藥學專業(yè)畢業(yè)論文外文翻譯--通過合成氟代嘧啶核苷競爭性抑制人多聚(a)特異性核糖核酸酶(parn)（英文）

1、See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/26244358Competitive Inhibition of Human Poly(A)-Specific Ribonuclease (PARN) by SyntheticFluoro-Pyranosyl Nucl

2、eosidesARTICLE in BIOCHEMISTRY · JULY 2009Impact Factor: 3.02 · DOI: 10.1021/bi900236k · Source: PubMedCITATIONS21READS739 AUTHORS, INCLUDING:Panagiotis MaragozidisUniversity of Thessaly9 PUBLICATIONS

3、67 CITATIONS SEE PROFILEStella MantaUniversity of Thessaly50 PUBLICATIONS 388 CITATIONS SEE PROFILEDimitrios AnastasakisUniversity of Patras4 PUBLICATIONS 42 CITATIONS SEE PROFILEDimitri KomiotisUniversity of T

4、hessaly89 PUBLICATIONS 808 CITATIONS SEE PROFILEAvailable from: Dimitrios VlachakisRetrieved on: 29 January 2016Article Biochemistry, Vol. 48, No. 26, 2009 6045nucleotides and nucleosides, and the active metabolites

5、target one or more enzymes, such as DNA and RNA polymerases. Ribonu- clease activities could also represent potential molecular targets for inhibition by nucleoside analogues. Such is the case of the RNase H domain of HI

6、V RNA-dependent DNA polymerase (26-28). During recent years, nucleoside analogues with a six-membered carbohydrate moiety, such as ketonucleosides, have been evalu- ated for their anticancer and antiviral potential (29-3

7、2). Further- more, the addition of a fluorine atom on the sugar ring and the addition of a benzoyl-group on the base improves the biological activityof the analogue (ref (31) and references therein).However, no molecular

8、 target of these compounds or their synthetic intermediates has been identified so far. The above-mentioned work, prompted us to study human PARN as a potential molecular target for inhibition by such nucleoside analogue

9、s. We focused on this enzyme given that its activity is reduced by natural nucleotides, its structure is known to a significant extent, and it exhibits the major deadenylation activity in mammals. We demonstrate that PAR

10、N can be efficiently inhibited by several nucleoside analogues bearing a six-membered fluoro-modified sugar moiety with or without the benzoyl group on the base (adenine or cytosine). We find that these analogues behave

11、as competitive inhibitors of PARN and more importantly, increased magnesium(II) ion concentrations could not restore the enzyme’s activity. To support our biochemical data, we performed mole- cular docking experiments fo

12、llowed by molecular dynamics (MD) simulations, and we predicted the docking conformation of the nucleosides into the active site. Both the biochemical and the in- silico 3D-model analyses revealed a crucial role of the t

13、hree hydroxyl groups of the sugar moiety for efficient inhibitory effect. Our data suggest that the analogues used in the present study could serve as lead compounds for the development of novel inhibitors of PARN and po

14、ssibly other essential deadenylases with potential use for novel therapeutic approaches.MATERIALS AND METHODSMaterials. All chemicals including purine ribonucleotides and deoxynucleotides, methylene blue, and polyadenyli

15、c acid potassium salt (average size 300 adenosines, A300) were from SigmaAldrich. Synthesis of Nucleosides Analogues. The fluoro-pyranosyl nucleosides were synthesized as previously described (31). Briefly, condensationo

16、f 1,2,4,6-tetra-O-acetyl-3-deoxy-3-fluoro-glucopyr- anose with cytosine, silyated N4-benzoyl cytosine, or N6-benzoyl adenine resulted in the production of [1-(20,40,60-tri-O-acetyl-30- deoxy-30-fluoro-β-D-glucopyranosyl)

17、-N4-benzoyl cytosine] (A5) or [9-(20,40,60-tri-O-acetyl-30-deoxy-30-fluoro-β-D-glucopyranosyl)-N6- benzoyl adenine] (A1), respectively, in the presence of trimethylsilyl trifluoromethane-sulfonate and tin chloride. Depro

18、tection of A5 and A1 with NaOH-ethanol-pyridine yielded [1-(30,40-dideoxy-30- fluoro-β-D-glucopyranosyl)] cytosine (C6), [1-(30,40-dideoxy-30- fluoro-β-D-glucopyranosyl)-N4-benzoyl cytosine] (A6), or [9-(30,40- dideoxy-3

19、0-fluoro-β-D-glucopyranosyl)-N6-benzoyl adenine] (A2), respectively. Treatment of A2 with 2,2-dimethoxypropane in dry N,N-dimethylformamide, followed by acetylation of the free hydroxyl group in the 20 position of the su

20、gar moiety with acetic anhydride/pyridine, removal of the isopropylidene group, and, finally, selective protection of the primary 60-hydroxyl group with a trityl group yielded compound A4. Oxidation of the fluoro acetyla

21、ted precursor A4 with pyridinium dichromate/acetic anhydride afforded [9-(30-deoxy-30-fluoro-60-O-trityl-β-D-glycero- hex-20-enopyranosyl-40-ulose)-N6-benzoyl adenine (A3).Expression and Purification of Recombinant PARN.

22、 The plasmid encoding full-size 74 kDa human PARN (kindly provided by Prof. A. Virtanen, Uppsala University, Sweden) (for expression of N-terminal His6-tagged polypeptide) was trans- formed into BL21(DE3) cells to expres

23、s the recombinant protein as described previously (33) with some modifications. Briefly, colonies were grown overnight at 37 ?C in the presence of kanamycin (50 μg/mL). The cultures were then diluted (1:100) in the same

24、medium and grown at 37 ?C induced by isopropyl-1- thio-β-D-galactopyranoside (IPTG) at a final concentration of 0.1 mM. Cultures for PARN expression were allowed to grow for 3 h at 37 ?C. Cells were harvested by centrifu

25、gation for 20 min at 4 ?C, and pellets werefrozen at -70 ?C. The expressed His-tagged soluble proteins were purified following previously described protocols (33). PARN Activity Assay and Kinetic Analysis. The enzy- mati

26、c activity was determined by the methylene blue assay as described before (34). Deadenylation rates as a function of time were determined with time-course assays (Supporting Informa- tion, Figure S1). Methylene blue buff

27、er was prepared by dissol- ving 1.2 mg of methylene blue into 100 mL of MOPS buffer (0.1 M MOPS-KOH, pH 7.5, and 2 mM EDTA). The standard reaction buffer contained 20 mM HEPES-KOH (pH 7.0), 1.5 mM MgCl2, 100 mM KCl, 0.1

28、U of RNasin, 0.2 mM EDTA, 0.25 mM DTT, 10% (v/v) glycerol, and 0.075-0.6 mM of poly(A). All ribonucleotides were dissolved in reaction buffer prior to use. The reactions were performed using 0.01-0.02 mM recombinant PARN

29、. For kinetic analysis, the substrate concentration [poly (A)] varied from 0.075 to 0.6 mM (19). The final reaction volume was 100 μL, and the reaction was performed at 30 ?C for 5- 10 min. The reaction was terminated by

30、 mixing the reaction solution with 900 μL of methylene blue buffer, and the mixed solution was incubated at 30 ?C for another 15 min in the dark in a water bath. The absorbance at 662 nm of 1 mL of sample was measured on

31、 a Spectronic Genesys 20 spectrophotometer. Coordinate Preparation. The coordinates of PARN were obtained from the known PARN crystal structure as deposited with the RCSB Protein databank (RCSB code: 2A1R). One of the tw

32、o identical active sites of the dimeric form was used for the docking calculations. All crystallographic water molecules were removed from the coordinate file prior to docking. Hydrogen atoms and partial charges were add

33、ed to the enzyme by the molecular operating environment (MOE) program (35) using the AMBER94 forcefield. MOE used the PEOE method to assign partial charges for all potential inhibitor compounds. After all non-hydrogen at

34、oms were fixed, the PARN molecule was energetically minimized by a combination of the steepest descent, conjugate gradient, and truncated Newton minimization meth- ods within MOE. Energy minimizations were also performed

35、 to relax the substrates prior to docking. Molecular Docking. The MOE suite was used to perform docking experiments and calculations of solvation and relative binding free energies. The Dock module of MOE utilizes a Mont

36、e Carlo simulated annealing (SA) method in docking calculations. A docking box of 60 ? 40 ? 40 points with a grid spacing of 0.375 A ?was placed around the active site of the protein for this purpose. The iteration limit

37、 was set to 20000, the number of cycles was set to 20, and the number of runs was set to 10. Molecular Dynamics Simulations. Molecular Dynamics (MD) simulations were performed using MOE and its built-in MD module using a