Drug inhibition assays

Drug treatment with TDP2 inhibitors JK-3-121 and SF-V-153⁴⁰ or with entecavir (ETV, Sigma-Aldrich, St Louis, MO) were performed in 96-well plates seeded with either PHH’s or hNTCP-eGFP HepG2 cells. TDP2 inhibitors were solubilized in 100% DMSO and were stored at −20 °C while ETV was solubilized in sterile 1× PBS. Prior to drug treatment, cells were treated with 0.5% DMSO for 24 h and then were challenged with HBV derived from HepG2.2.15 cells that had been heparin column purified. Once persistent HBV infection was established (day 16) drug treatment was started. A series of concentrations was used for each drug. For TDP2 inhibitors JK-3-121 and SV-F-153 concentrations of 1x10⁴, 1x10³, 1x10², and 10 nM, stocks of each TDP2 inhibitor were created in order to maintain the same level of DMSO. ETV was used at final concentrations of 250, 125, and 25 nM. Drug treatment was performed over 18 days during which the inhibitors were freshly supplied every 2 days at each media change with monitoring of HbsAg and hAlb levels over this period of time. At the end of the 18 days, cells were lysed with lysis buffer (50 mM Tris-Base, 50 mM EDTA, 1% SDS, 100 mM NaCl pH 8.0) or with Qiagen RLT supplemented with 2-Mercaptoethanol for 10 min at RT. Total HBV DNA, pgRNA, and cccDNA were quantified. As a control, a set of wells were only challenged with HBV and had no drug treatment administered over the course of the experiment. Ever condition was performed in sextuplets.

Prophylactic drug treatment with TDP2 inhibitors JK-3-121 and SF-V-153 or with ETV was performed in 96-well plates seeded with either PHH’s or hNTCP-eGFP HepG2 cells. TDP2 inhibitors were solubilized in 100% DMSO and were stored at −20 °C while ETV was solubilized in sterile 1× PBS. 1 day prior to HBV challenge cells were treated with respective concentration of either TDP2 inhibitors or ETV. Cells were then challenged with HBV derived from HepG2.2.15 cells that were heparin column purified in the presence of the respective drug. Every 2 days, media was collected and new media with the respective concentration of inhibitor was added in order to maintain a constant level of drug throughout the experiment. For TDP2 inhibitors, JK-3-121 and SV-F-153 concentrations of 1x10⁴, 1x10³, 1x10², and 10 nM, stocks of each TDP2 inhibitor were created in order to maintain the same level of DMSO. ETV was used at final concentrations of 250, 125, and 25 nM. Drug treatment was performed over 34 days for SACC-PHHs and for 19 days for hNTCP-eGFP HepG2 cells with monitoring of HbsAg and hAlb levels over this period of time. At the end of the 34 or 19 days, cells were lysed with lysis buffer (50 mM Tris-Base, 50 mM EDTA, 1% SDS, 100 mM NaCl pH 8.0) or with Qiagen RLT supplemented with 2-Mercaptoethanol for 10 min at RT. Total HBV DNA, pgRNA, and cccDNA were quantified. As a control, a set of wells were only challenged with HBV and had no drug treatment administered over the course of the experiment. Ever condition was performed in sextuplets.