DENV NS5 Polymerase Inhibitor Assay

The cell-based experiments were performed as previously described³³ (link). Briefly, the Huh-7 cells were transfected with 0.5 µg of p(+)RLuc-(–)DV-UTRΔC-FLuc and DENV NS5 expression vector pcDNA-NS5-Myc followed by compound treatment for 3 days. The RLuc and FLuc activities were analysed by Dual-Glo Luciferase Assay System³⁷ (link). The enzyme-based fluorescence-based alkaline phosphatase-coupled polymerase assay (FAPA) was performed as previously described³³ (link). Briefly, the template was amplified from the cDNA of DENV-2 minus strand 3′-UTR and its RNA was synthesised by the T7 Megascript kit³⁸ (link). The 100 nM DENV NS5 protein was incubated with 1, 5 or 10 µM test compound, 100 nM (−) 3′-UTR RNA, 20 µM CTP, GTP, UTP and 20 µM BBT-ATP (Jena Bioscience) in a total volume of 30 µL in assay buffer (50 mM Tris-HCl, pH 7.5, 10 mM KCl, 1 mM MgCl₂, 0.3 mM MnCl₂, 0.001% Triton X-100 and 10 µM cysteine) at 25 °C for 60 min. The 30 µL of 2.5× stop buffer (200 mM NaCl, 25 mM MgCl₂, 1.5 M diethanolamine, pH 10) with 25 nM calf intestinal alkaline phosphatase (Promega) was added to the reaction after 60-min incubation. The fluorescence signal was measured at excitation wavelength of 422 nm and emission wavelength of 566 nm, respectively.