A scintillation proximity assay for dengue virus NS5 2′-O-methyltransferase—kinetic and inhibition analyses

Lim, Siew Pheng; Wen, Daying; Yap, Thai Leong; Yan, Chung Ka; Lescar, Julien; Vasudevan, Subhash G.

doi:10.1016/j.antiviral.2008.08.005

Cited by 57 publications

(60 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nevertheless, the optimum conditions for NS5MTase DV described here are in contrast to a reported pH optimum of 29O-MTase activity of NS5MTase DV on another type of short RNA substrate, GpppAGAACCUG, reported recently (Kroschewski et al, 2008;Lim et al, 2008). Activity was found to be optimal at pH 10 under low-salt conditions and in the absence of divalent ions.…”

Section: Optimization Of Reaction Conditionscontrasting

confidence: 52%

“…Activity was found to be optimal at pH 10 under low-salt conditions and in the absence of divalent ions. Interestingly, Mg 2+ and Mn 2+ ions stimulated the activity and shifted the optimum pH to 9.0 and 7.5, respectively (Kroschewski et al., 2008;Lim et al, 2008 conditions found by different groups could be due to differences in the protein sequences, the position of the His tag, the purification procedures influencing the folding of the protein or different substrates. Note that the NS5MTase DV used here was purified in the same way as the protein used for structure determination (Egloff et al, 2002(Egloff et al, , 2007 Dependence of 2 §O-methylation and binding on substrate length…”

Section: Optimization Of Reaction Conditionsmentioning

confidence: 99%

“…Activity values were measured in triplicate and relative activity values ±SD were calculated, setting the optimum condition (Tris/HCl at pH 7.5) at 100 %. Addition of 10 mM KCl, 2 mM MgCl 2 , 2 mM MnCl 2 and 0.05 % CHAPS was tested in parallel in Tris/HCl at pH 7.5 and pH 10 as described by Lim et al (2008) and in glycine buffer at pH 10 as described by Zhou et al (2007). …”

Section: Optimization Of Reaction Conditionsmentioning

confidence: 99%

“…Enzymes involved in the viral capping pathway are increasingly considered as promising targets for potential anti-DENV drugs (Dong et al, 2008b;Lescar et al, 2008). With respect to the flavivirus MTase as a potential target of antivirals, reverse genetic experiments using replicon and genome-length RNA have shown that mutations of N7-and/or 29O-MTase catalytic residues block or attenuate replication (Ray et al, 2006;Zhou et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

“…The latter consists of a conserved catalytic tetrad [for the MTase domain of DENV NS5 (NS5MTase DV ), this is K61-D146-K181-E217], which was shown to be essential for 29O-MTase activity, whereas only one of the residues (D146 for NS5MTase DV ) was essential for N7-MTase activity (Zhou et al, 2007). The structure of NS5MTase DV was also determined in ternary complexes with AdoHcy in the cofactor site and GTP or cap analogues (Egloff et al, 2007) or the inhibitor ribavirin triphosphate (a GTP analogue) in a site (Benarroch et al, 2004a) that is expected to serve as the cap-binding site during 29O-methylation.Enzymes involved in the viral capping pathway are increasingly considered as promising targets for potential anti-DENV drugs (Dong et al, 2008b;Lescar et al, 2008). With respect to the flavivirus MTase as a potential target of antivirals, reverse genetic experiments using replicon and genome-length RNA have shown that mutations of N7-and/or 29O-MTase catalytic residues block or attenuate replication (Ray et al, 2006;Zhou et al, 2007).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Selisko

Peyrane

Canard

et al. 2009

Journal of General Virology

View full text Add to dashboard Cite

The flavivirus RNA genome contains a conserved cap-1 structure, 7Me GpppA 29OMe G, at the 59 end. Two mRNA cap methyltransferase (MTase) activities involved in the formation of the cap, the (guanine-N7)-and the (nucleoside-29O)-MTases (29O-MTase), reside in a single domain of nonstructural protein NS5 (NS5MTase). This study reports on the biochemical characterization of the 29O-MTase activity of NS5MTase of dengue virus (NS5MTase DV ) using purified, short, capped RNA substrates ( 7Me GpppAC n or GpppAC n ). NS5MTase DV methylated both types of substrate exclusively at the 29O position. The efficiency of 29O-methylation did not depend on the methylation of the N7 position. Using 7Me GpppAC n and GpppAC n substrates of increasing chain lengths, it was found that both NS5MTase DV 29O activity and substrate binding increased before reaching a plateau at n55. Thus, the cap and 6 nt might define the interface providing efficient binding of enzyme and substrate. K m values for 7Me GpppAC 5 and the co-substrate S-adenosyl-Lmethionine (AdoMet) were determined (0.39 and 3.26 mM, respectively). As reported for other AdoMet-dependent RNA and DNA MTases, the 29O-MTase activity of NS5MTase DV showed a low turnover of 3.25¾10 "4 s "1 . Finally, an inhibition assay was set up and tested on GTP and AdoMet analogues as putative inhibitors of NS5MTase DV , which confirmed efficient inhibition by the reaction product S-adenosyl-homocysteine (IC 50 0.34 mM) and sinefungin (IC 50 0.63 mM), demonstrating that the assay is sufficiently sensitive to conduct inhibitor screening and characterization assays. INTRODUCTIONMost eukaryotic and viral mRNAs are modified by the addition of a cap structure at the 59 end. The mRNA cap consists of an N7-methylguanosine linked to the first transcribed nucleotide by a 59-59 triphosphate bridge (cap-0 structure, 7Me GpppN) (Shuman, 2001). Methylation of the 29O positions of the ribose of the first or the second transcribed nucleotide leads to a cap-1 ( 7Me GpppN 29OMe ) or cap-2 ( 7Me GpppN 29OMe N 29OMe ) structure, respectively. The cap structure stabilizes mRNA and plays a vital role in its translation by controlling mRNA splicing, its transport across the nuclear membrane and its recognition by the eIF4E translation factor (Furuichi & Shatkin, 2000). In eukaryotic cells, the acquisition of the cap-0 structure is a co-transcriptional event. RNA capping implies three steps in which the 59 triphosphate end of RNA is hydrolysed to a 59 diphosphate by an RNA triphosphatase (RTPase), then capped with GMP by a guanylyltransferase (GTase) and finally methylated at the N7 position of the guanine by an S-adenosyl-L-methionine (AdoMet)-dependent (guanine-N7)-methyltransferase (N7-MTase) (Gu & Lima, 2005). In contrast to mRNAs of lower eukaryotes, which bear a cap-0 structure, the mRNAs of higher eukaryotes acquire a cap-1 structure via the action of a (nucleoside-29O)-MTase (29O-MTase) (Langberg & Moss, 1981). Compared with N7 methylation, this mRNA methylation step seems to be less important for binding, tr...

show abstract

Section: Optimization Of Reaction Conditionscontrasting

confidence: 52%

Section: Optimization Of Reaction Conditionsmentioning

confidence: 99%

Section: Optimization Of Reaction Conditionsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Selisko

Peyrane

Canard

et al. 2009

Journal of General Virology

View full text Add to dashboard Cite

show abstract

Enzymatic Assays to Explore Viral mRNA Capping Machinery

Kasprzyk

Jemielity

2021

ChemBioChem

View full text Add to dashboard Cite

In eukaryotes, mRNA is modified by the addition of the 7‐methylguanosine (m 7 G) 5′ cap to protect mRNA from premature degradation, thereby enhancing translation and enabling differentiation between self (endogenous) and non‐self RNAs (e. g., viral ones). Viruses often develop their own mRNA capping pathways to augment the expression of their proteins and escape host innate immune response. Insights into this capping system may provide new ideas for therapeutic interventions and facilitate drug discovery, e. g., against viruses that cause pandemic outbreaks, such as beta‐coronaviruses SARS‐CoV (2002), MARS‐CoV (2012), and the most recent SARS‐CoV‐2. Thus, proper methods for the screening of large compound libraries are required to identify lead structures that could serve as a basis for rational antiviral drug design. This review summarizes the methods that allow the monitoring of the activity and inhibition of enzymes involved in mRNA capping.

show abstract

Evaluation of Adamantane Derivatives as Inhibitors of Dengue Virus mRNA Cap Methyltransferase by Docking and Molecular Dynamics Simulations

Luzhkov

Decroly

Canard

et al. 2012

Molecular Informatics

View full text Add to dashboard Cite

Binding of the Dengue virus S-adenosyl-L-methionine (AdoMet)-dependent mRNA cap methyltransferase (NS5MTaseDV ) with adamantane derivatives was explored using molecular modeling methods and (nucleoside-2'O)-methyltransferase bioassay. The studied compounds include urea derivatives of adamantane and the antiviral drugs amantadine and rimantadine. The urea derivatives of adamantanes had previously been identified as inhibitors of NS5MTaseDV . The docking simulations using GOLD, Glide, and Dock give consistent binding modes and binding affinities of adamantanes in the AdoMet-binding site of NS5MTaseDV and, in particular, yield similar positions for the previously found inhibitors. Combined, they perfectly correspond to the bioassay measurements of nucleoside-2'O-methyltransferase activity of NS5TaseDV , which confirmed inhibitory properties of the active urea adamantane but did not show inhibitory activity for amantadine and rimantadine. We also employed microscopic molecular dynamics (MD) simulations and a linear interaction energy (LIE) method to verify the docking results. The MD/LIE binding free energies of selected protein-inhibitor complexes agree overall with the binding affinities from docking and demonstrate that amantadine and rimantadine only weakly bind at the explored site. The MD simulations also demonstrated the flexible character of a protein loop that is located between the β2 and β3 strands and is part of the AdoMet-binding pocket of NS5MTaseDV .

show abstract

A scintillation proximity assay for dengue virus NS5 2′-O-methyltransferase—kinetic and inhibition analyses

Cited by 57 publications

References 42 publications

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides 7MeGpppACn and GpppACn

Enzymatic Assays to Explore Viral mRNA Capping Machinery

Evaluation of Adamantane Derivatives as Inhibitors of Dengue Virus mRNA Cap Methyltransferase by Docking and Molecular Dynamics Simulations

Contact Info

Product

Resources

About