Streptavidin coated 384 well plates

Streptavidin-coated 384-well plates (Thermo Fisher Scientific) were used for HTS to find compounds with anti-RT activity. Biotinylated poly (rA)₅₀/oligo (dT)₈ templates (25 pmol/well) were bound to streptavidin-coated plates as described above. A total of 10 µM of compound and purified RT were incubated for 2 h at 37 °C in the reaction buffer containing DIG-dUTP. The reaction was stopped by addition of 50 mM EDTA. The following steps were performed in a manner similar to the protocol used for the detection of HBV RT polymerase, with the exception of the different buffer volume. For primary mass screening of compounds, all compounds that inhibited over 70% were considered first round hits (26/20,000 = 0.13%). For secondary screening, compounds that were filled as the selection standard to be active in the primary screening were further analyzed in a dose-dependent manner to validate hits. The compounds were used at 10 different concentrations (130, 43, 14, 4.8, 1.6, 0.5, 0.18, 0.06, 0.02, 0.007 µM). Three compounds that inhibited in a dose-dependent manner and showed under 10 µM of IC₅₀ values were selected for the cell-based analysis.