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 language | English (US) |
|---|---|
| Pages (from-to) | 201-210 |
| Number of pages | 10 |
| Journal | Journal of Biomolecular Screening |
| Volume | 16 |
| Issue number | 2 |
| DOIs | |
| State | Published - Feb 2011 |
| Externally published | Yes |
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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 journal › Article
}
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 -