Small Molecule Inhibitors Discovery And Studies Against Autophagy-related Protein ATG4B

Autophagy is an evolutionarily conserved process for the turnover of intracellular materials in eukaryotes.Transport a series of damaged organelles,misfolded proteins and in-tracellular pathogens to lysosomes for digestion and degradation via autophagy,thus providing the cells with the nutrients and materials needed for self-renewal.It is a very important biological phenomenon,involved in many stages of growth and development of eukaryotes like apoptosis and senescence.Autophagy is tightly controlled by a number of autophagy-related proteins and protein complexes,each of which is responsible for regulating different stages of autophagy initiation and formation.Among them,autophagy-related protein ATG4B plays an important role in regulating autophagy.Through cleavage the terminal amino acids of the autophagy core protein LC3 promotes lipidation and delipidation of LC3,which affects the formation of autophagosomes.Studies have shown that ATG4B is closely related to cancer cell growth and anti-cancer drug resistance by regulating autophagy.ATG4B protein activity inhibiting will lead to impaired autophagy process.Therefore,ATG4B is an attractive target for cancer treatment.ATG4B inhibitors have been reported,such as the covalent compound FMK9a discovered by Roche,S130 discovered by in-silico screening at Sun YAT-Sen University,etc.However,the research of ATG4B inhibitors is still at an early stage.On the one hand,the lack of efficient and standardized screening systems;on the other hand,some ATG4B small molecule inhibitors have drawbacks including weak potency,incomplete evaluation of cell activity and poor selectivity.Therefore,the purpose of this study is to find more promising new small molecule inhibitors targeting ATG4B protein.Based on the mechanism of ATG4B enzyme cleavage substrate LC3B,a high-through-put screening method of HTRF targeting ATG4B small molecule inhibitors was optimized and established in this study.By screening a tailored 2000 compounds with the HTRF assay,we have identified a novel ATG4B inhibitor named DC-ATG4in.The IC₅₀ value of DC-ATG4in was 3.08±0.47μM.The ability of DC-ATG4in inhibiting the cleavage of substrate LC3B by ATG4B was further verified by SDS-PAGE gel electrophoresis.Mass spectrometry,¹H-nuclear magnetic resonance and surface plasmon resonance confirmed that DC-ATG4in inhibitor was non-covalent with a binding affinity of 14.33±1.52μM.Moreover,western blot and immunofluorescence experiments confirmed that DC-ATG4in was an autophagy inhibitor,which could block the activation of autophagy induced by sorafenib.More importantly,DC-ATG4in combined with sorafenib synergically enhanced the inhibition activities in hepatocellular carcinoma cells.In conclusion,on the one hand,this study successfully established a high-throughput HTRF screening platform targeting ATG4B small molecule inhibitors,and obtained a novel compound DC-ATG4in,which proved that DC-ATG4in has the activity of inhibiting ATG4B cleavage substrate LC3B.The discovery of this inhibitor has enriched the structural diversity of existing ATG4B small molecule inhibitors,and the new chemical scaffold provides a new direction for the development of more effective ATG4B inhibitors.On the other hand,DC-ATG4in inhibited the process of autophagy and acted synergistically with Sorafenib in suppressing the growth of HCC cancer cells.This conclusion suggests that targeting ATG4B protein to inhibit autophagy induced by anticancer drugs and enhance the sensitivity of tumor cells to anticancer drugs may be a potential new clinical strategy for cancer treatment.