3.3. HIV-1 Reverse Transcriptase and HBV Polymerase Activity Assays

HIV-1 reverse transcriptase (HIV-1 RT) was purchased from Abcam (cat#ab63979). Recombinant HBV polymerase (Hepatitis B virus genotype D subtype ayw, full length) was cloned using the baculovirus system, expressed in sf9 cells, and purified with similar strategy and methods described by Lanford et al. [39]. A DNA primer (5′-CCGAGTAGTGTTGG-3′) was synthesized by IDTDNA and a 358-nt RNA template was synthesized in-house using Megascript T7 transcription kit (ThermoFisher, cat#AM1334). dNTPs were purchased from Thermo Fisher and ³H-dTTP from Perkin Elmer. Filter plates were purchased from Millipore (cat#MABN0V050) and microscint-20 was purchased from Perkin Elmer (cat#6013621).

The RNA-dependent DNA polymerization (RdDp) activity of HIV-1 RT was measured by the incorporation of radioactively labeled nucleotides by HIV-1 RT into acid-insoluble DNA products from the DNA primer primed RNA template. To test compound inhibition, the reactions were performed at 30 °C for 40 min in a reaction mixture containing reaction buffer (50 mM Tris-Cl, pH 7.5, 100 mM KCl, 12.5 mM MgCl₂, 4 mM DTT), 1 nM HIV-1 RT, 0.1 µM DNA primer, 0.02 µM RNA template, 10% DMSO, 0.1 µM dATP, 1 µM dGTP, 0.1 µM dCTP, 0.32 µM ³H-dTTP, and compounds at various concentrations. The 50 µL reactions were performed in 96-well plates. The reactions were quenched with a 60 µL cold mixture of 20% (w/v) TCA and 0.5 mM ATP and incubated at 4 °C for 1 h. The reactions were loaded onto 96-well filter plates. The filters on plates were washed three times with 10% TCA and once with 70% ethanol on a Millipore plate wash station with vacuum applied. The filters on the plate were air-dried and 40 μL Microscint-20 was added to each well. The acid-precipitated tritiated DNA products retained on the filters were detected by a Trilux MicroBeta scintillation reader (Perkin Elmer).

The RdDp activity of HBV polymerase was measured similarly as described for the HIV-1 RT. To test compound inhibition effect, the reactions were performed at 30 °C for 120 min in a reaction mixture containing reaction buffer (50 mM Tris-Cl, pH 7.5, 100 mM KCl, 12.5 mM MgCl₂, 4 mM DTT), 15 µg/mL polymerase, 0.5 µM DNA primer, 0.05 µM RNA template, 10% DMSO, 0.046 µM dATP, 0.057 µM dGTP, 0.017 µM dCTP, 0.32 µM ³H-dTTP, and compounds at various concentrations.

All data were analyzed with GraphPad Prism. The compound concentration at which the enzyme-catalyzed rate was reduced by 50% (IC₅₀) was calculated by fitting the data to the equation Y = % Min + (% Max − % Min) / (1 + 10^((logIC₅₀-X)*h)), where Y corresponds to the percent inhibition to the enzyme activity, % Min is the residual inhibition activity without compound, % Max is the maximum inhibition of enzyme activity at saturating compound concentration, and X corresponds to the log of compound concentrations, and h is the hillslope.