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-U(30) 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 IC(50) 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-C(30) RNA. The FAPA method presented here can be readily adapted for other nucleotide-dependent enzymes that generate PPi.
authors.affiliation: Novartis Institute for Tropical Diseases, Chromos, Singapore.
authors.forename: Pornwaratt
authors.initials: P
authors.lastname: Niyomrattanakit
authors.forename: Siti Nurdiana
authors.initials: SN
authors.lastname: Abas
authors.forename: Chin Chin
authors.initials: CC
authors.lastname: Lim
authors.forename: David
authors.initials: D
authors.lastname: Beer
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.forename: Yen-Liang
authors.initials: YL
authors.lastname: Chen
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000469:Alkaline Phosphatase / Q000378:metabolism*; D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D054852:Small Molecule Libraries; D013050:Spectrometry, Fluorescence; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D054852:Small Molecule Libraries; D017361:Viral Nonstructural Proteins; D000255:Adenosine Triphosphate; D012324:RNA-Dependent RNA Polymerase; D000469:Alkaline Phosphatase