Figure 4
Amodiaquine inhibits host cathepsin B.
(a) AQ does not inhibit PA binding to RAW264.7 cells. Cells were incubated with AQ for 1 h at 4 °C before addition of 1 μg/mL PA for an additional 1 h. Cells were lysed and analyzed by immunoblotting with a PA-specific antibody. (b) AQ neutralizes acidic vesicles. RAW264.7 cells were pre-treated with AQ or vehicle control for 1 h and then treated with 500 ng/ml of PA for an additional hour at 37 °C before addition of Lysosensor Green DND-189 for a further 10 min. Cells were then visualized by fluorescence microscopy. (c) AQ results in increased abundance of PA pores. Cells were pre-treated with AQ for 1 h at 37 °C and then were exposed to 1 μg/mL of PA at 37 °C for 1 h. Cells were lysed and analyzed by immunoblotting with a PA-specific antibody. (d). FRET assay showing the activity of purified human cathepsin B without drugs, or with addition of AQ, DEAQ, CQ, or DECQ at 4, 8, 16, 33, or 66 μM. (e) 1H-NMR spectra of AQ in the presence of cathepsin B. The reference spectrum of AQ with its atoms labeled (top) and the STD spectrum (bottom) are shown. The data were collected using a protein:ligand ratio of 1:100 with on- and off-resonance saturation at 0.04 ppm and 30 ppm, respectively. (f) Chemical structure of AQ showing the atom-specific magnitude of the STD effects. The STD effect was calculated according to the formula ASTD=(I0 − Isat)/I0. All STD effects are expressed as a percentage relative to the H4 atom (100%).
