affinity than the substrate utilize substrate binding energy to drive protein conformational changes to form caged substrate complexes. These protein cages provide strong stabilization of enzymatic transition states. Using part of the substrate binding energy to drive the protein conformational change avoids a similar strong stabilization of the Michaelis complex and irreversible ligand binding. A seminal step in the development of modern enzyme catalysts was the evolution of enzymes that couple substrate binding to a conformational change. These include enzymes that function in glycolysis (triosephosphate isomerase), the biosynthesis of lipids (glycerol phosphate dehydrogenase), the hexose monophosphate shunt (6-phosphogluconate dehydrogenase), and the mevalonate pathway (isopentenyl diphosphate isomerase), catalyze the final step in the biosynthesis of pyrimidine nucleotides (orotidine monophosphate decarboxylase), and regulate the cellular levels of adenine nucleotides (adenylate kinase). The evolution of enzymes that undergo ligand-driven conformational changes to form active protein-substrate cages is proposed to proceed by selection of variants, in which the selected side chain substitutions destabilize a second protein conformer that shows compensating enhanced binding interactions with the substrate. The advantages inherent to enzymes that incorporate a conformational change into the catalytic cycle provide a strong driving force for the evolution of flexible protein folds such as the TIM barrel. The appearance of these folds represented a watershed event in enzyme evolution that enabled the rapid propagation of enzyme activities within enzyme superfamilies.

P1301: BIOLOGICAL SIGNIFICANCE OF UCK2 IN HTLV-1-INFECTED CELLS IN ATL LEUKEMOGENESIS AND ACQUIRED RESISTANCE TO AZACITIDINE

Background: Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). We have reported that regional DNA hypermethylation in HTLV-1-infected T-cells reflects the disease status of ATL and the anti-ATL effects of DNA demethylating agents, azacitidine (AZA) and decitabine (Blood 2020, 136: 871-884). We recently generated AZA-resistant (AZA-R) cells from ATL cell lines via long-term drug exposure and found down-regulation of uridine cytidine kinase2 (UCK2) correlated with lower susceptibility to AZA (Int J Cancer 2022, 150: 1184-1197). UCK1 and UCK2 are involved in mono-phosphorylation of uridine and cytidine in pyrimidine nucleotide biosynthesis. Both proteins also catalyze mono-phosphorylation of AZA. Overexpression of UCK2 has been observed in several types of solid tumor tissues. Aims: Here, we aimed to understand the role of UCK2 in HTLV-1-infected cells during multi-step carcinogenesis of ATL and the biological significance of inactivation of UCK2 in AZA-R cells. Methods: All studies using human samples were performed in accordance with the guidelines set out in the Declaration of Helsinki and were approved by the institutional review board at Saga University (2018-03-02). Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood samples from healthy volunteers, HTLV-1 carriers and patients with ATL. ATL cell lines were cultured in RPMI-1640 medium containing 10% FBS. Results: We found that UCK2 gene expression was higher in HTLV-1-infected T-cells than in normal T-cells using the gene expression datasets GSE55851 and GSE33615. UCK2 protein expression was increased in HTLV-1-infected T-cells (CADM1+ cells) isolated from patients with ATL compared with uninfected T-cells (Fig. A). We also found UCK2 gene expression was upregulated via activation of T-cell receptor (TCR) signaling pathway in normal CD4+ T-cells (Fig. B). Somatic alterations and epigenetic modifications activating TCR signaling have been identified in ATL cells. Therefore, we think UCK2 protein is overexpressed through dysregulated TCR signaling in HTLV-1-infected cells during ATL leukemogenesis. We have successfully established AZA-R cell lines, whose UCK2 expression was lost or decreased. Although they proliferated normally in vitro, the knockdown of UCK2 dramatically suppressed cell growth of ATL cell lines (Fig. C). Since UCK2 is involved in pyrimidine nucleotide biosynthesis, we then performed metabolome analysis in AZA-R cells. Although uridine and cytidine were accumulated in AZA-R cells, possibly due to loss of UCK2, the amount of UTP and CTP was almost same with parental cells. On the other hands, dihydroorotic acid (DHO) was decreased and orotate was increased in AZA-R cells (Fig. D). DHODH catalyzes the oxidation of DHO to orotic acid and is a rate-limiting enzyme in de novo pyrimidine nucleotide synthesis. AZA-R cells were more susceptible to BAY2402234, a DHODH inhibitor (Fig. E), indicating the activation of pyrimidine de novo synthesis in AZA-R cells. Image:Summary/Conclusion: Taken together, we think UCK2 supports the vigorous cell proliferation not only in normal TCR-stimulated T-cells but also in HTLV-1-infected cells. Inactivation of UCK2 makes cells resistant to AZA but suppresses the cell growth possibly due to the pyrimidine nucleotide starvation. AZA-R cells with reduced UCK2 expression proliferate normally in vitro by the activation of pyrimidine de novo synthesis.

Filamentation modulates allosteric regulation of PRPS.

Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of E. coli PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.

MiRNA-mRNA analysis of sheep adrenal glands reveals the network regulating reproduction

BackgroundThe adrenal gland participates in the process of sheep reproduction. MicroRNAs (miRNAs), endogenous small noncoding RNAs, regulate gene expression at the posttranscriptional level. However, the miRNA-mRNA network profile of adrenal glands relating to reproduction in sheep is still not well-studied. As sheep with FecBBB genotype show higher lambing number compare with the sheep with FecB++ genotype. This research aims to compare gene expression by small RNA-seq in adrenal tissues at follicular (F) and luteal (L) phases in FecBBB (MM) and FecB++ (ww) sheep. After analysis of gene expression, significant differentially expressed microRNAs (DEMs) and corresponding target genes were identified.ResultsA total of 180 miRNAs were found in this study, of which 19 DEMs were expressed in the four comparison groups (MM_F_A vs. MM_L_A, MM_F_A vs. ww_F_A, MM_L_A vs. ww_L_A, ww_F_A vs. ww_L_A). Subsequently, 354 target genes of 19 DEMs were predicted by integrated analysis. Cluster analysis was performed by K_means_cluster, and the expression patterns of these DEMs were separated into four subclusters. Functional analysis of target genes was performed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). The results indicated that the target genes were involved mainly in the Notch signaling pathway, signal transduction, cell communication, innate immune response and amino acid metabolism. Specifically, the Notch signaling pathway, biosynthetic process and metabolic process of pyrimidine nucleotide and amino acid metabolism appear to play key regulatory roles in the sheep fertility trait. Furthermore, miRNA-mRNA interaction networks were constructed by differentially expressed genes combined with our previous study of transcriptome data. The results showed that several key genes, including TDRD3, ANAPC7, CCNL2, BRD2 and MUT, were related to the transformation from the follicular phase to the luteal phase. PLAC8L1, NFAT5, DDX24 and MBD1 were related to the high fecundity of small tail Han sheep.ConclusionsIn this study, the miRNA transcriptome profile was identified, and miRNA-mRNA interaction networks were constructed in adrenal gland tissue of small tail Han sheep, the interaction between miR-370-3p and its targets were considered to play a major role in the reproduction regulation process. The results enriched the number of known miRNAs in adrenal glands and provided novel ideas and further information to demonstrate posttranscriptional regulation mechanisms at follicular and luteal phases in different genotypes of small tail Han sheep.

Contribution of Model Organisms to Investigating the Far-Reaching Consequences of PRPP Metabolism on Human Health and Well-Being.

Phosphoribosyl pyrophosphate synthetase (PRS EC 2.7.6.1) is a rate-limiting enzyme that irreversibly catalyzes the formation of phosphoribosyl pyrophosphate (PRPP) from ribose-5-phosphate and adenosine triphosphate (ATP). This key metabolite is required for the synthesis of purine and pyrimidine nucleotides, the two aromatic amino acids histidine and tryptophan, the cofactors nicotinamide adenine dinucleotide (NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+), all of which are essential for various life processes. Despite its ubiquity and essential nature across the plant and animal kingdoms, PRPP synthetase displays species-specific characteristics regarding the number of gene copies and architecture permitting interaction with other areas of cellular metabolism. The impact of mutated PRS genes in the model eukaryote Saccharomyces cerevisiae on cell signalling and metabolism may be relevant to the human neuropathies associated with PRPS mutations. Human PRPS1 and PRPS2 gene products are implicated in drug resistance associated with recurrent acute lymphoblastic leukaemia and progression of colorectal cancer and hepatocellular carcinoma. The investigation of PRPP metabolism in accepted model organisms, e.g., yeast and zebrafish, has the potential to reveal novel drug targets for treating at least some of the diseases, often characterized by overlapping symptoms, such as Arts syndrome and respiratory infections, and uncover the significance and relevance of human PRPS in disease diagnosis, management, and treatment.

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 β-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.

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.

Community Structure and Microbial Function Responses of Biofilms Colonizing on Microplastics with Vertical Distribution in Urban Water

Microplastics have received increasing attention worldwide due to their carrier effects. In the aquatic environment, microplastics always show a vertical distribution, which thereby may change the structure and function of the attached microbial communities. However, few studies have focused on this alteration. In this study, the structural changes and functional expression responses of the attached bacterial communities to microplastics under vertical distribution were investigated in the field combined with high-throughput sequencing technology. Polyethylene terephthalate (PET) and polyvinyl chloride (PVC) were selected as the target microplastics, which were frequently detected in the aqueous environment. The results showed that the α-diversity of bacterial communities attached to PET microplastics was much higher than that of those attached to PVC microplastics. The abundance and diversity of the bacterial communities attached to PET and PVC both increased with the increase in water depth. The α-diversity index of bacteria attached to the two typical microplastics was significantly higher in deep water (90 cm) than that in water 30 cm and 60 cm deep. The Cyanobacteria, Proteobacteria, Planctomycetes, and Verrucomicrobia were the dominant phyla in the attached bacterial communities. In addition, the deep water distinctly altered the bacteria community attached to different microplastics. The results of functional prediction showed that the functional expression of pyrimidine metabolism, amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, and aminoacyl-tRNA biosynthesis were positively correlated with water depth. In addition, the functional responses of the bacterial communities attached to microplastics were also increased, especially in deep water. Further, the bacterial functions of those attached to PET were significantly higher than that of those attached to PVC. This suggests that both the microplastic polymer and the water depth could affect the structure and function of the attached bacterial communities and that the water depth was more important, which may be related to the difference in the vertical distribution of light and turbidity. The results of this study provide a new insight into the microbial response to and environmental risk of microplastic pollution.

A novel series of teriflunomide derivatives as orally active inhibitors of human dihydroorotate dehydrogenase for the treatment of colorectal carcinoma

Human dihydroorotate dehydrogenase (hDHODH) is a key enzyme in the de novo synthesis pathway of pyrimidine nucleotide in cells. The moderate efficiency of teriflunomide, an approved hDHODH inhibitor for the treatment of multiple sclerosis, limited its therapeutic application of malignancy. Herein, thirty-seven novel teriflunomide derivatives with a biphenyl scaffold were designed, synthesized and evaluated. As a result, the optimal compound A37 exhibited a potent hDHODH inhibitory activity with an IC50 value of 10.2 nM, which was about 40-fold stronger than that of teriflunomide (IC50 = 407.8 nM), and showed favorable antiproliferative activities against HCT116 cells with an IC50 value of 0.3 μM. Meanwhile, A37 displayed an acceptable safety profile at an oral dosage of 400 mg/kg in the acute toxicity assay, and exhibited a promising antitumor effect with tumor growth inhibition rate of 54.4% at an oral dosage of 30 mg/kg in HCT116 xenograft model. These results indicate that A37 is an efficacious hDHODH inhibitor with potential in the treatment of colorectal carcinoma.

Dihydroorotate dehydrogenase inhibition acts synergistically with tyrosine kinase inhibitors to induce apoptosis of mantle cell lymphoma cells.

Mantle cell lymphoma (MCL) is a non‐Hodgkin lymphoma that remains incurable with the treatment options available today. In the present study, we have identified the dihydroorotate dehydrogenase (DHODH), an essential enzyme for the de novo biosynthesis of pyrimidine‐based nucleotides, to be overexpressed in MCL in comparison to healthy peripheral blood mononuclear cells (PBMC). In vitro inhibition of the DHODH activity using a newly developed DHODH inhibitor, namely (R)‐HZ05, can induce MCL cell death in the nanomolar range independently than the P53 status of the investigated cell lines. Moreover, the combination of (R)‐HZ05 with tyrosine kinase inhibitor shows the synergistic activity on cell death. Pre‐clinical investigation on the efficacy of (R)‐HZ05 shows that it can be prolonged animal lifespan similar to ibrutinib. (R)‐HZ05 use in combination with tyrosine kinase inhibitor demonstrated a superior efficacy on tumor burden reduction and survival than either drug alone. We have demonstrated that the depletion of the pyrimidine nucleotide pool, using DHODH inhibitor, represents a new therapeutic strategy that may benefit MCL patients.

A novel and efficient synthesis of 1, 2, 3,4-tetrahydropyrimidine carboxamide derivatives by Biginelli reaction and their in vitro antibacterial activity

Introduction and Aim: An efficient aspect of two step synthesis of tetrahydro pyrimidine carboxamide derivatives were developed by Biginelli reaction. The two-step synthesized 1,2,3,4-tetrahydropyrimidinecarboxamide compounds were evaluated by In-vitro studies like antifungal and bacterial activities. Few compounds were shown excellent zone of inhibition against fungal and Microbial activities. In this study showed significant effect of antifungal and bacterial action against tetrahydro pyrimidine derivatives. Materials and Methods: In this procedure to synthesize 1,2,3,4-tetrahydropyrimidinecarboxamide derivatives, ethyl acetoacetate, cytosine, para-toluene sulphonic acid and ethanol were used. Results: The synthesized compounds of 1,2,3,4-tetrahydropyrimidine carboxamide derivatives through Biginelli reaction used an efficient catalyst such as para-toluene sulphonic acid and solvent ethanol. In this study tested compounds have developed into two step protocol for synthesis of diversely substituted novel pyrimidines. Conclusion: In this study the synthesized 1,2,3,4-tetrahydropyrimidine carboxamide (THPC) shows significant inhibiting action against Gram +ve, Gram –ve bacterial and fungal growth.

Structure activity relationship of 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as CD73 inhibitors

Adenosine in the tumor microenvironment acts on A2A and A2B adenosine receptors (ARs) on immune cells (T cells, dendritic cells, NK cells, macrophages and neutrophils) to prevent their activation. Therefore, a means of lowering extracellular adenosine would be beneficial in cancer immunotherapy either as a co‐therapy or monotherapy. Inhibitors of CD73 (ecto‐5’‐nucleotidase, the main extracellular adenosine‐forming enzyme) are already in clinical trials for cancer immunotherapy. Many purine (ADP) analogues have been shown to inhibit this enzyme, but we have explored pyrimidine (UDP/CDP) analogues. We recently reported N4‐substituted 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as potent CD73 inhibitors. We now expand the structure activity relationship (SAR) of pyrimidine nucleotides as human CD73 inhibitors, focusing mainly on cytidine functionalization at the N4 position. 4‐Chloro (MRS4598 16, Ki 0.673 nM) and 4‐iodo (MRS4620 18, Ki 0.436 nM) substitution of the N4‐benzyloxy group increased inhibitory potency Ki by ~20‐fold. Primary alkylamine derivatives coupled through a p‐amido group and varying methylene chain length (24and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. hCD73 X‐ray structures with two of the newly synthesized inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between C‐terminal and N‐terminal domains) as N4‐benzyl groups in potent adenine‐derived inhibitors. Molecular dynamics simulation identified stabilizing interactions and predicted conformational diversity around the N4‐benzyloxy moiety. Thus, by structure‐guided substitution of the N4‐benzyloxy moiety, we have greatly enhanced their inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in human head and neck squamous cell carcinoma (HNSCC) and palatine tonsils in situ.