Search

Your search keyword '"Scintillation proximity assay"' showing total 4 results
Start Over Descriptor "Scintillation proximity assay" Journal antiviral research
4 results on '"Scintillation proximity assay"'

1. A scintillation proximity assay for dengue virus NS5 2'-O-methyltransferase-kinetic and inhibition analyses

Author

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

Subjects
RNA capping, Adenosine, GTP', Biology, Biochemistry, Methylation, Sensitivity and Specificity, Sinefungin, Viral Proteins, Virology, Binding site, Enzyme Inhibitors, Pharmacology, chemistry.chemical_classification, Binding Sites, RNA, Methyltransferases, Dengue Virus, O-methyltransferase, S-Adenosylhomocysteine, Kinetics, Enzyme, Scintillation proximity assay, chemistry, biology.protein, Scintillation Counting, Guanosine Triphosphate
Abstract

Dengue virus (DENV) NS5 possesses methyltransferase (MTase) activity at its N-terminal amino acid sequence and is responsible for formation of a type 1 cap structure, m(7)GpppAm(2'-O) in the viral genomic RNA. Optimal in vitro conditions for DENV2 2'-O-MTase activity were characterized using purified recombinant protein and a short biotinylated GTP-capped RNA template. Steady-state kinetics parameters derived from initial velocities were used to establish a robust scintillation proximity assay for compound testing. Pre-incubation studies showed that MTase-AdoMet and MTase-RNA complexes were equally catalytically competent and the enzyme supports a random bi bi kinetic mechanism. The assay was validated with competitive inhibitory agents, S-adenosyl-homocysteine and two homologues, sinefungin and dehydrosinefungin. A GTP-binding pocket present at the N-terminal of DENV2 MTase was previously postulated to be the cap-binding site. Interestingly, inhibition of the enzyme by GTP was two-fold lower than with RNA cap analogues, G[5']ppp[5']A and m(7)G[5']ppp[5']A and about three-fold poorer than a two-way methylated analogue, m(7)G[5']ppp[5']m(7)G. This assay allows rapid and highly sensitive detection of 2'-O-MTase activity and can be readily adapted for high-throughput screening for inhibitory compounds. It is suitable for determination of enzymatic activities of a wide variety of RNA capping MTases.

Published
2008

2. Small molecule modulators of HIV Rev/Rev response element interaction identified by random screening

Author

Richard J. Hazen, Richard L. Chapman, Thomas B. Stanley, and Edward P. Garvey

Subjects
Molecular Sequence Data, Electrophoretic Mobility Shift Assay, Biology, Antiviral Agents, Binding, Competitive, Genes, env, Inhibitory Concentration 50, Structure-Activity Relationship, Virology, Humans, Electrophoretic mobility shift assay, Amino Acid Sequence, Surface plasmon resonance, Structural motif, Pharmacology, HIV, rev Gene Products, Human Immunodeficiency Virus, Surface Plasmon Resonance, Molecular biology, Binding constant, Small molecule, Scintillation proximity assay, Gene Products, rev, Biotinylation, Biophysics, RNA, Viral, Scintillation Counting, Macromolecule
Abstract

A high throughput scintillation proximity assay with biotinylated human immunodeficiency virus (HIV) Rev protein and tritiated Rev response element RNA was used to screen over 500,000 small molecules. Several chemical classes of inhibitors and two chemical classes of enhancers of binding were identified, with the molecular weight range being 400-600. The most common structural motif of inhibitor was an acidic moiety at the end of a linear aromatic system. Most of these modulators had EC(50) values in the 1-10 microM potency range, with several below 1 microM. Several classes displayed structure-activity relationships suggesting specific molecular interactions between small molecule and macromolecule. Several molecules were confirmed as inhibitors in a gel shift assay and by surface plasmon resonance analysis. Furthermore, one inhibitor was shown to bind the Rev protein with a binding constant equal to its IC(50) value, consistent with the mechanism of inhibition being binding Rev. Thus, small molecules can modulate this macromolecular protein-RNA interaction in vitro. However, no compound demonstrated HIV antiviral activity in a relevant cell-based assay.

Published
2002

3. Ten years of dengue drug discovery: progress and prospects.

Author

Lim SP, Wang QY, Noble CG, Chen YL, Dong H, Zou B, Yokokawa F, Nilar S, Smith P, Beer D, Lescar J, and Shi PY

Subjects
History, 21st Century, Humans, Singapore, Antiviral Agents isolation & purification, Dengue drug therapy, Dengue virology, Dengue Virus drug effects, Drug Discovery history, Drug Discovery trends
Abstract

To combat neglected diseases, the Novartis Institute of Tropical Diseases (NITD) was founded in 2002 through private-public funding from Novartis and the Singapore Economic Development Board. One of NITD's missions is to develop antivirals for dengue virus (DENV), the most prevalent mosquito-borne viral pathogen. Neither vaccine nor antiviral is currently available for DENV. Here we review the progress in dengue drug discovery made at NITD as well as the major discoveries made by academia and other companies. Four strategies have been pursued to identify inhibitors of DENV through targeting both viral and host proteins: (i) HTS (high-throughput screening) using virus replication assays; (ii) HTS using viral enzyme assays; (iii) structure-based in silico docking and rational design; (iv) repurposing hepatitis C virus inhibitors for DENV. Along the developmental process from hit finding to clinical candidate, many inhibitors did not advance beyond the stage of hit-to-lead optimization, due to their poor selectivity, physiochemical or pharmacokinetic properties. Only a few compounds showed efficacy in the AG129 DENV mouse model. Two nucleoside analogs, NITD-008 and Balapiravir, entered preclinical animal safety study and clinic trial, but both were terminated due to toxicity and lack of potency, respectively. Celgosivir, a host alpha-glucosidase inhibitor, is currently under clinical trial; its clinical efficacy remains to be determined. The knowledge accumulated during the past decade has provided a better rationale for ongoing dengue drug discovery. Though challenging, we are optimistic that this continuous, concerted effort will lead to an effective dengue therapy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Published
2013
Full Text
View/download PDF

4. Development of specific dengue virus 2'-O- and N7-methyltransferase assays for antiviral drug screening.

Author

Barral K, Sallamand C, Petzold C, Coutard B, Collet A, Thillier Y, Zimmermann J, Vasseur JJ, Canard B, Rohayem J, Debart F, and Decroly E

Subjects
Antiviral Agents pharmacology, Dengue drug therapy, Dengue Virus drug effects, Dengue Virus genetics, Dengue Virus metabolism, Enzyme Inhibitors pharmacology, Humans, Methyltransferases antagonists & inhibitors, Methyltransferases genetics, Methyltransferases metabolism, RNA Caps genetics, RNA Caps metabolism, RNA, Messenger genetics, RNA, Messenger metabolism, RNA, Viral genetics, RNA, Viral metabolism, Viral Nonstructural Proteins antagonists & inhibitors, Viral Nonstructural Proteins genetics, Viral Nonstructural Proteins metabolism, Dengue virology, Dengue Virus enzymology, Drug Evaluation, Preclinical methods, Enzyme Assays methods, Methyltransferases analysis, Viral Nonstructural Proteins analysis
Abstract

Dengue virus (DENV) protein NS5 carries two mRNA cap methyltransferase (MTase) activities involved in the synthesis of a cap structure, (7Me)GpppA(2'OMe)-RNA, at the 5'-end of the viral mRNA. The methylation of the cap guanine at its N7-position (N7-MTase, (7Me)GpppA-RNA) is essential for viral replication. The development of high throughput methods to identify specific inhibitors of N7-MTase is hampered by technical limitations in the large scale synthesis of long capped RNAs. In this work, we describe an efficient method to generate such capped RNA, GpppA(2'OMe)-RNA₇₄, by ligation of two RNA fragments. Then, we use GpppA(2'OMe)-RNA₇₄ as a substrate to assess DENV N7-MTase activity and to develop a robust and specific activity assay. We applied the same ligation procedure to generate (7Me)GpppA-RNA₇₄ in order to characterize the DENV 2'-O-MTase activity specifically on long capped RNA. We next compared the N7- and 2'-O-MTase inhibition effect of 18 molecules, previously proposed to affect MTase activities. These experiments allow the validation of a rapid and sensitive method easily adaptable for high-throughput inhibitor screening in anti-flaviviral drug development., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Published
2013
Full Text
View/download PDF

Catalog

Books, media, physical & digital resources
See catalog results