Figure 5
Selected betulin derivatives were used for HDV infection inhibition of NTCP-HepG2 cells. NTCP-HepG2 cells were preincubated for 5 min with the indicated concentrations of (a) betulinic acid (compound 4), (b) lupenone (compound 17), (c) betulonoyl dimethyl-L-aspartate (compound 19), (d) 20,29-dihydrobetulin (compound 2), and (g) the myr-preS12-48 peptide in DMEM at 37 °C. Then, cells were additionally inoculated with 700 genome equivalents/cell of HDV particles at 37 °C. After 6 h, cells were washed and further incubated with inhibitor- and virus-free medium, and medium was changed every 3–4 days. At day 9 post infection, cells were fixed and an immunostaining against the HDAg was performed, as a marker of HDV infection. The number of infected cells per well was determined by fluorescence microscopy. NTCP-HepG2 cells incubated without inhibitor were used as control (set to 100% infection rate). Infection experiments in the presence of 0.5 µM myr-preS12-48 peptide served as control for the 0% infection rate. Scale bars: 100 µm. Data represent means ± SEM of three independent experiments each with triplicate determinations (n = 9). (e, f) Cells were preincubated with 600 µM of compound 4 and compound 17, respectively, for intervals of 15 min and 6 h at 37 °C. Cells treated with solvent (DMEM) alone served as control (set to 100%) and data from cells without induction of NTCP expression were set to 0%. Subsequently, cells were washed three times with tempered PBS and [3H]TC (1 µM) uptake as well as [3H]preS1 (5 nM) binding were analyzed, respectively. Means of the 0% controls were subtracted and net [3H]TC transport rates and net [3H]preS1 binding rates are expressed as % of control at the y-axis. Data were combined from two independent experiments, each with quadruplicate determinations (n = 8). (h) Cell viability was determined with an LDH cytotoxicity assay. HepG2 cells treated with HGM w/o test compound were used as positive control and lysed cells as negative control. Data show means ± SD of three independent experiments each with triplicate determinations (n = 9). *Significantly different from control with p < 0.01 (two-way ANOVA with Dunnett's multiple comparison test).
