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Science (New York, N.Y.) Jan 2022The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat...
The COVID-19 pandemic has underscored the critical need for broad-spectrum therapeutics against respiratory viruses. Respiratory syncytial virus (RSV) is a major threat to pediatric patients and older adults. We describe 4′-fluorouridine (4′-FlU, EIDD-2749), a ribonucleoside analog that inhibits RSV, related RNA viruses, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with high selectivity index in cells and human airway epithelia organoids. Polymerase inhibition within in vitro RNA-dependent RNA polymerase assays established for RSV and SARS-CoV-2 revealed transcriptional stalling after incorporation. Once-daily oral treatment was highly efficacious at 5 milligrams per kilogram (mg/kg) in RSV-infected mice or 20 mg/kg in ferrets infected with different SARS-CoV-2 variants of concern, initiated 24 or 12 hours after infection, respectively. These properties define 4′-FlU as a broad-spectrum candidate for the treatment of RSV, SARS-CoV-2, and related RNA virus infections.
Topics: Administration, Oral; Animals; Antiviral Agents; COVID-19; Cell Line; Coronavirus RNA-Dependent RNA Polymerase; Disease Models, Animal; Female; Ferrets; Humans; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Mononegavirales; RNA-Dependent RNA Polymerase; Respiratory Mucosa; Respiratory Syncytial Virus Infections; Respiratory Syncytial Virus, Human; SARS-CoV-2; Transcription, Genetic; Uracil Nucleotides; Virus Replication; COVID-19 Drug Treatment
PubMed: 34855509
DOI: 10.1126/science.abj5508 -
Current Opinion in Pharmacology Apr 2022Extracellular uridine nucleotides regulate physiological and pathophysiological metabolic processes through the activation of P2Y, P2Y, P2Y and P2Y purinergic receptors,... (Review)
Review
Extracellular uridine nucleotides regulate physiological and pathophysiological metabolic processes through the activation of P2Y, P2Y, P2Y and P2Y purinergic receptors, which play a key role in adipogenesis, glucose uptake, lipolysis and adipokine secretion. Using adipocyte-specific knockout mouse models, it has been demonstrated that lack of the P2YR or P2YR can protect against diet-induced obesity and improve whole-body glucose metabolism. The P2YR facilitated adipogenesis and inflammation, and the loss of P2YR or P2YR raised the levels of the protective endocrine factor adiponectin. Hence, potent antagonists for these receptors may be tested to identify drug candidates for the treatment of obesity and type 2 diabetes. However, future studies are required to provide insight into purinergic regulation of brown adipocytes and their role in thermogenesis. This review summarizes the current studies on uridine nucleotide-activated P2YRs and their role in adipocyte function, diet-induced obesity and associated metabolic deficits.
Topics: Adipocytes; Animals; Diabetes Mellitus, Type 2; Humans; Mice; Obesity; Receptors, Purinergic; Uracil Nucleotides
PubMed: 35231671
DOI: 10.1016/j.coph.2022.102190 -
Stem Cell Research & Therapy Apr 2023Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation...
BACKGROUND
Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs.
METHODS
MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3 antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC RESULTS: The extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y purinoceptors prevented this effect.
CONCLUSIONS
Data suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.
Topics: Humans; Female; Aged; Adolescent; Young Adult; Adult; Osteogenesis; Postmenopause; Mesenchymal Stem Cells; Cell Differentiation; Uracil Nucleotides; Uridine Diphosphate; Adenosine Triphosphate; Bone Marrow Cells; Cells, Cultured
PubMed: 37076930
DOI: 10.1186/s13287-023-03315-6 -
Bioorganic & Medicinal Chemistry Jun 2008The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6)...
The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y(2), P2Y(4), and P2Y(6) receptors. The 2-thio modification, found previously to enhance P2Y(2) receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP, and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y(2) receptor, in the form of Up(4)-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include 9, alpha,beta-methylene-UDP, a P2Y(6) receptor agonist; 30, Up(4)-phenyl ester and 34, Up(4)-[1]glucose, selective P2Y(2) receptor agonists; dihalomethylene phosphonate analogues 16 and 41, selective P2Y(2) receptor agonists; 43, the 2-thio analogue of INS37217 (P(1)-(uridine-5')-P(4)-(2'-deoxycytidine-5')tetraphosphate), a potent and selective P2Y(2) receptor agonist.
Topics: Humans; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y2; Structure-Activity Relationship; Uracil Nucleotides
PubMed: 18514530
DOI: 10.1016/j.bmc.2008.05.013 -
The FEBS Journal Sep 2015RNA molecules are subjected to post-transcriptional modifications that might determine their maturation, activity, localization and stability. These alterations can... (Review)
Review
RNA molecules are subjected to post-transcriptional modifications that might determine their maturation, activity, localization and stability. These alterations can occur within the RNA molecule or at its 5'- or 3'- extremities, and are essential for gene regulation and proper function of the RNA. One major type of modification is the 3'-end addition of nontemplated nucleotides. Polyadenylation is the most well studied type of 3'-RNA modification, both in eukaryotes and prokaryotes. The importance of 3'-oligouridylation has recently gained attention through the discovery of several types of uridylated-RNAs, by the existence of enzymes that specifically add poly(U) tails and others that preferentially degrade these tails. Namely, Dis3L2 is a 3'-5' exoribonuclease from the RNase II/RNB family that has been shown to act preferentially on oligo(U)-tailed transcripts. Our understanding of this process is still at the beginning, but it is already known to interfere in the regulation of diverse RNA species in most eukaryotes. Now that we are aware of the prevalence of RNA uridylation and the techniques available to globally evaluate the 3'-terminome, we can expect to make rapid progress in determining the extent of terminal oligouridylation in different RNA populations and unravel its impact on RNA decay mechanisms. Here, we sum up what is known about 3'-RNA modification in the different cellular compartments of eukaryotic cells, the conserved enzymes that perform this 3'-end modification and the effectors that are selectively activated by this process.
Topics: Animals; Cell Compartmentation; Exoribonucleases; Humans; Metabolic Networks and Pathways; Models, Biological; Models, Molecular; Oligoribonucleotides; Poly U; Protein Conformation; RNA; RNA 3' End Processing; RNA Stability; Uracil Nucleotides
PubMed: 26183531
DOI: 10.1111/febs.13377 -
Annals of Medicine Dec 2023We sought to evaluate the expression of matrix metalloproteinase-9 (MMP-9) in dry eyes treated with 0.05% cyclosporin A and 3.0% diquafosol tetrasodium.
PURPOSE
We sought to evaluate the expression of matrix metalloproteinase-9 (MMP-9) in dry eyes treated with 0.05% cyclosporin A and 3.0% diquafosol tetrasodium.
METHODS
One-hundred ninety-five eyes of 195 patients with dry eye were divided into three groups as follows: group 1, cyclosporin group ( = 69); group 2, diquafosol group ( = 59); and group 3, artificial tears eyes ( = 67). All eyes were treated and followed up for three months. Schirmer I Test, corneal staining, tear-film break-up time (TBUT), and tear-film MMP-9 content were measured at three months and compared between groups. The expression of MMP-9 was confirmed using a point-of-care test device (InflammaDry®; RPS Diagnostics, Sarasota, FL, USA) and graded as zero to four points.
RESULTS
At the third month, MMP-9 expression was lower in group 1 as compared with in groups 2 and 3 ( 0.020 and 0.006, respectively). The mean MMP-9 grade according to point-of-care testing was also lower in group 1 than in groups 2 or 3 ( 0.002 and 0.038, respectively). MMP-9 showed a correlation with corneal staining in both groups 1 and 2 (all < 0.001) and with Schirmer I Test and TBUT in group 1 ( = 0.018 and 0.015, respectively).
CONCLUSIONS
MMP-9 expression and grade were lower after treatment with cyclosporin than after treatment with diquafosol in the dry eye disease. Anti-inflammatory treatment can decrease ocular MMP-9 levels in dry eye disease.
Topics: Humans; Cyclosporine; Matrix Metalloproteinase 9; Dry Eye Syndromes; Uracil Nucleotides
PubMed: 37354028
DOI: 10.1080/07853890.2023.2228192 -
Current Topics in Medicinal Chemistry 2004In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has... (Review)
Review
In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X(1) (derivatives of pyridoxal phosphates and suramin), P2X(3)(A-317491), P2X(7) (derivatives of the isoquinoline KN-62), P2Y(1)(nucleotide analogues MRS 2179 and MRS 2279), P2Y(2)(thiouracil derivatives such as AR-C126313), and P2Y(12)(nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y(1)receptors. The dinucleotide INS 37217 potently activates the P2Y(2)receptor. UTP-gamma-S and UDP-beta-S are selective agonists for P2Y(2)/P2Y(4)and P2Y(6)receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y(1)receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7.
Topics: Adenine Nucleotides; Animals; Humans; Ion Channel Gating; Ligands; Mutagenesis, Site-Directed; Purine Nucleotides; Purinergic P2 Receptor Agonists; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Uracil Nucleotides
PubMed: 15078212
DOI: 10.2174/1568026043450961 -
European Journal of Biochemistry Apr 1990The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these...
The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N-labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of L-[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.
Topics: Alanine; Animals; Intestinal Mucosa; Isotope Labeling; Kinetics; Liver; Male; Mice; Mice, Inbred Strains; Muscle, Smooth; Nitrogen Isotopes; Uracil Nucleotides
PubMed: 2338085
DOI: 10.1111/j.1432-1033.1990.tb15507.x -
Journal of Neurochemistry Nov 2006Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded...
Isolation and propagation of neural stem cells derived from human brain tissue uniquely enables the study of human neurogenesis in vitro. In addition, ex vivo-expanded human neural stem/precursor cells (NPCs) may offer novel therapeutic strategies. We investigated the effects of extracellular nucleotides on the proliferation and differentiation of human mesencephalic neural stem/precursor cells (hmNPCs). When combined with the mitogens epidermal growth factor and fibroblast growth factor 2, UTP (1 microm) boosted proliferation of hmNPCs as shown by increased expression of the proliferation marker proliferating cell nuclear antigen (330%). UTP-induced proliferation was abrogated by the preferential P2Y receptor blocker pyridoxal phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS). UTP also stimulated dopaminergic differentiation. Treatment with UTP (100 microm) increased the number of tyrosine hydroxylase (TH)-positive cells and TH protein by 267 and 319% respectively. UTP-stimulated dopaminergic differentiation of hmNPCs was blocked by the P2 receptor antagonists suramin (10 microm) and PPADS (100 microm). In addition, UDP (1 microm) enhanced TH protein expression by 194%. During differentiation, treatment with UTP stimulated the extracellular signal-regulated kinase (ERK) pathway. Both ERK1/2 phosphorylation and dopaminergic differentiation were inhibited by U0126, a selective ERK kinase inhibitor, as well as by suramin. When other P2 receptor agonists (ATP, ADP and adenosine 5'-O-(2-thiophosphate) (ADPbetaS); all 100 microm) were applied, both proliferation and dopaminergic differentiation of NPCs were compromised. We conclude that uracil nucleotides exert specific P2 receptor-mediated effects on midbrain-derived human NPCs, and may be used to enhance both proliferation and dopaminergic differentiation.
Topics: Adenine Nucleotides; Blotting, Western; Cell Count; Cell Death; Cell Differentiation; Cell Proliferation; Dopamine; Electrophysiology; Extracellular Signal-Regulated MAP Kinases; Fluorescent Antibody Technique; Humans; Mesencephalon; Nerve Tissue Proteins; Neurons; Oligonucleotide Array Sequence Analysis; Patch-Clamp Techniques; Pyridoxal Phosphate; RNA; Receptors, Purinergic P2; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Suramin; Uracil Nucleotides; Uridine Triphosphate
PubMed: 17076658
DOI: 10.1111/j.1471-4159.2006.04132.x -
Journal of Neuroinflammation Sep 2014During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive...
BACKGROUND
During cerebral inflammation uracil nucleotides leak to the extracellular medium and activate glial pyrimidine receptors contributing to the development of a reactive phenotype. Chronically activated microglia acquire an anti-inflammatory phenotype that favors neuronal differentiation, but the impact of these microglia on astrogliosis is unknown. We investigated the contribution of pyrimidine receptors to microglia-astrocyte signaling in a chronic model of inflammation and its impact on astrogliosis.
METHODS
Co-cultures of astrocytes and microglia were chronically treated with lipopolysaccharide (LPS) and incubated with uracil nucleotides for 48 h. The effect of nucleotides was evaluated in methyl-[3H]-thymidine incorporation. Western blot and immunofluorescence was performed to detect the expression of P2Y6 receptors and the inducible form of nitric oxide synthase (iNOS). Nitric oxide (NO) release was quantified through Griess reaction. Cell death was also investigated by the LDH assay and by the TUNEL assay or Hoechst 33258 staining.
RESULTS
UTP, UDP (0.001 to 1 mM) or PSB 0474 (0.01 to 10 μM) inhibited cell proliferation up to 43 ± 2% (n = 10, P <0.05), an effect prevented by the selective P2Y6 receptor antagonist MRS 2578 (1 μM). UTP was rapidly metabolized into UDP, which had a longer half-life. The inhibitory effect of UDP (1 mM) was abolished by phospholipase C (PLC), protein kinase C (PKC) and nitric oxide synthase (NOS) inhibitors. Both UDP (1 mM) and PSB 0474 (10 μM) increased NO release up to 199 ± 20% (n = 4, P <0.05), an effect dependent on P2Y6 receptors-PLC-PKC pathway activation, indicating that this pathway mediates NO release. Western blot and immunocytochemistry analysis indicated that P2Y6 receptors were expressed in the cultures being mainly localized in microglia. Moreover, the expression of iNOS was mainly observed in microglia and was upregulated by UDP (1 mM) or PSB 0474 (10 μM). UDP-mediated NO release induced apoptosis in astrocytes, but not in microglia.
CONCLUSIONS
In LPS treated co-cultures of astrocytes and microglia, UTP is rapidly converted into UDP, which activates P2Y6 receptors inducing the release of NO by microglia that causes astrocyte apoptosis, thus controlling their rate of proliferation and preventing an excessive astrogliosis.
Topics: Animals; Animals, Newborn; Apoptosis; Astrocytes; Cell Cycle; Cell Proliferation; Cells, Cultured; Coculture Techniques; Enzyme Inhibitors; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Lipopolysaccharides; Microglia; Nitric Oxide; Rats; Rats, Wistar; Receptors, Purinergic P2; Thymidine; Time Factors; Tritium; Uracil Nucleotides
PubMed: 25178395
DOI: 10.1186/s12974-014-0141-3