A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-Dependent RNA polymerase

Pornwaratt Niyomrattanakit, Siti Nurdiana Abas, Chin Chin Lim, David Beer, Pei-Yong Shi, Yen Liang Chen

Research output: Contribution to journalArticle

33 Citations (Scopus)

Abstract

The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3′UTR-U30 RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBT PPi, which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3′dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC50 values of 0.13 μM and 0.01 μM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 μM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3′UTR-C30 RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.

Original languageEnglish (US)
Pages (from-to)201-210
Number of pages10
JournalJournal of Biomolecular Screening
Volume16
Issue number2
DOIs
StatePublished - Feb 2011
Externally publishedYes

Fingerprint

RNA Replicase
Dengue Virus
Viruses
Alkaline Phosphatase
Assays
Fluorescence
Nucleotides
Adenosine Triphosphate
RNA
High-Throughput Screening Assays
Guanosine Triphosphate
Fluorophores
Inhibitory Concentration 50
Substrates
Reaction kinetics
Adenosine
Byproducts
Screening
Throughput
Enzymes

Keywords

  • dengue virus
  • FAPA
  • HTS
  • NS5
  • RNA-dependent RNA polymerase

ASJC Scopus subject areas

  • Analytical Chemistry
  • Drug Discovery
  • Pharmacology
  • Biochemistry
  • Molecular Medicine
  • Biotechnology

Cite this

A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-Dependent RNA polymerase. / Niyomrattanakit, Pornwaratt; Abas, Siti Nurdiana; Lim, Chin Chin; Beer, David; Shi, Pei-Yong; Chen, Yen Liang.

In: Journal of Biomolecular Screening, Vol. 16, No. 2, 02.2011, p. 201-210.

Research output: Contribution to journalArticle

Niyomrattanakit, Pornwaratt ; Abas, Siti Nurdiana ; Lim, Chin Chin ; Beer, David ; Shi, Pei-Yong ; Chen, Yen Liang. / A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-Dependent RNA polymerase. In: Journal of Biomolecular Screening. 2011 ; Vol. 16, No. 2. pp. 201-210.
@article{6bb679c76edc4433a8a6215d73bc8aa7,
title = "A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-Dependent RNA polymerase",
abstract = "The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3′UTR-U30 RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBT PPi, which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3′dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC50 values of 0.13 μM and 0.01 μM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 μM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3′UTR-C30 RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.",
keywords = "dengue virus, FAPA, HTS, NS5, RNA-dependent RNA polymerase",
author = "Pornwaratt Niyomrattanakit and Abas, {Siti Nurdiana} and Lim, {Chin Chin} and David Beer and Pei-Yong Shi and Chen, {Yen Liang}",
year = "2011",
month = "2",
doi = "10.1177/1087057110389323",
language = "English (US)",
volume = "16",
pages = "201--210",
journal = "Journal of Biomolecular Screening",
issn = "1087-0571",
publisher = "SAGE Publications Inc.",
number = "2",

}

TY - JOUR

T1 - A fluorescence-based alkaline phosphatase-coupled polymerase assay for identification of inhibitors of dengue virus RNA-Dependent RNA polymerase

AU - Niyomrattanakit, Pornwaratt

AU - Abas, Siti Nurdiana

AU - Lim, Chin Chin

AU - Beer, David

AU - Shi, Pei-Yong

AU - Chen, Yen Liang

PY - 2011/2

Y1 - 2011/2

N2 - The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3′UTR-U30 RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBT PPi, which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3′dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC50 values of 0.13 μM and 0.01 μM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 μM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3′UTR-C30 RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.

AB - The flaviviral RNA-dependent RNA polymerase (RdRp) is an attractive drug target. To discover new inhibitors of dengue virus RdRp, the authors have developed a fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) for high-throughput screening (HTS). A modified nucleotide analogue (2′-[2-benzothiazoyl]-6′-hydroxybenzothiazole) conjugated adenosine triphosphate (BBT-ATP) and 3′UTR-U30 RNA were used as substrates. After the polymerase reaction, treatment with alkaline phosphatase liberates the BBT fluorophore from the polymerase reaction by-product, BBT PPi, which can be detected at excitation and emission wavelengths of 422 and 566 nm, respectively. The assay was evaluated by examining the time dependency, assay reagent effects, reaction kinetics, and signal stability and was validated with 3′dATP and an adenosine-nucleotide triphosphate inhibitor, giving IC50 values of 0.13 μM and 0.01 μM, respectively. A pilot screen of a diverse compound library of 40,572 compounds at 20 μM demonstrated good performance with an average Z factor of 0.81. The versatility and robustness of FAPA were evaluated with another substrate system, BBT-GTP paired with 3′UTR-C30 RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.

KW - dengue virus

KW - FAPA

KW - HTS

KW - NS5

KW - RNA-dependent RNA polymerase

UR - http://www.scopus.com/inward/record.url?scp=79951988793&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=79951988793&partnerID=8YFLogxK

U2 - 10.1177/1087057110389323

DO - 10.1177/1087057110389323

M3 - Article

VL - 16

SP - 201

EP - 210

JO - Journal of Biomolecular Screening

JF - Journal of Biomolecular Screening

SN - 1087-0571

IS - 2

ER -