Abstract: To verify the effects of two compounds extracted from Saururus chinensis (Lour.) Baill,XGN56 and XGN59,on the enzyme activity of autophagy key protein ATG4B and the regulation of autophagy.The hydrogen bond conjugation of free ATG4B and ATG4B-LC3 complex with compounds were verified by molecular docking;SDS-PAGE and fluorescence resonance energy transfer method (FRET) were used to determine the IC₅₀ value of these two compounds (10 μmol/L) inhibiting ATG4B;LC3 fusion with GFP fluorescent tag protein was used to detect the effect of these two compounds (10 μmol/L) on the accumulation of LC3 puncta,and cells were divided into control group,compounds group and compounds coupled with Baf group (0.5 μmol/L);WT-MEF and ATG5^-/--MEF cells overexpressed with GFP-LC3 were employed to detect the inducment of LC3 puncta by these two compounds..Results showed that XGN56 and XGN59 can form hydrogen bonds with both free ATG4B and ATG4B-LC3 complex respectively.Furthermore,both compounds could effectively inhibit the enzymatic activity of ATG4B in a concentration-dependent manner,with IC₅₀ of 7.74 and 8.00 μmol/L respectively,and could promote the formation of GFP-labeled autophagosomes (P<0.001).All these results indicate that XGN56 and XGN59 promote the autophagy activity probably via the inhibition of ATG4B to a certain extent.

Key words: ATG4B, Saururus chinensis L., molecular docking, inhibitors, autophagy