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Frontiers in Chemistry 2019A series of -((3-phenyl-1-(phenylsulfonyl)-1-pyrazol-4-yl)methyl)anilines and , structurally related to previously synthesized and tested...
A series of -((3-phenyl-1-(phenylsulfonyl)-1-pyrazol-4-yl)methyl)anilines and , structurally related to previously synthesized and tested (-(1,3-diphenyl-1-pyrazol-4-yl)methyl)anilines (), were designed and synthesized. The new derivatives were evaluated in cell-based assays for their cytotoxicity and antiviral activity against a large panel of RNA and DNA viruses of public health significance. Generally, the tested compounds did not display cytotoxicity toward the cell lines used. The majority of derivatives - were able to interfered with YFV and RSV replication in the micromolar range showing a marked improvement in potency and selectivity with respect to the reference inhibitors 6-azauridine and ribavirin, respectively. The introduction of a -methoxy substituent on the phenylsulfonyl group (compounds ) completely abolished the anti-RSV activity and reduced or eliminated the potency against YFV. On the contrary, several -methoxy analogs were able to interfere with BVDV replication with a comparable (, and ) or better ( and ) potency than the reference inhibitor, ribavirin. Compound , selected for time of addition experiments on BHK-21 cell cultures infected with YFV, achieved the highest reduction of virus titer when added 2 h post infection and maintained up to 4 h post infection.
PubMed: 31024899
DOI: 10.3389/fchem.2019.00214 -
The Journal of Biological Chemistry Sep 1980The cytostatic effect of 6-azauridine on cell growth is generally regarded to be a consequence of the inhibition of de novo pyrimidine biosynthesis by the metabolite,...
The cytostatic effect of 6-azauridine on cell growth is generally regarded to be a consequence of the inhibition of de novo pyrimidine biosynthesis by the metabolite, 6-azauridine 5'-monophosphate. We show here that wheat embryonic axes further metabolize 6-azauridine to the 5'-triphosphate and incorporate the analogue into RNA, thus offering an alternative mechanism for growth inhibition. At a level of 6-azauridine required to maximally inhibit UTP biosynthesis, the ratio of 6-azaUTP to UTP is about 2:1 and substitution of 6-azauridine for uridine in new RNA is on the order of 1 in 18. The new metabolites of 6-azauridine are identified by high pressure and thin layer chromatography coupled with enzyme treatments.
Topics: Azauridine; Chromatography, High Pressure Liquid; Plants; RNA; Ribonucleotides; Triticum; Uracil Nucleotides; Uridine Triphosphate
PubMed: 6157685
DOI: No ID Found -
Journal of Virology Mar 2020Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition,...
Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition, IAV can cause pandemic infections with great consequences when new viruses are introduced into humans. In this study, we evaluated 10 previously described compounds with antiviral activity against mammarenaviruses for their ability to inhibit IAV infection using our recently described bireporter influenza A/Puerto Rico/8/34 (PR8) H1N1 (BIRFLU). Among the 10 tested compounds, eight (antimycin A [AmA], brequinar [BRQ], 6-azauridine, azaribine, pyrazofurin [PF], AVN-944, mycophenolate mofetil [MMF], and mycophenolic acid [MPA]), but not obatoclax or Osu-03012, showed potent anti-influenza virus activity under posttreatment conditions [median 50% effective concentration (EC) = 3.80 nM to 1.73 μM; selective index SI for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, >28.90 to 13,157.89]. AmA, 6-azauridine, azaribine, and PF also showed potent inhibitory effect in pretreatment (EC = 0.14 μM to 0.55 μM; SI-MTT = 70.12 to >357.14) or cotreatment (EC = 34.69 nM to 7.52 μM; SI-MTT = 5.24 to > 1,441.33) settings. All of the compounds tested inhibited viral genome replication and gene transcription, and none of them affected host cellular RNA polymerase II activities. The antiviral activity of the eight identified compounds against BIRFLU was further confirmed with seasonal IAVs (A/California/04/2009 H1N1 and A/Wyoming/3/2003 H3N2) and an IBV (B/Brisbane/60/2008, Victoria lineage), demonstrating their broad-spectrum prophylactic and therapeutic activity against currently circulating influenza viruses in humans. Together, our results identified a new set of antiviral compounds for the potential treatment of influenza viral infections. Influenza viruses are highly contagious pathogens and are a major threat to human health. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always guarantee complete protection against drifted or, more noticeably, shifted influenza viruses. Although U.S. Food and Drug Administration (FDA) drugs are approved for the treatment of influenza infections, influenza viruses resistant to current FDA antivirals have been reported and continue to emerge. Therefore, there is an urgent need to find novel antivirals for the treatment of influenza viral infections in humans, a search that could be expedited by repurposing currently approved drugs. In this study, we assessed the influenza antiviral activity of 10 compounds previously shown to inhibit mammarenavirus infection. Among them, eight drugs showed antiviral activities, providing a new battery of drugs that could be used for the treatment of influenza infections.
Topics: A549 Cells; Animals; Antiviral Agents; Cell Proliferation; Dogs; Drug Evaluation, Preclinical; Genome, Viral; HEK293 Cells; Host-Pathogen Interactions; Humans; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza B virus; Madin Darby Canine Kidney Cells; Virus Replication
PubMed: 31941776
DOI: 10.1128/JVI.02149-19 -
Scientific Reports Jun 2021Reverse genetics is an important tool in the elucidation of viral replication and the development of countermeasures; however, these methods are impeded by laborious and...
Reverse genetics is an important tool in the elucidation of viral replication and the development of countermeasures; however, these methods are impeded by laborious and inefficient replicon delivery methods. This paper demonstrates the use of a baculovirus to facilitate the efficient delivery of autonomous CHIKV replicons into mosquito and mammalian cells in vitro as well as adult mosquitoes in vivo. The efficacy of this approach was verified via co-localization among an eGFP reporter, nsP1, and dsRNA as well as through the inhibition of an RNA-dependent RNA polymerase (RdRp) null mutation (DDAA) in nsP4, or the treatment of a known antiviral compound (6-azauridine). We also investigated the correlation between CHIKV replicon-launched eGFP expression and the effectiveness of CHIKV replicon variants in inducing IFN-β expression in human cell lines. This delivery method based on a single vector is applicable to mosquito and mammalian cells in seeking to decipher the mechanisms underlying CHIKV replication, elucidate virus-host interactions, and develop antivirals. This study presents an effective alternative to overcome many of the technological issues related to the study and utilization of autonomous arbovirus replicons.
Topics: Aedes; Animals; Antiviral Agents; Chikungunya Fever; Chikungunya virus; Chlorocebus aethiops; Culicidae; Humans; Mosquito Vectors; RNA, Viral; RNA-Dependent RNA Polymerase; Vero Cells; Viral Nonstructural Proteins; Virus Replication
PubMed: 34112897
DOI: 10.1038/s41598-021-91830-y -
Antimicrobial Agents and Chemotherapy Jun 2014No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the...
No antiviral therapies are available for the tick-borne flaviviruses associated with hemorrhagic fevers: Kyasanur Forest disease virus (KFDV), both classical and the Alkhurma hemorrhagic fever virus (AHFV) subtype, and Omsk hemorrhagic fever virus (OHFV). We tested compounds reported to have antiviral activity against members of the Flaviviridae family for their ability to inhibit AHFV replication. 6-Azauridine (6-azaU), 2'-C-methylcytidine (2'-CMC), and interferon alpha 2a (IFN-α2a) inhibited the replication of AHFV and also KFDV, OHFV, and Powassan virus. The combination of IFN-α2a and 2'-CMC exerted an additive antiviral effect on AHFV, and the combination of IFN-α2a and 6-azaU was moderately synergistic. The combination of 2'-CMC and 6-azaU was complex, being strongly synergistic but with a moderate level of antagonism. The antiviral activity of 6-azaU was reduced by the addition of cytidine but not guanosine, suggesting that it acted by inhibiting pyrimidine biosynthesis. To investigate the mechanism of action of 2'-CMC, AHFV variants with reduced susceptibility to 2'-CMC were selected. We used a replicon system to assess the substitutions present in the selected AHFV population. A double NS5 mutant, S603T/C666S, and a triple mutant, S603T/C666S/M644V, were more resistant to 2'-CMC than the wild-type replicon. The S603T/C666S mutant had a reduced level of replication which was increased when M644V was also present, although the replication of this triple mutant was still below that of the wild type. The S603 and C666 residues were predicted to lie in the active site of the AHFV NS5 polymerase, implicating the catalytic center of the enzyme as the binding site for 2'-CMC.
Topics: Amino Acid Substitution; Antiviral Agents; Cell Line; Cytidine; Cytopathogenic Effect, Viral; Drug Resistance, Viral; Flavivirus; Hemorrhagic Fevers, Viral; Humans; Models, Molecular; Mutation; Tick-Borne Diseases; Virus Replication
PubMed: 24663025
DOI: 10.1128/AAC.02393-14 -
European Journal of Biochemistry Nov 19821. Slices of spleen from anaemic mice were incubated with [14C]bicarbonate in the presence and absence of 6-azauridine and the amounts of 14C that entered the de novo...
1. Slices of spleen from anaemic mice were incubated with [14C]bicarbonate in the presence and absence of 6-azauridine and the amounts of 14C that entered the de novo pyrimidine biosynthetic pathway were assessed and compared. Compounds analyzed included carbamoylaspartate, dihydroorotate, orotate plus its derivatives, acid-soluble uracil and cytosine 5'-nucleotides, nucleic acid pyrimidines, free pyrimidine bases and nucleosides. As the intracellular levels of carbamoyl phosphate and acid-soluble deoxyribonucleotides are known to be relatively low, the radioactivities of these compounds were not measured. Degradation of labelled uridine was limited in this tissues, therefore the radioactivity of degradative products of pyrimidines was not considered. 2. When the slices were incubated with 0.5 mM 6-azauridine for 10 min and then with [14C]bicarbonate for an additional 10 min and 30 min, the sum of radioactivity found in the above compounds, which represents the total amount of 14C that entered the pyrimidine pathway, was 2.1 and 2.3 times greater than when the tissue slices were incubated in the absence of the analogue. 3. When the 14C distribution among the carbon atoms of the molecules of labelled carbamoylaspartate and uracil was investigated, we found that more than 90% of the total 14C in these compounds derived directly from carbamoyl phosphate and the remaining portion was from aspartate, either in the presence or absence of 6-azauridine. 4. There was no indication that 6-azauridine altered [14C]bicarbonate permeation through the cell membrane or its intracellular metabolism. 5. These results, along with the pattern of early intermediate accumulation seen in the presence of 6-azauridine, indicate that 6-azauridine stimulates the production of carbamoyl phosphate for the pyrimidine biosynthetic pathway in the mouse spleen. 6. Of the radioactive early intermediates which accumulated, only orotate, its derivatives (orotidine and orotidine 5'-monophosphate) or both appeared in the medium, presumably the result of leakage through the cell membranes. 7. Stimulation of the pyrimidine pathway was not observed in the case of Ehrlich ascites tumour cells incubated under similar conditions with 6-azauridine.
Topics: Animals; Azauridine; Bicarbonates; Carbamates; Carbamyl Phosphate; Carbon Radioisotopes; In Vitro Techniques; Kinetics; Male; Mice; Mice, Inbred Strains; Pyrimidines; Spleen
PubMed: 6185334
DOI: 10.1111/j.1432-1033.1982.tb07009.x -
Organic & Biomolecular Chemistry Jan 2017To display favorable fluorescent properties, the non-emissive native nucleosides need to be modified. Here we present a motif that relies on conjugating 5-membered...
To display favorable fluorescent properties, the non-emissive native nucleosides need to be modified. Here we present a motif that relies on conjugating 5-membered aromatic heterocycles (e.g., thiophene) to a 6-azapyrimidine (1,2,4-triazine) core. Synthetic accessibility and desirable photophysical properties make these nucleosides attractive candidates for enzymatic incorporation and biochemical assays. While 6-azauridine triphosphate is known to be poorly tolerated by polymerases in RNA synthesis, we illustrate that conjugating a thiophene ring at position 5 overcomes such limitations, facilitating its T7 RNA polymerase-mediated in vitro transcription incorporation into RNA constructs. We further show that the modified transcripts can be ligated to longer oligonucleotides to form singly modified RNAs, as illustrated for an A-site hairpin model RNA construct, which was employed to visualize aminoglycoside antibiotics binding.
Topics: Azauridine; DNA-Directed RNA Polymerases; Fluorescence; RNA; Viral Proteins
PubMed: 27981333
DOI: 10.1039/c6ob02080a -
The Journal of Biological Chemistry Jun 1979Cells resistant to pyrazofurin and 6-azauridine have been selected from a simian virus 40-transformed Syrian hamster line and from a Chinese hamster lung line. By... (Comparative Study)
Comparative Study
Cells resistant to pyrazofurin and 6-azauridine have been selected from a simian virus 40-transformed Syrian hamster line and from a Chinese hamster lung line. By increasing the concentrations of inhibitors in several steps, mutant cells from both lines have been obtained which resist high concentrations (1 to 5 mM) of the two inhibitors separately or together. Orotidine-5'-phosphate decarboxylase (EC 4.1.1.23), the sixth and last enzyme in UMP biosynthesis, is inhibited by the nucleoside monophosphates derived from pyrazofurin or 6-azauridine. The activity of this enzyme is increased in each resistant cell line tested. Furthermore, there is a parallel increase in each case in the activity of the fifth enzyme of the pathway, orotate phosphoribosyltransferase (EC 2.4.2.10), which is not inhibited by pyrazofurin or 6-azauridine monophosphates, and the amount of increase is up to 67 times the level found in wild type cells. In contrast, the activities of the first three enzymes of UMP biosynthesis remain essentially unchanged in the mutants. Resistant Chinese hamster cells remain sensitive to 5-fluorouridine; this indicates that uridine kinase, the enzyme necessary to convert 6-azauridine to the monophosphate, is still functional.
Topics: Amides; Animals; Antibiotics, Antineoplastic; Azauridine; Carboxy-Lyases; Cell Line; Cell Transformation, Viral; Cricetinae; Drug Resistance; Kidney; Mesocricetus; Mutation; Orotate Phosphoribosyltransferase; Orotidine-5'-Phosphate Decarboxylase; Pentosyltransferases; Pyrazoles; Ribonucleosides; Ribose; Simian virus 40
PubMed: 220255
DOI: No ID Found -
Fertility and Sterility 1967
Topics: Alkaloids; Animals; Antimetabolites; Colchicine; Embryo Implantation; Female; Fertility; Fetus; Haplorhini; Nucleosides; Pregnancy; Pregnancy, Animal; Purines; Rabbits; Vinblastine
PubMed: 4959820
DOI: 10.1016/s0015-0282(16)36181-7 -
Proceedings of the National Academy of... Aug 2022Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by...
Earlier work has shown that siRNA-mediated reduction of the SUPT4H or SUPT5H proteins, which interact to form the DSIF complex and facilitate transcript elongation by RNA polymerase II (RNAPII), can decrease expression of mutant gene alleles containing nucleotide repeat expansions differentially. Using luminescence and fluorescence assays, we identified chemical compounds that interfere with the SUPT4H-SUPT5H interaction and then investigated their effects on synthesis of mRNA and protein encoded by mutant alleles containing repeat expansions in the huntingtin gene (), which causes the inherited neurodegenerative disorder, Huntington's Disease (HD). Here we report that such chemical interference can differentially affect expression of mutant alleles, and that a prototypical chemical, 6-azauridine (6-AZA), that targets the SUPT4H-SUPT5H interaction can modify the biological response to mutant gene expression. Selective and dose-dependent effects of 6-AZA on expression of alleles containing nucleotide repeat expansions were seen in multiple types of cells cultured in vitro, and in a animal model for HD. Lowering of mutant HD protein and mitigation of the "rough eye" phenotype associated with degeneration of photoreceptor neurons in vivo were observed. Our findings indicate that chemical interference with DSIF complex formation can decrease biochemical and phenotypic effects of nucleotide repeat expansions.
Topics: Alleles; Animals; Azauridine; Cells, Cultured; DNA Repeat Expansion; Disease Models, Animal; Drosophila melanogaster; Humans; Huntingtin Protein; Huntington Disease; Luminescent Measurements; Mutant Proteins; Mutation; Nuclear Proteins; Phenotype; Photoreceptor Cells, Invertebrate; Repressor Proteins; Transcriptional Elongation Factors
PubMed: 35914128
DOI: 10.1073/pnas.2204779119