ABSTRACT
This study demonstrates the inhibitory effect of 42 pyrimidonic pharmaceuticals (PPs) on the 3-chymotrypsin-like protease of SARS-CoV-2 (3CLpro) through molecular docking, molecular dynamics simulations, and free binding energies by means of molecular mechanics-Poisson Boltzmann surface area (MM-PBSA) and molecular mechanics-generalized Born surface area (MM-GBSA). Of these tested PPs, 11 drugs approved by the US Food and Drug Administration showed an excellent binding affinity to the catalytic residues of 3CLpro of His41 and Cys145: uracil mustard, cytarabine, floxuridine, trifluridine, stavudine, lamivudine, zalcitabine, telbivudine, tipiracil, citicoline, and uridine triacetate. Their percentage of residues involved in binding at the active sites ranged from 56 to 100, and their binding affinities were in the range from -4.6 ± 0.14 to -7.0 ± 0.19 kcal/mol. The molecular dynamics as determined by a 200 ns simulation run of solvated docked complexes confirmed the stability of PP conformations that bound to the catalytic dyad and the active sites of 3CLpro. The free energy of binding also demonstrates the stability of the PP-3CLpro complexes. Citicoline and uridine triacetate showed free binding energies of -25.53 and -7.07 kcal/mol, respectively. Therefore, I recommend that they be repurposed for the fight against COVID-19, following proper experimental and clinical validation.
Subject(s)
COVID-19 Drug Treatment , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Papain-Like Proteases/antagonists & inhibitors , Drug Repositioning/methods , Protease Inhibitors/pharmacology , SARS-CoV-2/drug effects , Acetates/chemistry , Acetates/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Cytidine Diphosphate Choline/chemistry , Cytidine Diphosphate Choline/pharmacology , Drug Evaluation, Preclinical , Humans , Molecular Docking Simulation , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Uridine/analogs & derivatives , Uridine/chemistry , Uridine/pharmacologyABSTRACT
A new uridine derivative 11457 A (1), and a new indole derivative 11457B (2), together with a known compound 1H-indole-2-carbaldehyde (3), were characterized from the fermentation broth of the actinomycete Pseudonocardia sp. SCSIO 11457, an isolate associated with the scleractinian coral Galaxea fascicularis. Upon detailed spectroscopic analysis, 11457 A (1) was identified as a uridine analog, and 11457B (2) was elucidated as an indole derivative 2-hydroxy-1-(1H-indol-2-yl)pentane-1,4-dione. Biological evaluation indicated that none of compounds 1-3 showed antibacterial activities against pathogenic bacteria and cytotoxic activities against human cancer cell lines.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , Indoles/chemistry , Pseudonocardia/chemistry , Uridine/chemistry , Animals , Anthozoa/microbiology , Anti-Bacterial Agents/chemistry , Antineoplastic Agents/chemistry , Cell Line, Tumor , Drug Evaluation, Preclinical , Fermentation , Humans , Microbial Sensitivity Tests , Molecular Structure , Pseudonocardia/metabolismABSTRACT
The content of selected major nitrogen compounds including nucleosides and their derivatives was evaluated in 75 samples of seven varieties of honey (heather, buckwheat, black locust, goldenrod, canola, fir, linden) by targeted ultra-high performance liquid chromatography-diode array detector - high-resolution quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QqTOF-MS) and determined by UHPLC-DAD. The honey samples contained nucleosides, nucleobases and their derivatives (adenine: 8.9 to 18.4 mg/kg, xanthine: 1.2 to 3.3 mg/kg, uridine: 17.5 to 51.2 mg/kg, guanosine: 2.0 to 4.1 mg/kg; mean amounts), aromatic amino acids (tyrosine: 7.8 to 263.9 mg/kg, phenylalanine: 9.5 to 64.1 mg/kg; mean amounts). The amounts of compounds significantly differed between some honey types. For example, canola honey contained a much lower amount of uridine (17.5 ± 3.9 mg/kg) than black locust where it was most abundant (51.2 ± 7.8 mg/kg). The presence of free nucleosides and nucleobases in different honey varieties is reported first time and supports previous findings on medicinal activities of honey reported in the literature as well as traditional therapy and may contribute for their explanation. This applies, e.g., to the topical application of honey in herpes infections, as well as its beneficial activity on cognitive functions as nootropic and neuroprotective, in neuralgia and is also important for the understanding of nutritional values of honey.
Subject(s)
Amino Acids, Aromatic/chemistry , Fagopyrum/chemistry , Honey , Nitrogen Compounds/chemistry , Adenine/chemistry , Chromatography, High Pressure Liquid , Mass Spectrometry , Nucleosides/chemistry , Phenylalanine/chemistry , Tilia/chemistry , Tyrosine/chemistry , Uridine/chemistry , Xanthine/chemistryABSTRACT
A novel ß-D-2'-deoxy-2'-α-fluoro-2'-ß-C-(fluoromethyl)uridine phosphoramidate prodrug (1) has been synthesized. This compound exhibits submicromolar-level antiviral activity in vitro against HCV genotypes 1b, 1a, 2a, and S282T replicons (EC50 = 0.18-1.13 µM) with low cytotoxicity (CC50 > 1000 µM). Administered orally, prodrug 1 is well tolerated at doses of up to 4 g/kg in mice, and produces a high level of the corresponding triphosphate in rat liver.
Subject(s)
Hepacivirus/drug effects , Hepatitis C/drug therapy , Organophosphorus Compounds/pharmacology , Prodrugs/pharmacology , Uridine/analogs & derivatives , Administration, Oral , Animals , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Hepacivirus/genetics , Humans , Liver/drug effects , Liver/virology , Mice , Mice, Inbred Strains , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Structure , Organophosphorus Compounds/administration & dosage , Organophosphorus Compounds/chemistry , Prodrugs/administration & dosage , Prodrugs/chemical synthesis , Prodrugs/chemistry , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Uridine/administration & dosage , Uridine/chemistry , Uridine/pharmacology , Virus Replication/drug effectsABSTRACT
Selenium is present in proteins in the form of selenocysteine, where this amino acid serves catalytic oxidoreductase functions. The use of selenocysteine in nature is strongly associated with redox catalysis. However, selenium is also found in a 2-selenouridine moiety at the wobble position of tRNAGlu, tRNAGln and tRNALys. It is thought that the modifications of the wobble position of the tRNA improves the selectivity of the codon-anticodon pair as a result of the physico-chemical changes that result from substitution of sulfur and selenium for oxygen. Both selenocysteine and 2-selenouridine have widespread analogs, cysteine and thiouridine, where sulfur is used instead. To examine the role of selenium in 2-selenouridine, we comparatively analyzed the oxidation reactions of sulfur-containing 2-thiouracil-5-carboxylic acid (s2c5Ura) and its selenium analog 2-selenouracil-5-carboxylic acid (se2c5Ura) using 1H-NMR spectroscopy, 77Se-NMR spectroscopy, and liquid chromatography-mass spectrometry. Treatment of s2c5Ura with hydrogen peroxide led to oxidized intermediates, followed by irreversible desulfurization to form uracil-5-carboxylic acid (c5Ura). In contrast, se2c5Ura oxidation resulted in a diselenide intermediate, followed by conversion to the seleninic acid, both of which could be readily reduced by ascorbate and glutathione. Glutathione and ascorbate only minimally prevented desulfurization of s2c5Ura, whereas very little deselenization of se2c5Ura occurred in the presence of the same antioxidants. In addition, se2c5Ura but not s2c5Ura showed glutathione peroxidase activity, further suggesting that oxidation of se2c5Ura is readily reversible, while oxidation of s2c5Ura is not. The results of the study of these model nucleobases suggest that the use of 2-selenouridine is related to resistance to oxidative inactivation that otherwise characterizes 2-thiouridine. As the use of selenocysteine in proteins also confers resistance to oxidation, our findings suggest a common mechanism for the use of selenium in biology.
Subject(s)
Selenium/metabolism , Selenocysteine/metabolism , Sulfur/metabolism , Uracil/metabolism , Glutathione/metabolism , Hydrogen Peroxide/pharmacology , Magnetic Resonance Spectroscopy , Organoselenium Compounds/chemistry , Organoselenium Compounds/metabolism , Oxidation-Reduction , Oxidative Stress , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Selenium/chemistry , Selenocysteine/chemistry , Sulfur/chemistry , Uracil/analogs & derivatives , Uracil/chemistry , Uridine/analogs & derivatives , Uridine/chemistry , Uridine/metabolismABSTRACT
Reported is an efficient synthesis of adenyl and uridyl 5'-tetrachlorophthalimido-5'-deoxyribonucleosides, and guanylyl 5'-azido-5'-deoxyribonucleosides, which are useful in solid-phase synthesis of phosphoramidate and ribonucleic guanidine oligonucleotides. Replacement of 5'-hydroxyl with tetrachlorophthalimido group was performed via Mitsunobu reaction for adenosine and uridine. An alternative method was applied for guanosine which replaced the 5'-hydroxyl with an azido group. The resulting compounds were converted to 5'-amino-5'-deoxyribonucleosides for oligonucleotide synthesis. Synthetic intermediates were tested as antimicrobials against six bacterial strains. All analogs containing the 2',3'-O-isopropylidine protecting group demonstrated antibacterial activity against Neisseria meningitidis, and among those analogs with 5'-tetrachlorophthalimido and 5'-azido demonstrated increased antibacterial effect.
Subject(s)
Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Adenosine/chemistry , Anti-Bacterial Agents/chemical synthesis , Azides/chemistry , Chemistry Techniques, Synthetic , Deoxyribonucleosides/chemical synthesis , Deoxyribonucleosides/pharmacology , Drug Evaluation, Preclinical/methods , Microbial Sensitivity Tests , Neisseria meningitidis/drug effects , Phthalimides/chemistry , Uridine/chemistryABSTRACT
Further exploration of substrate diversity of the sansaninycin biosynthetic pathway using available halogen- and methyl-phenylalanines led to the generation of diverse sansanmycin derivatives, either at the single C- or N-terminus alone or at both C- and N-termini. The structures of all of these derivatives were determined by MS/MS spectra, and amongst them, the structures of [2-Cl-Phe]-sansanmycin H (1) and [2-Cl-Phe]-sansanmycin A (2) were further identified by NMR. Both the C-terminal derivative I and the N-terminal derivative 2 were assayed for their antibacterial activitiesi and compound 1 exhibited moderate activity against P. aeruginosa and ΔtolC mutant E. coli.
Subject(s)
Oligopeptides/chemistry , Oligopeptides/metabolism , Peptide Synthases/metabolism , Uridine/analogs & derivatives , Molecular Structure , Substrate Specificity , Uridine/chemistry , Uridine/metabolismABSTRACT
On December 11, 2015, the FDA approved uridine triacetate (VISTOGARD; Wellstat Therapeutics Corporation) for the emergency treatment of adult and pediatric patients following a fluorouracil or capecitabine overdose regardless of the presence of symptoms, and of those who exhibit early-onset, severe, or life-threatening toxicity affecting the cardiac or central nervous system, and/or early onset, unusually severe adverse reactions (e.g., gastrointestinal toxicity and/or neutropenia) within 96 hours following the end of fluorouracil or capecitabine administration. Uridine triacetate is not recommended for the nonemergent treatment of adverse reactions associated with fluorouracil or capecitabine because it may diminish the efficacy of these drugs, and the safety and efficacy of uridine triacetate initiated more than 96 hours following the end of administration of these drugs has not been established. The approval is based on data from two single-arm, open-label, expanded-access trials in 135 patients receiving uridine triacetate (10 g or 6.2 g/m(2) orally every 6 hours for 20 doses) for fluorouracil or capecitabine overdose, or who exhibited severe or life-threatening toxicities within 96 hours following the end of fluorouracil or capecitabine administration. Ninety-six percent of patients met the major efficacy outcome measure, which was survival at 30 days or survival until the resumption of chemotherapy, if prior to 30 days. The most common adverse reactions were vomiting, nausea, and diarrhea. This article summarizes the FDA review of this New Drug Application, the data supporting approval of uridine triacetate, and the unique regulatory situations encountered by this approval. Clin Cancer Res; 22(18); 4545-49. ©2016 AACR.
Subject(s)
Acetates/pharmacology , Acetates/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Drug Approval , Neoplasms/therapy , Uridine/analogs & derivatives , Acetates/chemistry , Animals , Antineoplastic Agents/chemistry , Capecitabine/administration & dosage , Capecitabine/adverse effects , Clinical Trials as Topic , Drug Evaluation, Preclinical , Fluorouracil/administration & dosage , Fluorouracil/adverse effects , Humans , Neoplasms/diagnosis , Prescription Drug Overuse , Research Design , Treatment Outcome , United States , United States Food and Drug Administration , Uridine/chemistry , Uridine/pharmacology , Uridine/therapeutic useABSTRACT
Selenium-derivatized RNAs are powerful tools for structure and function studies of RNAs and their protein complexes. By taking the advantage of selenium modifications, researchers can determine novel RNA structures via convenient SAD and MAD phasing. As one of the naturally occurring tRNA modifications, 2-seleno-uridine, which presents almost exclusively at the wobble position of anticodon loop in various bacterial tRNAs (Ching et al., Proc Natl Acad Sci U S A 82:347, 1985; Dunin-Horkawicz et al., Nucleic Acids Res 34:D145-D149, 2006), becomes one of the most promising modifications for crystallographic studies. Our previous studies have demonstrated many unique properties of 2-seleno-uridine, including stability (Sun et al., RNA 19:1309-1314, 2013), minimal structural perturbation (Sun et al., Nucleic Acids Res 40:5171-5179, 2012), and enhanced base-pairing fidelity (Sun et al., Nucleic Acids Res 40:5171-5179, 2012). In this protocol, we present the efficient chemical synthesis of 2-seleno-uridine triphosphate ((Se)UTP) and the facile transcription and purification of (Se)U-containing RNAs ((Se)U-RNA).
Subject(s)
Crystallography, X-Ray/methods , Nucleic Acids/chemistry , Nucleotides/chemistry , RNA/chemistry , Base Pairing , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Hydrolysis , Organoselenium Compounds/chemistry , RNA, Transfer/chemistry , Selenium/chemistry , Solvents/chemistry , Transcription, Genetic , Uridine/analogs & derivatives , Uridine/chemistryABSTRACT
The objective of the present study was to evaluate the antibacterial properties of a plant secondary metabolite - caffeine. Caffeine is present in over 100 plant species. Antibacterial activity of caffeine was examined against the following plant-pathogenic bacteria: Ralstonia solanacearum (Rsol), Clavibacter michiganesis subsp. sepedonicus (Cms), Dickeya solani (Dsol), Pectobacterium atrosepticum (Pba), Pectobacterium carotovorum subsp. carotovorum (Pcc), Pseudomonas syringae pv. tomato (Pst), and Xanthomonas campestris subsp. campestris (Xcc). MIC and MBC values ranged from 5 to 20 mM and from 43 to 100 mM, respectively. Caffeine increased the bacterial generation time of all tested species and caused changes in cell morphology. The influence of caffeine on the synthesis of DNA, RNA and proteins was investigated in cultures of plant pathogenic bacteria with labelled precursors: [(3)H]thymidine, [(3)H]uridine or (14)C leucine, respectively. RNA biosynthesis was more affected than DNA or protein biosynthesis in bacterial cells treated with caffeine. Treatment of Pba with caffeine for 336 h did not induce resistance to this compound. Caffeine application reduced disease symptoms caused by Dsol on chicory leaves, potato slices, and whole potato tubers. The data presented indicate caffeine as a potential tool for the control of diseases caused by plant-pathogenic bacteria, especially under storage conditions.
Subject(s)
Anti-Bacterial Agents/chemistry , Bacteria/drug effects , Caffeine/chemistry , Plant Diseases/microbiology , Cichorium intybus/microbiology , DNA/chemistry , Dose-Response Relationship, Drug , Leucine/chemistry , Microbial Sensitivity Tests , Pectobacterium/drug effects , Pectobacterium carotovorum/drug effects , Plant Leaves/microbiology , Pseudomonas syringae/drug effects , RNA/chemistry , Ralstonia/drug effects , Solanum tuberosum/microbiology , Temperature , Thymidine/chemistry , Uridine/chemistry , Xanthomonas campestris/drug effectsABSTRACT
We describe the formation and structure of nucleolipid/dendrimer multilayer films controlled by non-covalent interactions to obtain biomaterials that exhibit molecular recognition of nucleic acids. Layers of cationic poly(amidoamine) (PAMAM) dendrimers of generation 4 and the anionic nucleolipids 1,2-dilauroyl-sn-glycero-3-phosphatidylnucleosides (DLPNs) based on uridine (DLPU) and adenosine (DLPA) were first formed at the silica-water interface. The PAMAM/DLPN layers were then exposed to short oligonucleotides, polynucleotides and single stranded DNA (ssDNA). The interfacial properties were characterized using quartz crystal microbalance with dissipation monitoring, attenuated total reflection Fourier transform infrared spectroscopy and neutron reflectometry. Both types of DLPN were found to adsorb as aggregates to preadsorbed PAMAM monolayers with a similar interfacial structure and composition before rinsing with pure aqueous solution. Nucleic acids were found to interact with PAMAM/DLPA layers due to base pairing interactions, while the PAMAM/DLPU layers did not have the same capability. This was attributed to the structure of the DLPA layer, which is formed by aggregates that extend from the interface towards the bulk after rinsing with pure solvent, while the DLPU layer forms compact structures. In complementary experiments using a different protocol, premixed PAMAM/DLPN samples adsorbed to hydrophilic silica only when the mixtures contained positively charged aggregates, which is rationalized in terms of electrostatic forces. The PAMAM/DLPA layers formed from the adsorption of these mixtures also bind ssDNA although in this case the adsorption is mediated by the opposite charges of the film and the nucleic acid rather than specific base pairing. The observed molecular recognition of nucleic acids by dendrimers functionalized via non-covalent interactions with nucleolipids is discussed in terms of biomedical applications such as gene vectors and biosensors.
Subject(s)
Adenosine/chemistry , Dendrimers/chemistry , Lipids/chemistry , Uridine/chemistry , DNA/chemistry , Polynucleotides/chemistry , Silicon Dioxide/chemistry , Water/chemistryABSTRACT
Unlike DNA, in addition to the 2'-OH group, uracil nucleobase and its modifications play essential roles in structure and function diversities of non-coding RNAs. Non-canonical Uâ¢U base pair is ubiquitous in non-coding RNAs, which are highly diversified. However, it is not completely clear how uracil plays the diversifing roles. To investigate and compare the uracil in U-A and Uâ¢U base pairs, we have decided to probe them with a selenium atom by synthesizing the novel 4-Se-uridine ((Se)U) phosphoramidite and Se-nucleobase-modified RNAs ((Se)U-RNAs), where the exo-4-oxygen of uracil is replaced by selenium. Our crystal structure studies of U-A and Uâ¢U pairs reveal that the native and Se-derivatized structures are virtually identical, and both U-A and Uâ¢U pairs can accommodate large Se atoms. Our thermostability and crystal structure studies indicate that the weakened H-bonding in U-A pair may be compensated by the base stacking, and that the stacking of the trans-Hoogsteen Uâ¢U pairs may stabilize RNA duplex and its junction. Our result confirms that the hydrogen bond (O4( )H-C5) of the Hoogsteen pair is weak. Using the Se atom probe, our Se-functionalization studies reveal more insights into the Uâ¢U interaction and U-participation in structure and function diversification of nucleic acids.
Subject(s)
RNA, Untranslated/chemistry , Selenium/chemistry , Uracil/chemistry , Base Pairing , Crystallography, X-Ray , Models, Molecular , Nucleic Acid Denaturation , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/chemistry , RNA, Untranslated/chemical synthesis , Uridine/chemistryABSTRACT
Natural RNAs, especially tRNAs, are extensively modified to tailor structure and function diversities. Uracil is the most modified nucleobase among all natural nucleobases. Interestingly, >76% of uracil modifications are located on its 5-position. We have investigated the natural 5-methoxy (5-O-CH(3)) modification of uracil in the context of A-form oligonucleotide duplex. Our X-ray crystal structure indicates first a H-bond formation between the uracil 5-O-CH(3) and its 5'-phosphate. This novel H-bond is not observed when the oxygen of 5-O-CH(3) is replaced with a larger atom (selenium or sulfur). The 5-O-CH(3) modification does not cause significant structure and stability alterations. Moreover, our computational study is consistent with the experimental observation. The investigation on the uracil 5-position demonstrates the importance of this RNA modification at the atomic level. Our finding suggests a general interaction between the nucleobase and backbone and reveals a plausible function of the tRNA 5-O-CH(3) modification, which might potentially rigidify the local conformation and facilitates translation.
Subject(s)
Uridine/analogs & derivatives , Crystallography, X-Ray , DNA, A-Form/chemistry , Hydrogen Bonding , Models, Molecular , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Phosphates/chemistry , Selenium/chemistry , Sulfur/chemistry , Uracil/chemistry , Uridine/chemistryABSTRACT
5-Methylaminomethyl-2-selenouridine (mnm(5)Se(2)U) is found in the first position of the anticodon in certain tRNAs from bacteria, archaea and eukaryotes. This selenonucleoside is formed in Escherichia coli from the corresponding thionucleoside mnm(5)S(2)U by the monomeric enzyme YbbB. This nucleoside is present in the tRNA of Methanococcales, yet the corresponding 2-selenouridine synthase is unknown in archaea and eukaryotes. Here we report that a bipartite ybbB ortholog is present in all members of the Methanococcales. Gene deletions in Methanococcus maripaludis and in vitro activity assays confirm that the two proteins act in trans to form in tRNA a selenonucleoside, presumably mnm(5)Se(2)U. Phylogenetic data suggest a primal origin of seleno-modified tRNAs.
Subject(s)
Archaeal Proteins/metabolism , Methanococcus/genetics , Organoselenium Compounds/metabolism , RNA, Transfer/metabolism , Selenium/metabolism , Thiosulfate Sulfurtransferase/metabolism , Uridine/analogs & derivatives , Amino Acid Sequence , Animals , Archaeal Proteins/chemistry , Archaeal Proteins/classification , Archaeal Proteins/genetics , Ligases/chemistry , Ligases/classification , Ligases/genetics , Ligases/metabolism , Methanococcus/metabolism , Models, Molecular , Molecular Sequence Data , Organoselenium Compounds/chemistry , Phylogeny , Protein Structure, Tertiary , RNA, Transfer/classification , RNA, Transfer/genetics , Sequence Alignment , Thiosulfate Sulfurtransferase/chemistry , Thiosulfate Sulfurtransferase/genetics , Uridine/chemistry , Uridine/genetics , Uridine/metabolismABSTRACT
Beginning with a known 3-oxabicyclo[3.1.0]hexane scaffold (I), the relocation of the fused cyclopropane ring bond and the shifting of the oxygen atom to an alternative location engendered a new 2-oxabicyclo[3.1.0]hexane template (II) that mimics more closely the tetrahydrofuran ring of conventional nucleosides. The synthesis of this new class of locked nucleosides involved a novel approach that required the isocyanate II (B = NCO) with a hydroxyl-protected scaffold as a pivotal intermediate that was obtained in 11 steps from a known dihydrofuran precursor. The completion of the nucleobases was successfully achieved by quenching the isocyanate with the lithium salts of the corresponding acrylic amides that led to the uracil and thymidine precursors in a single step. Ring closure of these intermediates led to the target, locked nucleosides. The anti-HIV activity of 29 (uridine analogue), 31 (thymidine analogue), and 34 (cytidine analogue) was explored in human osteosarcoma (HOS) cells or modified HOS cells (HOS-313) expressing the herpes simplex virus 1 thymidine kinase (HSV-1 TK). Only the cytidine analogue showed moderate activity in HOS-313 cells, which means that the compounds are not good substrates for the cellular kinases.
Subject(s)
Pyrimidine Nucleosides/chemical synthesis , Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Cell Line, Tumor/drug effects , Cell Line, Tumor/virology , Cytidine/analogs & derivatives , Cytidine/chemistry , Drug Design , Drug Evaluation, Preclinical/methods , Herpesvirus 1, Human/enzymology , Herpesvirus 1, Human/genetics , Humans , Magnetic Resonance Spectroscopy , Molecular Conformation , Molecular Structure , Pyrimidine Nucleosides/chemistry , Stereoisomerism , Thymidine/analogs & derivatives , Thymidine/chemistry , Thymidine Kinase/genetics , Thymidine Kinase/metabolism , Uridine/analogs & derivatives , Uridine/chemistryABSTRACT
Incorporation of 5-bromouridine (5BrdU) into DNA makes it sensitive to UV and ionizing radiation, which opens up a prospective route for the clinical usage of 5-bromouridine and other halonucleosides. In the present work the polymerase chain reaction (PCR) protocol, which enables a long DNA fragment (resembling DNA synthesized in the cell in the presence of halonucleosides) to be completely substituted with 5BrdU, was optimized. Using HPLC coupled to enzymatic digestion, it was demonstrated that the actual amounts of native nucleosides and 5BrdU correspond very well to those calculated from the sequence of PCR products. The synthesized DNA is photosensitive to photons of 300nm. HPLC analysis demonstrated that the photolysis of labeled PCR products leads to a significant decrease in the 5BrdU signal and the simultaneous occurrence of a uridine peak. Agarose and polyacrylamide gel electrophoresis suggest that single strand breaks and cross-links are formed as a result of UV irradiation. The PCR protocol described in the current paper may be employed for labeling DNA not only with BrdU but also with other halonucleosides.
Subject(s)
DNA/chemistry , Drug Compounding/methods , Polymerase Chain Reaction/methods , Uridine/analogs & derivatives , Bromouracil/analogs & derivatives , Clinical Trials as Topic , DNA/metabolism , DNA-Directed DNA Polymerase/analysis , DNA-Directed DNA Polymerase/metabolism , Drug Evaluation, Preclinical , Halogenation , Humans , Nucleosides/chemistry , Nucleosides/metabolism , Oligonucleotides/analysis , Oligonucleotides/chemistry , Oligonucleotides/metabolism , Photosensitizing Agents/chemistry , Photosensitizing Agents/metabolism , Plasmids/genetics , Uridine/chemistry , Uridine/metabolism , Uridine Triphosphate/chemistry , Uridine Triphosphate/metabolismABSTRACT
INTRODUCTION: Xuebijing injection (XBJ) is a traditional Chinese herbal prescription widely used in the treatment of sepsis. This is the first report concerning the identification of XBJ constituents. In addition, to evaluate XBJ's quality, partial least square discrimination analysis (PLS-DA) was performed on chemical fingerprint data. OBJECTIVE: Establish an LC-MS method to identify the components in XBJ for the purpose of quality control. METHODOLOGY: Compounds were separated by HPLC using a C(18) column and gradient elution of acetonitrile-methanol (60:40, v/v) and water-acetic acid (100 : 0.5, v/v) in 80 min. HPLC equipped with diode array detector (DAD) coupled with time-of-flight (TOF) tandem mass spectrometry and HPLC electrospray ionisation (ESI) multi-stage tandem ion-trap mass spectrometry (IT-MS(n) ) method was developed to analyse XBJ's major components. Both positive and negative ionisation modes were employed. RESULTS: Twenty-one compounds including amino acids, phenolic acids, flavonoid glycoside, terpene glycoside and phthalide were identified or tentatively characterised. Their retention times, UV and MS spectra were compared with those of authentic compounds or literature data. The score plot of PLS-DA clearly revealed variations among samples produced in different commercial batches. CONCLUSIONS: The analytical method developed is highly effective for the discrimination and quality control of XBJ.
Subject(s)
Chromatography, High Pressure Liquid/methods , Drugs, Chinese Herbal/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Benzofurans/analysis , Benzofurans/chemistry , Chalcone/analogs & derivatives , Chalcone/analysis , Chalcone/chemistry , Drugs, Chinese Herbal/analysis , Gallic Acid/analysis , Gallic Acid/chemistry , Molecular Structure , Pigments, Biological/analysis , Pigments, Biological/chemistry , Quinones/analysis , Quinones/chemistry , Uridine/analysis , Uridine/chemistryABSTRACT
OBJECTIVE: To study the chemical constituents from aerial parts of Gynura divaricata. METHODS: The constituents were isolated on silica gel column chromatography, preparative TLC and Sephadex LH-20 column chromatography, identified by physicochemical properties and the structures were elucidated by spectral analysis. RESULTS: 10 compounds were isolated and identified as 2-(1', 2', 3', 4'-tetrahydroxybutyl)-6-(2", 3", 4"-trihydroxybutyl)-pyrazine (1), 2-(1', 2', 3', 4'-tetrahydroxybutyl)-5-(2", 3", 4"-trihydroxybutyl) -pyrazine (2), nicotinic acid (3), 5-hydroxy-picolinic acid(4), methyl-5-hydroxy-2- pyridinecarboxylate (5), adenosine (6), uridine (7), stigmasterol-5-O- beta-D-glucoside (8), dibutyl terephthalate (9), methyl chlorogenate (10). CONCLUSION: Compounds 1, 2, 5, 9, 10 are obtained from this genus for the first time, Compounds 3, 4 are obtained from this plant for the first time.
Subject(s)
Asteraceae/chemistry , Niacin/isolation & purification , Picolinic Acids/isolation & purification , Plant Components, Aerial/chemistry , Plants, Medicinal/chemistry , Adenosine/chemistry , Adenosine/isolation & purification , Chlorogenic Acid/analogs & derivatives , Chlorogenic Acid/chemistry , Chlorogenic Acid/isolation & purification , Magnetic Resonance Spectroscopy , Molecular Structure , Niacin/chemistry , Picolinic Acids/chemistry , Uridine/chemistry , Uridine/isolation & purificationABSTRACT
Cultivated faba bean (Vicia faba) is widely used as human food, especially in Europe, Northern Africa and China. In view of its superior feeding value over field peas or other legumes, it is also widely used as animal feed for a variety of species. V. faba also contains medically important components such as 3,4-dihydroxyphenylalanine (levo-DOPA, L-DOPA), the principal treatment used for Parkinson's disease patients. However, this species also contains several antinutritional components, including the pyrimidine glycosides vicine and convicine; phytates; and the sucrose galactosides including raffinose, stachyose and verbascose. We have undertaken a genomic project to provide publicly available expressed sequence tag sequences (EST) prepared from early to mid developing embryo in an attempt to identify genes that are likely to be involved in the biosynthesis of L-DOPA and the vicine group of compounds. As initial examples of the utility of this approach, we describe the complete sequence of fabatin, new defensins, type 4 metallothioneins and a variety of other key genes which were identified in this EST library. No candidate sequences corresponding to the biosynthesis of L-DOPA or the vicine group could be identified at this early stage of seed development.
Subject(s)
Genomics/methods , Plant Proteins/genetics , Seeds/genetics , Vicia faba/genetics , Amino Acid Sequence , Defensins/genetics , Defensins/metabolism , Dihydroxyphenylalanine/biosynthesis , Dihydroxyphenylalanine/chemistry , Dihydroxyphenylalanine/therapeutic use , Dopamine Agents/chemistry , Dopamine Agents/therapeutic use , Expressed Sequence Tags , Gene Library , Genetic Engineering/methods , Genetic Engineering/trends , Glucosides/biosynthesis , Glucosides/chemistry , Humans , Metallothionein/genetics , Metallothionein/metabolism , Molecular Sequence Data , Oligosaccharides/biosynthesis , Oligosaccharides/chemistry , Parkinson Disease/prevention & control , Plant Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Plants, Medicinal/genetics , Plants, Medicinal/metabolism , Pyrimidinones/chemistry , Seeds/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Uridine/analogs & derivatives , Uridine/biosynthesis , Uridine/chemistry , Vicia faba/metabolismABSTRACT
The Sm-like protein Hfq promotes the association of small antisense RNAs (sRNAs) with their mRNA targets, but the mechanism of Hfq's RNA chaperone activity is unknown. To investigate RNA annealing and strand displacement by Hfq, we used oligonucleotides that mimic functional sequences within DsrA sRNA and the complementary rpoS mRNA. Hfq accelerated at least 100-fold the annealing of a fluorescently labeled molecular beacon to a 16-nt RNA. The rate of strand exchange between the oligonucleotides increased 80-fold. Therefore, Hfq is very active in both helix formation and exchange. However, high concentrations of Hfq destabilize the duplex by preferentially binding the single-stranded RNA. RNA binding and annealing were completely inhibited by 0.5 M salt. The target site in DsrA sRNA was 1000-fold less accessible to the molecular beacon than an unstructured oligonucleotide, and Hfq accelerated annealing with DsrA only 2-fold. These and other results are consistent with recycling of Hfq during the annealing reaction, and suggest that the net reaction depends on the relative interaction of Hfq with the products and substrates.