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Joanne Stubbe - One of the best experts on this subject based on the ideXlab platform.
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binding of cob ii alamin to the adenosylcobalamin dependent ribonucleotide reductase from lactobacillus leichmannii identification of dimethylbenzimidazole as the axial ligand
Journal of Biological Chemistry, 1999Co-Authors: Christopher C Lawrence, Gary J Gerfen, Vicente Samano, Rainer Nitsche, Morris J Robins, Janos Retey, Joanne StubbeAbstract:Abstract The ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii catalyzes the reduction of nucleoside 5′-triphosphates to 2′-deoxynucleoside 5′-triphosphates and uses coenzyme B12, adenosylcobalamin (AdoCbl), as a cofactor. Use of a mechanism-based inhibitor, 2′-deoxy-2′-methylenecytidine 5′-triphosphate, and isotopically labeled RTPR and AdoCbl in conjunction with EPR spectroscopy has allowed identification of the lower axial ligand of cob(II)alamin when bound to RTPR. In common with the AdoCbl-dependent enzymes catalyzing irreversible heteroatom migrations and in contrast to the enzymes catalyzing reversible carbon skeleton rearrangements, the dimethylbenzimidazole moiety of the cofactor is not displaced by a protein histidine upon binding to RTPR.
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gemcitabine 5 triphosphate is a stoichiometric mechanism based inhibitor of lactobacillus leichmannii ribonucleoside triphosphate reductase evidence for thiyl radical mediated nucleotide radical formation
Biochemistry, 1998Co-Authors: Domingos J Silva, Joanne Stubbe, Vicente Samano, Morris J RobinsAbstract:Ribonucleoside triphosphate reductase (RTPR) from Lactobacillus leichmannii utilizes adenosylcobalamin and catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. One equivalent of 2‘,2‘-difluoro-2‘-deoxycytidine 5‘-triphosphate, F2dCTP, rapidly inactivates RTPR. Analysis of the reaction products reveals that inactivation is accompanied by release of two fluoride ions and 0.84 equiv of 5‘-deoxyadenosine and attachment of 1 equiv of corrin covalently to an active-site cysteine residue of RTPR. No cytosine release was detected. Proteolysis of corrin-labeled RTPR with endoproteinase Glu-C and peptide mapping at pH 5.8 revealed that C419 was predominantly modified. The kinetics of the inactivation have been examined by stopped-flow (SF) UV−vis spectroscopy and rapid freeze quench (RFQ) electron paramagnetic resonance (EPR) spectroscopy. Monitoring ΔA525 nm shows that cob(II)alamin is formed with an apparent kobs of 50 s-1, only 2.5-fold slower than a similar experiment carried o...
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cloning sequencing and expression of the adenosylcobalamin dependent ribonucleotide reductase from lactobacillus leichmannii
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Squire J Booker, Joanne StubbeAbstract:Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii.
Phillip A. Furman - One of the best experts on this subject based on the ideXlab platform.
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the mechanism of action of β d 2 deoxy 2 fluoro 2 c methylcytidine involves a second metabolic pathway leading to β d 2 deoxy 2 fluoro 2 c methyluridine 5 triphosphate a potent inhibitor of the hepatitis c virus rna dependent rna polymerase
Antimicrobial Agents and Chemotherapy, 2008Co-Authors: Eisuke Murakami, Congrong Niu, Haiying Bao, Holly Micolochick M Steuer, Tony Whitaker, Tammy Nachman, Michael A Sofia, Peiyuan Wang, Michael J Otto, Phillip A. FurmanAbstract:beta-D-2'-Deoxy-2'-fluoro-2'-C-methylcytidine (PSI-6130) is a potent inhibitor of hepatitis C virus (HCV) RNA replication in an HCV replicon assay. The 5'-triphosphate of PSI-6130 is a competitive inhibitor of the HCV RNA-dependent RNA polymerase (RdRp) and acts as a nonobligate chain terminator. Recently, it has been shown that the metabolism of PSI-6130 also results in the formation of the 5'-triphosphate of the uridine congener, beta-D-2'-deoxy-2'-fluoro-2'-C-methyluridine (PSI-6206; RO2433). Here we show that the formation of the 5'-triphosphate of RO2433 (RO2433-TP) requires the deamination of PSI-6130 monophosphate and that RO2433 monophosphate is subsequently phosphorylated to the corresponding di- and triphosphates by cellular UMP-CMP kinase and nucleoside diphosphate kinase, respectively. RO2433-TP is a potent inhibitor of the HCV RdRp; however, both enzymatic and cell-based assays show that PSI-6130 triphosphate is a more potent inhibitor of the HCV RdRp than RO2433-TP.
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the anti hepatitis b virus activities cytotoxicities and anabolic profiles of the and enantiomers of cis 5 fluoro 1 2 hydroxymethyl 1 3 oxathiolan 5 yl cytosine
Antimicrobial Agents and Chemotherapy, 1992Co-Authors: Phillip A. Furman, Michelle G Davis, D C Liotta, M T Paff, Lloyd Frick, Donald J Nelson, R E Dornsife, J A Wurster, L J Wilson, James A FyfeAbstract:The anti-hepatitis B (anti-HBV) activities of the (-) and (+) enantiomers of cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]cytosine (29-deoxy-39-thia-5-fluorocytosine [FTC]) were studied by using an HBV-transfected cell line (HepG2 derivative 2.2.15, subclone P5A). The (-) isomer was found to be a potent inhibitor of viral replication, with an apparent 50% inhibitory concentration of 10 nM, while the (+) isomer was found to be considerably less active. Both isomers showed minimal toxicity to HepG2 cells (50% inhibitory concentration, > 200 microM) and showed minimal toxicity in the human bone marrow progenitor cell assay. In accord with the cellular antiviral activity data, the 59-triphosphate of (-)-FTC inhibited viral DNA synthesis in an endogenous HBV DNA polymerase assay, while the 59-triphosphate of the (+) isomer was inactive. Unphosphorylated (-)-FTC did not inhibit product formation in the endogenous assay, suggesting that the antiviral activity of the compound is dependent on anabolism to the 59-triphosphate. Both (-)- and (+)-FTC were anabolized to the corresponding 59-triphosphates in chronically HBV-infected HepG2 cells. The rate of accumulation and the steady-state concentration of the 59-triphosphate of (-)-FTC were greater. Also, (-)-FTC was not a substrate for cytidine deaminase and, therefore, is not subject to deamination and conversion to an inactive uridine analog. The (+) isomer is, however, a good substrate for cytidine deaminase. Images
G.p. Connolly - One of the best experts on this subject based on the ideXlab platform.
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structure activity relationship of a pyrimidine receptor in the rat isolated superior cervical ganglion
British Journal of Pharmacology, 1995Co-Authors: G.p. Connolly, Paul J. HarrisonAbstract:Abstract 1. The effects of pyrimidines and purines on the d.c. potential of the rat isolated superior cervical ganglion (SCG) have been examined by a grease-gap technique to determine the structure-activity requirements of the receptor activated by pyrimidines, i.e. a pyrimidinoceptor. 2. 5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl (ZTP), the pyrimidines, cytidine 5'-triphosphate (CTP), uridine 5'-triphosphate (UTP) and thymidine 5'-triphosphate (TTP) and the purines, adenosine 5'-triphosphate (ATP; in the presence of an A1-purinoceptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) (1 microM)), adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), guanosine 5'-triphosphate (GTP), inosine 5'-triphosphate (1TP) depolarized ganglia in a concentration-dependent manner. The relative order of ZTP and purine 5'-triphosphates in depolarizing ganglia was ZTP > or = ATP gamma S > > ATP > or = ITP = GTP, and for the pyrimidine 5'-triphosphates UTP > TTP > or = CTP. Depolarizations evoked by ATP gamma S were followed by concentration-dependent hyperpolarizations at 100 and 1000 microM. 3. At concentrations of between 0.1 microM and 1 mM, uridine 5'-diphosphate (UDP), uridine 5'-diphosphoglucose (UDPG) and uridine 5'-diphosphoglucuronic acid (UDPGA) evoked significant and concentration-dependent depolarizations, whereas uridine 5'-monophosphate (UMP), uridine and uracil were inactive or produced small ( or = UTP > UDPG > UDPGA > > uracil > or = UMP = pseudouridine > or = uridine. At 3 and 10 mM, uridine produced concentration-dependent hyperpolarizations. Nikkomycin Z, a nucleoside resembling UTP (viz. the triphosphate chain at the 5'-position on the ribose moiety being replaced by a peptide), was inactive between 1 microM and 1 mM. Generally, a concentration of 10 mM was required before thymidine, 6-azathymine, 6-azauracil or 6-azauridine depolarized ganglia. 4. Suramin (300 microM), a P2-purinoceptor antagonist, significantly depressed depolarizations evoked by alpha, beta-methylene-ATP (alpha, beta-MeATP; 100 microM), ATP gamma S (100 microM), CTP (1 mM), GTP (1 mM), ZTP (30 microM) and ATP (300 microM) in the presence of DPCPX (1 microM). Suramin reversed a small depolarization evoked by UMP (1 mM) into a small hyperpolarization. In contrast depolarizations evoked by UDP, UTP, UDPG (all at 100 microM) and TTP (300 microM) were unaltered or enhanced by suramin. 5. It is concluded that the rat SCG contains distinct nucleotide receptors including a P2-purinoceptor (activated by alpha, beta-MeATP, ATP, GTP, ITP and ZTP) and a pyrimidinoceptor (activated by UTP, UDP, UDPG, UDPGA and TTP). The pyrimidinoceptor on rat SCG neurones had specific structure activity requirements with the di- and triphosphates of uridine being the most effective depolarizing agonists examined.
Nerea Ferreirós - One of the best experts on this subject based on the ideXlab platform.
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Quantitation of endogenous nucleoside triphosphates and nucleosides in human cells by liquid chromatography tandem mass spectrometry.
Analytical and bioanalytical chemistry, 2015Co-Authors: Dominique Thomas, Nikolas Herold, Oliver T. Keppler, Gerd Geisslinger, Nerea FerreirósAbstract:Nucleosides and nucleoside triphosphates are the building blocks of nucleic acids and important bioactive metabolites, existing in all living cells. In the present study, two liquid chromatography tandem mass spectrometry methods were developed to quantify both groups of compounds from the same sample with a shared extraction procedure. After a simple protein precipitation with methanol, the nucleosides were separated with reversed phase chromatography on an Atlantis T3 column while for the separation of the nucleoside triphosphates, an anion exchange column (BioBasic AX) was used. No addition of ion pair reagent was required. A 5500 QTrap was used as analyzer, operating as triple quadrupole. The analytical method for the nucleoside triphosphates has been validated according to the guidelines of the US Food and Drug Administration. The lower limit of quantification values were determined as 10 pg on column (0.5 ng/mL in the injection solution) for deoxyadenosine triphosphate and deoxyguanosine triphosphate, 20 pg (1 ng/mL) for deoxycytidine triphosphate and thymidine triphosphate, 100 pg (5 ng/mL) for cytidine triphosphate and guanosine triphosphate, and 500 pg (25 ng/mL) for adenosine triphosphate und uridine triphosphate respectively. This methodology has been applied to the quantitation of nucleosides and nucleoside triphosphates in primary human CD4 T lymphocytes and macrophages. As expected, the concentrations for ribonucleosides and ribonucleoside triphophates were considerably higher than those obtained for the deoxy derivatives. Upon T cell receptor activation, the levels of all analytes, with the notable exceptions of deoxyadenosine triphosphate and deoxyguanosine triphosphate, were found to be elevated in CD4 T cells.
Chris Meier - One of the best experts on this subject based on the ideXlab platform.
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Membrane-permeable Triphosphate Prodrugs of Nucleoside Analogues.
Angewandte Chemie (International ed. in English), 2016Co-Authors: Tristan Gollnest, Thiago Dinis De Oliveira, Anna K. Rath, Ilona Hauber, Dominique Schols, Jan Balzarini, Chris MeierAbstract:The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.
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interactions of conformationally biased north and south 2 fluoro 2 3 dideoxynucleoside 5 triphosphates with the active site of hiv 1 reverse transcriptase
Biochemistry, 2000Co-Authors: Stefan G Sarafianos, Chris Meier, Marc C Nicklaus, Pamela Russ, Maqbool A Siddiqui, Harry Ford, Hiroaki Mitsuya, Eiichi Kodama, Tina Knispel, Lynne AndersonAbstract:Molecular dynamics simulations of a ternary complex of HIV-1 reverse transcriptase (RT), double-stranded DNA, and bound dideoxynucleoside-5‘-triphosphate (RT−DNA−ddNTP), utilizing the ddNTPs ddATP, βFddATP, and αFddATP, explain the experimentally observed order of potency of these 5‘-triphosphates as inhibitors of RT: ddATP > βFddATP > αFddATP. On the basis of RT's known preference to bind the incoming dNTP (or ddNTP) with a north conformation at the polymerase site, αFddATP, which in solution prefers almost exclusively a north conformation, was predicted to be the most potent inhibitor. However, Tyr115, which appears to function as a steric gate to preclude the binding of ribonucleoside 5‘-triphosphates, prevents the effective binding of αFddATP in its preferred north conformation. The south-biased βFddATP, while able to bind to RT without hindrance by Tyr115, has to pay a high energy penalty to be flipped to the active north conformation at the polymerase site. Finally, the more flexible and less confo...
