Identification,Validation And Mechanism Investigation Of Small Molecular Inhibitors Targeting Key Autophagy-Related Proteins

Autophagy is a universal and dynamic process whereby eukaryotic cells remove dispensable or toxic cytoplasmic material through lysosomal degradation.Central to autophagy is the formation of double-membraned vesicles,autophagosome.Cellular components are sequestered by autophagosomes,which then fuse with lysosomes to generate autolysosomes and proceed to degradation.Autophagy is among the hottest field in current life scientific research.The Japanese scientist Yoshinori Ohsumi has won the 2016 Nobel Prize in Physiology or Medicine because of his outstanding contributions to the research of the mechanism of autophagy.In this thesis,we focused on small molecules drug discovery targeting the autophagy biomarker LC3B and the proteinase ATG4B.We established the first high through-put screening platform and combined it with docking based virtual screening.After the“hit”obtained,we conducted cross-validation experiments to test the robustness of the hit and then we synthesized derivatives and figured out its mechanism of action in cell based models.We firstly set up a fluorescence based high through-put screening platform targeting LC3-LIR interaction interface.Combined with computer aided drug design approach,docking based virtual screening,we identified the first small molecule targeting LC3B.After several rounds of biophysical methods cross-validation,we are convinced that it was a highly selective covalent inhibitor of LC3B.And we have successfully determined a high-resolution crystal structure of the LC3B-inhibitor complex.We subsequently synthesized several series of derivatives under the guidance of structure based drug design.Among them,one molecule shows a 30-fold more potent affinity than the“hit”,with a K_i value of double digit nano molar.Followed by protein mass spectra analysis,protein-site mutation and comparison with the negative compound with 2-D scaffold highly similar to the“candidate”,we prove that the“candidate”selectively modifies the Lysine 49 of LC3B.It occupies the L-site of LC3B and blocks the interaction of ATG4B and ATG7 with LC3B.Single-digit micro molar“candidate”inhibited the transformation of LC3B both from pro-LC3B to LC3B-I,and from LC3B-I to LC3B-II.The autophagosome formation was reduced and rare autpophagy was observed under the Torin-1 treatment or nutrition deprivation.This study provides an easy to operate,stable and repeatable,low-cost and productive high through-put screening platform for small molecules screening targeting LC3B.And it is the first one up-to-date.The“candidate”shows a valuable profile to be a chemical probe interfering the complex function of LC3B and it potentiates a novel lead for LC3B and autophagy inhibition with a novel scaffold.We also established the first HTRF based platform for small molecular drug discovery of ATG4B.We identified two novel small molecular inhibitors of ATG4B.One“hit”is a covalent allosteric inhibitor,it selectively reacts with one of the cysteine of ATG4B and subsequently inhibits the enzymatic activity of ATG4B to LC3B.The other one is a reversible inhibitor and it showed a competitive inhibitory effect of ATG4B’s cleavage of C-terminal tail of LC3B with the same concentration as our“candidate”of LC3B.This molecule is up-to-date the first covalent allosteric inhibitor of ATG4B,which should raise some interest for the dynamic interaction of ATG4B with LC3B.As there is no small molecular inhibitor showed an inhibitory activity in cell models,the latter“hit”and its analogues in this study would be the positive control for the scientific research of ATG4B’s cellular functions.