Figure 1. NVP-BHG712 is a small molecule compound that targets CTSK.
A, Chemical formulas of the ligands KWZ, OLC, I37 and 7AS for the molecular conformation of CTSK. B, Images of molecular conformations of CTSK (3KWZ, 4DMY, 4X6H and 5TDI) bound to ligands (KWZ, OLC, I37 and 7AS). C, Molecular docking diagram of the small molecule compound NVP-BHG712 with the four molecular conformations of CTSK. D, Chemical structure of NVP-BHG712.
Figure 2.NVP-BHG712 inhibited RANKL-induced osteoclastogenesis and bone resorption in vitro.
A, CCK-8 analysis of the cytotoxicity of NVP-BHG712 in RAW264.7 cells and BMMs. Experiments were repeated 3 times. B, C Formation of TRAP-positive cells from RANKL-treated BMMs treated with or without different concentrations of NVP-BHG712 (0, 0.1, 0.2, 0.4, 0.8, or 1.6 μM) for 7 days (B) and quantification of osteoclast numbers (C). D, E Representative images of pit formation by osteoclasts derived from RANKL-treated BMMs with or without different concentrations of NVP-BHG712 (0, 0.1, 0.2, or 0.4 μM) (D) and quantification of pit area (E). The data are presented as the mean ± SD (n=3) (***P <.001)
Figure 3.NVP-BHG712 inhibited osteoclastogenesis and F-actin ring formation in mature osteoclasts at the early phase in vitro.
A, B Representative images of the formation of TRAP-positive cells treated with 0.4 μM NVP-BHG712 (Group 1: without NVP-BHG712) at different time points and four durations (Group 2-5: day 1-3, day 3-5, day 5-6, day 1-6, respectively) (A) and quantification of osteoclast numbers (B). C, D Representative immunofluorescence images of the F-actin ring structures of mature osteoclasts derived from RANKL-induced BMMs treated with 0.4 μM NVP-BHG712 (Group 1: without NVP-BHG712) at different time points and for durations (Group 2-5: day 1-3, day 3-5, day 5-6, day 1-6, respectively) (C) and quantification of F-actin ring formation of osteoclasts (D). Group C:M-CSF, Group R: M-CSF+RANKL, Group N: M-CSF+RANKL+NVP-BHG712. The data are presented as the mean ± SD (n=3) (*P <.05, **P <.01, ***P <.001).
Figure 4.NVP-BHG712 inhibited the RANKL-induced expression of osteoclast differentiation-related genes. A-D, Representative Western blots of the protein expression of CTSK, MMP9 and CTR in osteoclasts derived from RANKL-treated BMMs treated with or without 0.4 μM NVP-BHG712 (A) and the quantification of CTSK (B), MMP9 (C) and CTR (D) expression. E-F, Representative quantification of the mRNA expression of MMP9 (E) and CTR (F) in osteoclasts derived from RANKL-treated BMMs treated with or without 0.4 μM NVP-BHG712 by qPCR.
G, H Representative Western blots of the protein expression of CTSK in osteoclasts derived from RANKL-treated BMMs treated with or without different concentrations of NVP-BHG712 (0, 0.1, 0.2, or 0.4 μM) (G) and the quantification of CTSK (H) expression. I, J Representative Western blots of the protein expression of CTSK in osteoclasts derived from RANKL-treated BMMs treated with or without 0.4 μM NVP-BHG712 for different time periods (days 1, 2, 3, and 4) (I) and the quantification of CTSK (J) expression. Group C:M-CSF, Group R: M-CSF+RANKL, Group N: M-CSF+RANKL+NVP-BHG712. The data are presented as the mean ± SD (n=3) (*P <.05, *P <.01, ***P <.001).